Methods for measuring electronegative LDL biomarkers

Abstract

The invention relates generally to methods of measuring biomarkers of electronegative low-density lipoproteins (LDL(-)) in the blood.

Description

METHODS FOR MEASURING ELECTRONEGATIVE LDL BIOMARKERSCROSS-REFERENCE TO RELATED APPLICATION

[0001] Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 730,876 filed December 11, 2024, the disclosure of which is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The invention relates generally to methods of measuring biomarkers of electronegative low-density lipoproteins (LDL(-)) in the blood.BACKGROUND OF THE INVENTION

[0003] Both elevated blood lipid levels and markers of inflammation are known to predict the risk for atherosclerotic cardiovascular disease (heart attack and stroke), the leading cause of death worldwide. However, these measures fail to identify a considerable proportion of individuals who subsequently experience these clinical events. It has been shown previously that a unique minor subspecies of lipid-carrying low density lipoproteins (LDLs) with an abnormally high negative charge (electronegative LDL or LDL(-)) has properties that promote inflammation in arterial tissue, and there is emerging evidence that levels of this subspecies are substantially elevated in patients with heart attack and stroke independent of standard risk markers. The current methodology for measuring electronegative LDL requires ion exchange chromatography and is extremely complex. This renders the current methodology unsuitable for clinical laboratory use.

[0004] It would be an advancement in the art to measure the electronegative LDL content in the blood of a subject by a method amendable to routine use in clinical laboratories. Such methods, as well as other inventions, are described herein.SUMMARY OF THE INVENTION

[0005] In one aspect, the invention provides a method for determining a cardiovascular condition, or a predisposition to develop the cardiovascular condition, of a patient, the method comprising: (a) separating lipoprotein particles having an average density of fromabout 1 .020 g / mL to about 1 .060 g / mL from the blood of said patient, wherein the product of step (a) comprises LDL(-) biomarkers; (b) converting the LDL(-) biomarkers into charged LDL(-) biomarkers; (c) determining the charged LDL(-) biomarkers concentration; and (d) determining said cardiovascular disease, or a predisposition to develop said cardiovascular condition, of the patient. In an exemplary embodiment, the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”). In an exemplary embodiment, the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent. In an exemplary embodiment, the LDL(-) biomarkers have a diameter from about 28.0 nm to about 32.0 nm as measured by ion mobility (“IDL-sized”). In an exemplary embodiment, the invention further comprises prescribing and / or administering a cardiovascular condition therapy to the subject when the cardiovascular condition is determined by the product of step (d). In an exemplary embodiment, the invention further comprises prescribing and / or administering a cardiovascular condition prophylaxis to the subject when the predisposition to develop said cardiovascular condition is determined by the product of step (d). In an exemplary embodiment, the charged LDL(-) biomarkers from the patient are classified on the basis of mobility by a differential mobility analyzer, and the mobility classification is scanned to create the charged LDL(-) biomarkers concentration. In an exemplary embodiment, the charged LDL(-) biomarkers concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition. In an exemplary embodiment, the invention further comprises, between step (c) and step (d), (c’) comparing the product of step (c) to a charged LDL(-) biomarkers reference concentration. In an exemplary embodiment, the charged LDL(-) biomarkers reference concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition.

[0006] In another aspect, the invention provides a method for determining a level of therapeutic responsiveness to a cardiovascular drug or other therapeutic intervention directed at a cardiovascular condition of a patient, comprising: (a) administering said cardiovascular drug or other therapeutic intervention to said patient; (b) obtaining lipoprotein particles from said patient; (c) separating lipoprotein particles having an average density of from about 1.020 g / mL to about 1.060 g / mL from the product of step (b) wherein the product of step (c) comprises LDL(-) biomarkers; (d) converting theLDL(-) biomarkers into charged LDL(-) biomarkers; (e) determining the charged LDL(-) biomarkers concentration; and (f) determining the level of therapeutic responsiveness to a cardiovascular drug or other therapeutic intervention directed at a cardiovascular condition of a patient. In an exemplary embodiment, the invention further comprises (g) reducing the amount of said cardiovascular drug or other therapeutic intervention to said patient when the product of step (f) is above the desired therapeutic responsiveness; or increasing the amount of said cardiovascular drug or other therapeutic intervention to said patient when the product of step (f) is below the desired therapeutic responsiveness. In an exemplary embodiment, the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”). In an exemplary embodiment, the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent. In an exemplary embodiment, the LDL(-) biomarkers have a diameter from about 28.0 nm to about 32.0 nm as measured by ion mobility (“IDL- sized”). In an exemplary embodiment, the charged LDL(-) biomarkers are classified on the basis of mobility by a differential mobility analyzer, and the mobility classification is scanned to create the charged LDL(-) biomarkers concentration. In an exemplary embodiment, the charged LDL(-) biomarkers concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition. In an exemplary embodiment, the invention further comprises between step (e) and step (f), (e’) comparing the product of step (e) to a charged LDL(-) biomarkers reference concentration. In an exemplary embodiment, the charged LDL(-) biomarkers reference concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition.

[0007] In another aspect, the invention provides a method for determining a cardiovascular condition, or a predisposition to develop the cardiovascular condition, of a patient, the method comprising: (a) obtaining a charged LDL(-) biomarkers concentration from the patient; and (b) determining the cardiovascular condition or the predisposition to develop the cardiovascular condition, of the patient. In an exemplary embodiment, the invention further comprises prescribing and / or administering a cardiovascular condition therapy to the subject when the cardiovascular condition is determined by the product of step (b). In an exemplary embodiment, the invention further comprises prescribing and / or administering a cardiovascular conditionprophylaxis to the subject when the predisposition to develop said cardiovascular condition is determined by the product of step (b). In an exemplary embodiment, the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”). In an exemplary embodiment, the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent. In an exemplary embodiment, the LDL(-) biomarkers have a diameter from about 28.0 nm to about 32.0 nm as measured by ion mobility (“IDL-sized”). In an exemplary embodiment, at least one of the steps of (b) and (c) is carried out by a deterministic algorithm. In an exemplary embodiment, the charged LDL(-) biomarkers concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition. In an exemplary embodiment, the invention further comprises, between step (a) and step (b), (a’) comparing the product of step (a) to a charged LDL(-) biomarkers reference concentration. In an exemplary embodiment, at least one of the charged LDL(-) biomarkers reference concentrations correlates with the cardiovascular condition, or the predisposition to develop the cardiovascular condition.

[0008] In another aspect, the invention provides a method for assessing lipid -related health risk comprising the steps of: (a) separating lipoprotein particles having an average density of from about 1.020 g / mL to about 1.060 g / mL from the blood of said patient, wherein the product of step (a) comprises LDL(-) biomarkers; (b) adding to the product of step (a) one or more diluents, thereby forming a diluted sample; (c) electrospraying said diluted sample; (d) reducing the electrosprayed sample to a uniform charge; (e) passing said uniformly charged diluted sample to a volatilizing chamber to evaporate said diluents, forming individual uniformly charged LDL(-) biomarker ions; (f) transporting said individual uniformly charged LDL(-) biomarker ions into a size- selectable differential mobility analyzer; (g) counting the number of individual uniformly charged LDL(-) biomarker ions, dn+, in a defined sampling time, dt, at each size selection, s, of d»+the selectable differential mobility analyzer,resulting in a size output; (h) scanning the size output over a range of size selections, resulting in an output histogram of charged LDL(-) biomarkers counted in the defined sampling time versus size; and (i)assessing the lipid-related health risk. In an exemplary embodiment, the invention further comprises prescribing and / or administering a lipid-related health risk therapy to the subject when the lipid-related health risk therapy is determined by the product of step (i). In an exemplary embodiment, the range of size selections is from about 13.0 nm to about 18.0 nm. In an exemplary embodiment, the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent. In an exemplary embodiment, the range of size selections is from about 28.0 nm to about 32.0 nm. In an exemplary embodiment, the output histogram of charged LDL(-) biomarkers correlates with the lipid-related health risk. In an exemplary embodiment, the invention further comprises between step (h) and step (i), (h’) comparing the product of step (h) to a reference output histogram of charged LDL(-) biomarkers counted in the defined sampling time versus size. In an exemplary embodiment, the reference output histogram of charged LDL(-) biomarkers correlates with the lipid-related health risk.

[0009] In another aspect, the invention provides a method for determining a cardiovascular condition, or a predisposition to develop the cardiovascular condition, of a patient, the method comprising: (a) precipitating non-lipoprotein proteins from the blood of said patient, wherein the product of step (a) comprises a supernatant fraction and the supernatant fraction comprises LDL(-) biomarkers; (b) converting the LDL(-) biomarkers into charged LDL(-) biomarkers; (c) determining the charged LDL(-) biomarkers concentration; and (d) determining said cardiovascular disease, or a predisposition to develop said cardiovascular condition, of the patient wherein the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”). In an exemplary embodiment, the invention further comprises prescribing and / or administering a cardiovascular condition therapy to the subject when the cardiovascular condition is determined by the product of step (d). In an exemplary embodiment, the invention further comprises prescribing and / or administering a cardiovascular condition prophylaxis to the subject when the predisposition to develop said cardiovascular condition is determined by the product of step (d). In an exemplary embodiment, the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent. In an exemplary embodiment, the charged LDL(-) biomarkers concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition. In an exemplary embodiment,the invention further comprises, between step (c) and step (d), (c’) comparing the product of step (c) to a charged LDL(-) biomarkers reference concentration. In an exemplary embodiment, the charged LDL(-) biomarkers reference concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition.

[0010] In another aspect, the invention provides a method for determining a level of therapeutic responsiveness to a cardiovascular drug or other therapeutic intervention directed at a cardiovascular condition of a patient, comprising: (a) administering said cardiovascular drug or other therapeutic intervention to said patient; (b) precipitating non-lipoprotein proteins from the blood of said patient, wherein the product of step (a) comprises a supernatant fraction and the supernatant fraction comprises LDL(-) biomarkers; (c) converting the LDL(-) biomarkers into charged LDL(-) biomarkers; (d) determining the charged LDL(-) biomarkers concentration; and (e) determining the level of therapeutic responsiveness to a cardiovascular drug or other therapeutic intervention directed at a cardiovascular condition of a patient wherein the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”). In an exemplary embodiment, the invention further comprises (f) reducing the amount of said cardiovascular drug or other therapeutic intervention to said patient when the product of step (e) is above the desired therapeutic responsiveness; or increasing the amount of said cardiovascular drug or other therapeutic intervention to said patient when the product of step (e) is below the desired therapeutic responsiveness. In an exemplary embodiment, the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent. In an exemplary embodiment, the charged LDL(-) biomarkers concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition. In an exemplary embodiment, the invention further comprises, between step (e) and step (f), (e’) comparing the product of step (e) to a charged LDL(-) biomarkers reference concentration. In an exemplary embodiment, the charged LDL(-) biomarkers reference concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition.

[0011] In another aspect, the invention provides a method for assessing lipid-related health risk comprising the steps of: (a) precipitating non-lipoprotein proteins from the blood of said patient, wherein the product of step (a) comprises a supernatant fraction andthe supernatant fraction comprises LDL(-) biomarkers; (b) adding to the product of step (a) one or more diluents, thereby forming a diluted sample; (c) electrospraying said diluted sample; (d) reducing the electrosprayed sample to a uniform charge; (e) passing said uniformly charged diluted sample to a volatilizing chamber to evaporate said diluents, forming individual uniformly charged LDL(-) biomarker ions; (f) transporting said individual uniformly charged LDL(-) biomarker ions into a size- selectable differential mobility analyzer; (g) counting the number of individual uniformly charged LDL(-) biomarker ions, dn+, in a defined sampling time, dt, at each size selection, s, of the selectable differential mobility analyzer,resulting in a size output; (h) scanning the size output over a range of size selections, resulting in an output histogram of charged LDL(-) biomarkers counted in the defined sampling time versus size; and (i) assessing the lipid-related health risk wherein the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”). In an exemplary embodiment, the invention further comprises prescribing and / or administering a lipid-related health risk therapy to the subject when the lipid-related health risk therapy is determined by the product of step (i). In an exemplary embodiment, the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent. In an exemplary embodiment, the output histogram of charged LDL(-) biomarkers correlates with the lipid-related health risk. In an exemplary embodiment, the invention further comprises, between step (h) and step (i), (h’) comparing the product of step (h) to a reference output histogram of charged LDL(-) biomarkers counted in the defined sampling time versus size. In an exemplary embodiment, the reference output histogram of charged LDL(-) biomarkers correlates with the lipid-related health risk.

[0012] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG 1 shows a method for quantitation of LDL(-) (Chu et al„ Biomedicines 8:254, 2020).

[0014] FIG 2 shows L5 is markedly increased in Ml and stroke patients, and enriched in metabolic syndrome (Chu et al., Biomedicines 8:254, 2020).

[0015] FIG 3 shows a metabolic model for generation of LDL(-) (Musliner, Michenfelder, Krauss, J. Lipid Res. 29: 349, 1988; Musliner et al., J.Lipid Res. 32:903, 1991).

[0016] FIG 4 shows ion mobility separates individual lipoprotein particles by size and directly measures their concentrations (Caulfield et al. Clin Chem 54:1307, 2008).

[0017] FIG 5 shows ion mobility quantifies levels of lipoprotein particle concentrations as a function of size and detects a novel minor species in the midzone between HDL and LDL.

[0018] FIG 6 shows in plasma, midzone peak concentration is correlated with distinct LDL & IDL - sized subspecies.

[0019] FIG 7 shows that surprisingly, both midzone and IDL-sized particles are found in narrow LDL subfractions isolated by ultracentrifugation.

[0020] FIG 8 shows that among 11 narrow ultracentrifugally isolated LDL density subfractions, molecular mass of IDL sized particles is exactly two-fold that of LDL in these fractions. This is consistent with IDL-sized particles within the LDL density range (i.e., not true IDL) formed by dimerization of LDL.

[0021] FIG 9 shows that among 11 narrow ultracentrifugally isolated LDL subfractions, both midzone diameter and concentration are highly correlated with the respective values for LDL (1-0.98). Thus the midzone is a biomarker for properties of subsets of plasma LDL particles throughout their density range.

[0022] FIG 10 shows the midzone is a biomarker of the metabolic model for generating LDL(-). Musliner, Michenfelder, Krauss, J. Lipid Res. 29: 349, 1988; Musliner et al., J. Lipid Res. 32:903, 1991.

[0023] FIG 11 shows the size of midzone particles corresponds to dimensions of apoAl- containing discs dissociated from VLDL:LDL complex by lipid-poor HDL. Thus the midzone in plasma may represent Al-containing discs formed during generation of electronegative LDL.

[0024] FIG 12 provides a summary for the midzone lipoprotein concentration as a biomarker for LDL(-). It appears homologous to discoidal ApoAl particles formed in the course of generating charge-modified LDL and IDL-sized LDL dimers from VLDL-1DL complexes in the presence of FFA and HDL. It correlates with LDL and IDL-sized species in both isolated plasma and LDL subfractions - consistent with their co-ordinate formation in the metabolic model for LDL(-) production.

[0025] FIG 13 provides that the midzone has the strongest correlation with hsCRP among lipoprotein particles.

[0026] FIG 14 provides a summary of midzone lipoprotein concentration as a biomarker for LDL(-).

[0027] FIG 15 shows schematic procedures of L5 LDL isolation.

[0028] FIG 16 shows evidence for the production of electronegative LDL(-) by the metabolic model in FIG 3. Incubation of VLDL, LDL and HDL was performed in the presence or absence of lipoprotein lipase (LPL), which catalyzes lipolysis of VLDL triglycerides. LDL(-) was identified using ion exchange chromatography (peak L5). Production and release of LDL(-) is shown to be dependent on LPL-mediated VLDL triglyceride lipolysis.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0029] "Centrifugation" means separation or analysis of substances in a solution as a function of density and density-related molecular weight by subjecting the solution to a centrifugal force generated by high-speed rotation in an appropriate instrument.

[0030] "Differential Mobility Analyzer" means a device for classifying charged particles on the basis of their ion electrical mobility. When the particles have a known uniform charge, the size of the particles classified may be determined from their mobility.

[0031] As used herein, "purify" and like terms refer to an increase in the relative concentration of a specified component with respect to other components. For example without limitation, removal of lipid from a lipoprotein solution constitutes purification of the lipoprotein fraction, at e.g. the expense of the lipid fraction. It is understood that "purifying" and like terms in the context of centrifugation refers to sufficient separation in a centrifuge tube following centrifugation to allow extraction of the separated components by methods well known in the art including, without limitation, aspiration and / or fractionation. It has been found that reducing the density of lipoprotein-containing solutions prior to centrifugation for example, without limitation, by reducing the salt concentration thereof, results in enhanced recovery of certain fractions of lipoprotein, including LDL and HDL fractions.

[0032] "Predisposition" as used herein is substantially synonymous with risk, inclination, tendency, predilection, or susceptibility.

[0033] "Distribution" means a generalized function of one or more variables, and commonly depicted as a scatter chart, graph, plot, or histogram.

[0034] The terms "marker," "biochemical marker" and like terms as used herein refer to naturally occurring biomolecules (or derivatives thereof) with known correlations to a disease, condition, or lipoprotein particle.

[0035] The term "about" as used herein in the context of a numerical value represents the value + / - 10% thereof.

[0036] The terms "lipoprotein" and "lipoprotein particle" as used herein refer to particles obtained from mammalian blood which include apolipoproteins biologically assembled with noncovalent bonds to package for example, without limitation, cholesterol and other lipids. Lipoproteins preferably refer to biological particles having a size range of about 7 to 120 nm, and include VLDL (very low density lipoproteins), IDL (intermediate density lipoproteins), LDL(-), LDL(-) biomarkers, LDL (low density lipoproteins), Lp(a) [lipoprotein (a)], HDL (high density lipoproteins) and chylomicrons as defined herein.

[0037] As used herein, "chylomicrons" means biological particles of size 70-120 nm, with corresponding densities of less than 1.006 g / mL. Chylomicrons have not been found to have any clinical significance in the prediction of heart disease, for example CHD.

[0038] The term "apolipoprotein" as used herein refers to lipid-binding proteins which constitute lipoproteins. Apolipoproteins are classified in five major classes: ApoA, ApoB, ApoC, ApoD, and ApoE, as known in the art.

[0039] "ApoA" as known in the art is a protein component of HDL. "ApoB" is a protein component of LDL, IDL, Lp(a), and VLDL, and indeed is the primary apolipoprotein of lower density lipoproteins, having human genetic locus 2p24-p23, as known in the art.

[0040] As used herein, "albumin" refers to ubiquitous proteins constituting approximately 60% of plasma, having density of about 1.35 g / mL, as known in the art.

[0041] LDL(-), as defined herein, are a heterogeneous subspecies of LDL particles. Certain LDL(-) particles are characterized as having a higher amount of triglycerides (from about 110% to about 300%) as compared to LDL particles. Certain LDL(-) particles are characterized as having a higher amount of free fatty acids (from about 180% to about 400%) as compared to LDL particles. Certain LDL(-) particles are characterized as having multiple apolipoproteins as compared to LDL particles. LDL(-) particles can be obtained by ion exchange chromatography as described in Chu et al, Biomedicines 2020, 8, 254; doi:10.3390 / biomedicines8080254 as ‘L5’.

[0042] LDL(-) biomarkers, as defined herein, are molecules with known correlations to LDL(-) particles. In certain embodiments, a LDL(-) biomarker is a lipoprotein particle having a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”). Such a midzone LDL(-) biomarker can be ascertained by centrifugation (e.g. ultracentrifugation) or differential precipitation. In certain embodiments, a LDL(-) biomarker is a lipoprotein particle having a diameter from about 28.0 nm to about 32.0 nm as measured by ion mobility (“IDL-sized”). Such an IDL-sized LDL(-) biomarker can be ascertained by centrifugation (e.g. ultracentrifugation).Introduction

[0043] The present invention contemplates apparatus and methods for use in differential charged-particle mobility, and preparation of samples for differential charged-particle mobility. Differential charged-particle mobility utilizes the principle that particles of a given size and charge state behave in a predictable manner when carried in a laminar-air flow passed through an electric field. Accordingly, differential charged-particle mobilityanalysis is a technique to determine the size of a charged particle undergoing analysis when the charged particle is exposed to an electric field.

[0044] Electrical mobility is a physical property of an ion and is related to the velocity an ion acquires when it is subjected to an electrical field. Electrical mobility, Z, is defined aswhere V=terminal velocity and E=electrical field causing particle motion. Particle diameter can be obtained fromwhere n=number of charges on the particle (in this case a single charge), e= 1.6. x 10’19coulombs / charge, Cc=particle size dependent slip correction factor, n=gas viscosity, and d=particle diameter. Accordingly, solving for d, provides the following relationship:

[0045] Thus, an explicit relationship for particle diameter as a function of known parameters results. By setting the parameters to different values, different particle diameters of the charged particles may be selected as further described below and known in the art. In preferred methods of differential charged-particle mobility analysis, the electric field strength E acting upon the charged particle is varied during analysis.

[0046] In differential charged-particle mobility analysis, particles (e.g.. lipoproteins and the like) are carried through the system using a series of laminar airflows. The lipoproteins in a volatile solution are introduced to an electrospray chamber containing approximately 5% CO2 wherein the lipoproteins desolvate. In the electrospray chamber the desolvated, charged lipoproteins are neutralized by ionized air, introduced for example without limitation by an alpha particle emitter in the chamber. Based on Fuch's formula, a predictable proportion of particles emerge from the chamber carrying a single charge and are transported from the chamber to the Differential Mobility Analyzer(DMA). For details on Fuch's formula, reference is made to Fuchs, N.A.: The Mechanicsof Aerosols, Macmillan, 1964. "Differential Mobility Analyzer," "DMA" and like terms refer to devices for classifying charged particles on the basis of ion electrical mobility, as known in the art and described herein. In differential charged-particle mobility analysis, when particles have a known uniform charge, the size of the particles classified may be determined from the mobility thereof. In the DMA the particles enter at the top outer surface of the chamber and are carried in a fast flowing laminar-air flow, (i.e„ "the sheath flow"). The sheath flow is filtered (to remove particles) air that constantly recirculates through the DMA at a constant velocity of 20 L / min. As the particles pass through the DMA (carried in the sheath flow) the electric potential across the chamber is ramped up at a known rate. As the electrical potential changes, particles of different diameter are collected via a slit at the bottom inner surface of the chamber. Particles follow a nonlinear path through the DMA depending on their charge and diameter. At any given electrical potential, particles of known size will follow a path that will allow them to pass through the collecting slit. Particles passing through the collecting slit are picked up by another, separate laminar-flow air stream and are carried to a particle counter. The particle counter enlarges the particles by condensation to a size that can be detected and counted for example by a laser detection system. Knowing the electrical potential being applied to the DMA when the particle was collected permits the accurate determination of the particle diameter and the number of particles present at that size. This data is collected and stored in bins as a function of time for different particle size. In this way the number of particles of any given size range can be determined and converted to a concentration of particles based on the time required to collect the data, the flow rate of sample being introduced into the electrospray device, and the number of charged particles at that size.Separating lipoprotein particles via centrifugation

[0047] In certain embodiments, the invention provides separating lipoprotein particles having an average density of from about 1.020 g / mL to about 1.060 g / mL from the blood of said patient, wherein the product comprises LDL(-) biomarkers. In an exemplary embodiment, the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”). In an exemplary embodiment, the LDL(-)biomarkers have a diameter from about 28.0 nm to about 32.0 nm as measured by ion mobility (“IDL-sized”).

[0048] In methods of the present invention contemplating separation (isolation and / or purification) of lipoproteins, initial sample collection and preparation may be conducted by methods well known in the art. Typically, a 2 to 5 ml fasting blood specimen is initially taken. Chylomicrons are not typically present in subjects who have been fasting for a period of at least 12 hours; thus, overlap of VLDL sizes and chylomicron sizes is eliminated by fasting. The specimen is then initially spun in a centrifuge (e.g., clinical centrifuge) preferably for approximately 10 minutes at approximately 2000 xG which centrifugation is sufficient to remove the cellular components from the specimen. During this process, the more dense cellular components stratify at the bottom of the sample. A remaining less dense plasma specimen containing lipoproteins on top is then drawn off using methods well known in the art, e.g., aspiration.

[0049] Historically, in preparation for centrifugation, a plasma specimen could be density adjusted to a specific density (e.g. average density of from about 1.020 g / mL to about 1.060 g / mL from the plasma sample) using high purity solutions or solids of inorganic salts, e.g., sodium chloride (NaCl), sodium bromide (NaBr) and the like. In some previous protocols, the specific density would be chosen to be greater than or equal to the highest density of the lipoprotein material to be analyzed, so that the lipoprotein material would float when density stratified. "Density stratified" and like terms refer to the layering of components in a solution subjected to centrifugation. These densities are tabulated in Table 1. The density-adjusted sample could then be ultracentrifuged for example for approximately 18 hours at 100,000 xG to separate the nonlipoprotein proteins from the lipoproteins. Non-lipoprotein proteins, particularly albumin, could be removed from the plasma specimen, preferably by ultracentrifugation. The lipoproteins would float to the top of the sample during ultracentrifugation. Accordingly, by sequentially centrifuging from lowest density to highest density of the density adjustment, the various classes and subclasses of lipoproteins could be sequentially extracted. Typically, a dialysis step would be required following extraction of a centrifuged sample to remove salts introduced for adjustment of density, which dialysis step would typically require 4-12 hrs under conditions well known in the art.

[0050] Conditions for centrifugation for lipoprotein-containing samples described herein are well known in the art of biochemical separation. For example without limitation, samples are typically centrifuged at 10°C for 1-4 hrs at 223,000 xG. In some embodiments, centrifugation employs centrifugal force of 50,000-100,000, 100,000- 120,000, 120,000-150,000, 150,000-200,000, 200,000-230,000, 230,000-250,000 xG, or even higher force. In some embodiments, the time of centrifugation is 1, 2, 2.2, 2.4, 2.6, 2.8, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 hr, or even longer. Prior to analysis by differential charged-particle mobility, an aliquot of the lipid fraction is removed ( e.g„ 10-200 pL) from the top of the centrifuge tube and diluted (e.g., 1:800) in 25 mM ammonium acetate (AA), 0.5 mM ammonium hydroxide, pH 7.4. Advantageously, in some embodiments described herein, a dialysis step is not necessary in conjunction with methods of the invention, resulting in less time required for analysis.

[0051] In some embodiments of aspects provided herein which contemplate lipoproteins, the lipoproteins may derive from a plasma specimen, obtained by methods well known in the art or as described herein. The terms "biological specimen," "biological sample" and like terms refer to explanted, withdrawn or otherwise collected biological tissue or fluid including, for example without limitation, whole blood, serum and plasma. The term "plasma" in the context of blood refers to the fluid obtained upon separating whole blood into solid and liquid components. The term "serum" in the context of blood refers to the fluid obtained upon separating whole blood into solid and liquid components after it has been allowed to clot. In some embodiments of any of the aspects of the present invention, the biological specimen is of human origin. In some embodiments of any of aspects provided herein, the biological specimen is serum. In some embodiments of any of the aspects provided herein, the biological specimen is plasma.

[0052] In some embodiments of the invention which contemplate centrifugation, the centrifugation does not reach equilibrium. "Centrifugation equilibrium" and like terms refers to centrifugation conducted for sufficient time and at sufficient centrifugal force such that the components of the solution being centrifuged have reached neutral density buoyancy, as well known in the art. Surprisingly, it has been found that foreshortenedcentrifugation protocols, as described herein wherein centrifugal equilibrium is not reached, can nonetheless provide significant purification of lipoproteins.

[0053] In some embodiments of the invention which contemplate centrifugation of sample containing lipoproteins and non-lipoprotein components, purified lipoprotein is collected from the top portion of the centrifuge tube following centrifugation. "Top portion of the centrifuge tube" and like terms refer to the liquid in the upper portion of a centrifuge tube when viewed outside of the centrifuge rotor which may, but does not necessarily, include liquid at the very top.

[0054] Lipoprotein density can be determined directly by a variety of physical biochemical methods well known in the art, including without limitation equilibrium density ultracentrifugation and analytic ultracentrifugation. Lipoprotein density may also be determined indirectly based on particle size and a known relationship between particle size and density. Lipoprotein size may be determined by a variety of biochemical methods well known in the art including, without limitation, methods described herein.Separating lipoprotein particles via differential precipitation

[0055] In certain embodiments, the invention provides precipitating non-lipoprotein proteins from the blood of said patient, wherein the product comprises a supernatant fraction and the supernatant fraction comprises LDL(-) biomarkers having a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”).

[0056] In certain embodiments, whole plasma samples are pre- treated with blue dextran reagent (2.5 mg / mL - 5.0 mg / mL Blue Dextran, 2.0 mg / mL - 3.0 mg / mL Dextran Sulfate, 0.25 mg / mL - 0.75 mg / mL disodium EDTA) in a 1:2 to 1:6 plasma: reagent ratio for between 30 sec to 30 min on ice. After pretreatment, 10 pL - 30 pL of the mixture is layered on top of 100 uL - 150 uL of deuterium oxide into ultracentrifuge tubes (17x20 mm) and centrifuged in a Beckman 42.2Ti rotor at 35,000 - 50,000 rpm for 1-3 hours and 30 sec to 30 min. After centrifugation, the top 70 - 110 uL (“top fraction”) is collected from each centrifuge tube and mixed to homogenize. The top fraction is diluted 100-300 fold with dilution buffer (10 mM - 40 mM Ammonium Acetate, 0.25 mM - 0.75 mM Ammonium Hydroxide, 10pg / mL -50 pg / mL Dextran Sulfate) and analyzed by ion mobility.

[0057] In certain embodiments, plasma is treated with 10-30% ethanol which removes >97% of fibrinogen, and lipoproteins are then precipitated with dextran sulfate (1 mg / mL - 3 mg / mL) and calcium (0.1 M - 0.2 M). Precipitated lipoproteins are harvested on paramagnetic particles, washed to remove free salt and proteins and then resuspended in 10 mM - 40 mM ammonium acetate for analysis by ion mobility.

[0058] In some embodiments, the sample further comprises a compound which can act as a precipitant for selected lipoprotein components therein, as known in the art. "Precipitant" refers to a compound which may cause or promote precipitation of a biomolecule upon addition to a solution of such biomolecule. A precipitant may require an additional agent to afford precipitation. "Additional agent to afford precipitation" and like terms refer to compounds which act with a precipitant and may be required to afford precipitation by the precipitant. Exemplary precipitants include, without limitation, salts of charged inorganic ions, preferably ammonium sulfate, antibodies, charged polymers ( e.g., DS and the like) optionally in the presence of ionic species (e.g., divalent cations), lectins, and the like. In some embodiments, the precipitant is present albeit under conditions (e.g., pH, concentration, lack of necessary additional agents, and the like) wherein lipoproteins are not precipitated. In some embodiments, the precipitant is DS. In some embodiments, the precipitant is DS, and the necessary additional agent is a divalent cation. In some embodiments, the lipoprotein-containing sample comprises DS but lacks divalent cations. Without wishing to be bound by any theory, it is believed that DS binds to particles which contain lipids in the presence of divalent cations, and that DS binding may interfere with non-specific binding interactions with resulting enhancement of recovery of certain lipoproteins. For example without limitation, it is observed that inclusion of DS significantly improved recovery of LDL from some preparations described herein.

[0059] In some embodiments of aspects provided herein which contemplate centrifugation of sample containing lipoproteins and non-lipoprotein components, the sample further comprises an albumin-binding compound under conditions suitable to allow formation of a complex comprising albumin and albumin-binding compound. Representative albumin-binding compounds include, without limitation, aromatic albumin-binding dyes. The aromatic albumin binding dye may comprise a diazo dye; an alkali metal salt, alkaline earth metal salt, or amine salt of said diazo dye; a sulfonic aciddye; a physiologically-acceptable alkali metal salt, alkaline earth metal salt, or amine salt of said sulfonic acid dye; or mixtures thereof. Aromatic albumin binding dyes particularly useful in the present invention include Reactive Blue 2, Evans Blue, Trypan Blue, Bromcresol Green, Bromcresol Purple, Methyl Orange, Procion red HE 3B, and the like. In certain embodiments, the albumin-binding compound is an analog of nicotinamide adenine dinucleotide (NAD). Representative NAD analogs suitable for use as albuminbinding compounds include, without limitation, RG 19, and Cibacrom Blue 3GA (CB 3GA).

[0060] In embodiments of the method contemplating the use of albumin-binding compounds, after mixture of the albumin-binding compound with a lipoproteincontaining sample, the sample is centrifuged as described herein. In some embodiments, the albumin-binding compound is conjugated with a chromatographic medium, which conjugate promotes facile removal of albumin complexed with albumin-binding compound for example, without limitation, by filtration. In some embodiments, the conjugated albumin-binding compound is observed to stratify at the bottom of the centrifuge tube, thereby facilitating removal ( e.g., by aspiration, etc.) of the lipoproteincontaining fraction. In some embodiments wherein the albumin-binding compound is conjugated with a chromatographic medium, the chromatographic medium may be paramagnetic particles, dextran, agarose or Sephadex®, preferably dextran "Paramagnetic particle" as known in the art refers to particles having a magnetite core coated with a ligand, for example without limitation, streptavidin. The affinity of biotin for streptavidin (Kj = 10’15M) is one of the strongest and most stable interactions in biology. Thus, paramagnetic particles combine convenient magnetic separation technology with the versatility and high affinity of the interactions such as the biotinstreptavidin interaction. It is observed that dextran conjugated albumin-binding compounds tend to remain soluble longer than other conjugate chromatographic media described herein. Without wishing to be bound by any theory, it is believed that the longer an albumin-binding compound can interact with albumin in a lipoproteincontaining sample, the more albumin-containing complex will be formed, thereby increasing purity and recovery of lipoprotein.

[0061] In further embodiments of the method contemplating the use of albumin-binding compounds, the albumin-binding compound is present during centrifugation at a concentration of up to 50 mg / mL, or even higher, without significant change in the quantity and relative proportion of the lipoproteins recovered from a plasma sample. In other embodiments, the concentration of albumin-binding compound is for example, without limitation, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45 or even 50 mg / mL.

[0062] In certain embodiments, the invention provides for the use of an albumin- binding compound in combination with DS. Also of significance is the increased recovery of LDL. Without wishing to be bound by any theory, results herein suggest that DS present in the extraction and diluent affords the best recovery and reproducibility.

[0063] In certain embodiments, purified lipoprotein-containing sample obtained by methods of the invention are further diluted prior to differential charged-particle mobility analysis.

[0064] In certain aspects and embodiments, the invention contemplates methods employing an albumin-binding compound conjugated with chromatographic media in combination with DS, and further in combination with a D20 solution under and adjacent a lipoprotein-containing sample in a centrifuge tube.

[0065] In some embodiments of aspects provided herein which contemplate centrifugation of sample containing lipoproteins and non-lipoprotein components, the sample further comprises a non-lipoprotein capture ligand capable of binding nonlipoprotein component to form a nonlipoprotein / non-lipoprotein capture ligand complex, further wherein the centrifugation causes the non-lipoprotein / non-lipoprotein capture ligand complex to be separated from the lipoprotein components. "Non-lipoprotein capture ligand" and like terms refer to compounds which bind plasma components which are not lipoproteins. Exemplary non-lipoprotein capture ligands include, without limitation, antibodies and aptamers as understood in the art. For example without limitation, separation of antibody (i.e., as non-lipoprotein capture ligand) from antigen (i.e., non-lipoprotein) can be realized with a variety of methods including modulation of temperature, pH, salt concentration and the like. For further example without limitation, separation of aptamer (i.e., as non-lipoprotein capture ligand) from aptamer target (i.e.,nonlipoprotein) can be realized with a variety of methods including modulation of temperature, pH, salt concentration, DNase or RNase and the like.

[0066] In some embodiments of aspects provided herein which contemplate centrifugation of sample containing lipoproteins and non-lipoprotein components, the sample further comprises a lipoprotein-capture ligand capable of binding lipoprotein component to form a lipoprotein / lipoprotein-capture ligand complex, further wherein the centrifugation causes the lipoprotein / lipoprotein-capture ligand complex to be separated from the non-lipoprotein components. "Lipoprotein-capture ligand" and like terms refer to compounds which bind lipoproteins. Exemplary lipoprotein-capture ligands include, without limitation, antibodies and aptamers as understood in the art. In preferred embodiments, the lipoprotein-capture ligand is an antibody.

[0067] In some embodiments of aspects provided herein which do not contemplate centrifugation of sample containing lipoproteins and non-lipoprotein components, the method contemplates a lipoprotein-capture ligand capable of binding lipoprotein component to form a lipoprotein / lipoprotein-capture ligand complex.

[0068] The term "aptamer" refers to macromolecules composed of nucleic acid, such as RNA or DNA, that bind tightly to a specific molecular target. The terms "bind," "binding" and the like refer to an interaction or complexation resulting in a complex sufficiently stable so as to permit separation. In some embodiments, the aptamer specifically bind Apo Al, Apo B, or Apo(a). Methods for the production and screening of aptamers useful for the present invention are well known in the art; see e.g., Griffin et al., United Stated Patent No. 5,756,291, incorporated herein by reference in its entirety and for all purposes.

[0069] As practiced in the art, the method of selection (i.e., training) of aptamer requires a pool of single stranded random DNA oligomers comprising both random sequences and flanking regions of known sequence to serve as primer binding sites for subsequent polymerase chain reaction (PCR) amplification. Such DNA oligomers are generated using conventional synthetic methods well known in the art. As an initial and optional step, PCR amplification is conducted by conventional methods, and the amplified pool is left as duplex DNA, or used as single stranded DNA after strand separation. Optionally, transcription into RNA can be conducted. The term "oligomer pool" in this context refersto such single stranded or duplex DNA, or RNA transcribed therefrom. The term "refined oligomer pool" refers to an oligomer pool which has been subjected to at least one round of selection as described herein.

[0070] Further the aforementioned aptamer training, a "selection" step is conducted employing a column or other support matrix (i.e., target-coupled support) having target molecule attached thereon. Attachment, well known in the art, may be by covalent or non-covalent means. The oligomer pool, or refined oligomer pool, and target-coupled support are incubated in order to permit formation of oligonucleotide-target complex, and the uncomplexed fraction of the oligomer pool or refined oligomer pool is removed from the support environment by, for example, washing by methods well known in the art. Subsequent removal of oligonucleotide by methods well known in the art results in a refined oligomer pool fraction having enhanced specificity for target relative to a predecessor oligomer pool or refined oligomer pool.

[0071] Alternatively, the aforementioned aptamer training can employ a "reverse selection" step wherein aptamer is selected to bind to other constituents of the biological sample. In this case, a column or other support matrix is employed (i.e., constituent- coupled support) having other constituents of the biological sample attached thereon. The oligomer pool, or refined oligomer pool, and constituent-coupled support are incubated in order to permit formation of oligonucleotide-constituent complex, and the uncomplexed fraction of the oligomer pool or refined oligomer pool is removed from the support environment by, for example, washing by methods well known in the art. Subsequent removal of oligonucleotide by methods well known in the art results in a refined oligomer pool fraction having enhanced specificity for other constituents of the biological sample relative to a predecessor oligomer pool or refined oligomer pool. Examples of other constituents of the biological sample used in the reverse selection step include, without limitation, immunoglobulins and albumins.

[0072] In a typical production training scheme, oligonucleotide recovered after complexation with target or other constituent of the biological sample is subjected to PCR amplification. The selection / amplification steps are then repeated, typically three to six times, in order to provide refined oligomer pools with enhanced binding and specificity to target or other constituent of the biological sample. Amplified sequences soobtained can be cloned and sequenced. Optionally, when a plurality of individual aptamer sequence specific for a target having been obtained and sequenced, pairwise and multiple alignment examination, well known in the art, can result in the elucidation of "consensus sequences" wherein a nucleotide sequence or region of optionally contiguous nucleotides are identified, the presence of which correlates with aptamer binding to target. When a consensus sequence is identified, oligonucleotides that contain the consensus sequence may be made by conventional synthetic or recombinant means.

[0073] The term "antibody" refers to an immunoglobulin which binds antigen ( e.g., lipoprotein or other component of the sample) with high affinity and high specificity. In this context "high affinity" refers to a dissociation constant of, for example without limitation, 1 p , 100 nM, 10 nM, 1 nM, 100 pM, or even more affinity, characterizing the binding reaction of antibody with antigen to which the antibody has been raised. The term "raised" refers to the production of high affinity antibody by methods long known in the art. Further in this context, the term "high specificity" refers to a preference of binding of a target antigen by a test antibody relative to nontarget antigen characterized by a ratio of dissociation constants of, for example without limitation, 1, 2, 5, 10, 20, 50, 100,200, 500, 1000, 10000, or more, in favor of binding of the target antigen to which the test antibody has been raised.

[0074] Methods of derivatization of antibodies and aptamers contemplated by the present invention include, for example without limitation, biotinylation. In some embodiments, the antibody or aptamer is biotinylated such that subsequent isolation on an avidin conjugated matrix, for example without limitation, an avidin chromatography column, affords facile separation by methods well known in the art of biochemical purification. In some embodiments, the biotinylated antibody or aptamer in complex with a lipoprotein is further subjected to streptavidin-conjugated magnetic beads. The ternary lipoprotein- biotinylated affinity reagent streptavidin conjugated magnetic bead complex is then isolated by immunomagnetic methods well known in the art.

[0075] In some embodiments of this aspect, the lipoprotein-capture ligand is linked to a solid support by use of appropriate linkers well known in the art. Exemplary solid supports include, without limitation, paramagnetic particles, beads, gel matrix material (e.g., agarose, Sephadex®), and the like.Ion mobility

[0076] In an exemplary embodiment, the invention comprises converting the LDL(-) biomarkers into charged LDL(-) biomarkers. In an exemplary embodiment, the invention comprises determining the charged LDL(-) biomarkers concentration. Both of the above embodiments can be ascertained by ion mobility. Ion mobility, also known as ion electrical mobility or charged-particle mobility, analysis offers an advantage over the other methods described herein in that it not only measures the particle size accurately based on physical principles but also directly counts the number of particles present at each size, thereby offering a direct measurement of lipoprotein size and concentration for each lipoprotein. Ion mobility analysis has been used routinely in analyzing particles in aerosols, and analyzers suitable for ion mobility analysis have been adapted to analyze large biological macromolecules. Ion mobility analysis is a very sensitive and accurate methodology with, nonetheless, a drawback that ion mobility analysis measures all particles introduced into the system. Accordingly, it is of prime importance to isolate and / or purify the compounds of interest prior to analysis. Lipoproteins are candidates for this method because lipoproteins can be isolated from other serum proteins based on density and other features described herein.

[0077] In exemplary differential charged-particle mobility experiments, serum samples (25 uL) are overlaid on a cushion (200 uL) of four different density salt (KBr) solutions. The densities of the solutions span a range, such as between 1.020 g / mL to about 1.060 g / m. Each sample is ultracentrifuged for a period of 3.7 hr at 223,000 xG The top 100 uL after the centrifugation is removed. Fractionated lipoprotein samples from each density are dialyzed overnight against ammonium acetate (25 mM), ammonium hydroxide (0.5 mM), pH 7.4. Following dialysis each sample is analyzed by differential charged-particle mobility.

[0078] In exemplary charts, the abscissa is the particle size (i.e., diameter), and the ordinate is an arbitrarily scaled mass. The area under the curves, in a particle mass versus independent variable (such as size, density, mobility, etc.) distribution, is directly representative of the lipoprotein particle mass. The measurement technique relies on counting individual particles as a function of size (diameter). It is therefore possible to convert the number of particles at a specific size into a mass value using the volume anddensity of the particles. The density of lipoproteins is a well-known function of particle size and is obtainable for example from the literature. The mass values associated with the figure are simply scaled to indicate relative values but can be converted to actual mass of lipoproteins in plasma using dilution factors along with flow rates of sample and air passing through the ion mobility spectrometer. Accordingly, in some embodiments adjusting the density of a lipoprotein-containing solution prior to non-equilibrium centrifugation to a value lower than expected to separate the higher density lipoproteins (e.g., HDL) actually results in separation of HDL and LDL. Advantageously, the method of reducing the density of the lipoprotein-containing sample also results in increased separation from albumin.Charged LDL(-) biomarkers reference concentration

[0079] A charged LDL(-) biomarkers reference concentration can be obtained by methods known to one of skill in the art. In an exemplary embodiment, the charged LDL(-) biomarkers reference concentration is as described in Chu et al, Biomedicines 2020, 8, 254; doi:10.3390 / biomedicines8080254, the content of which is herein incorporated by reference in its entirety for all purposes. This is illustrated in FIG 15. In FIG 15, EDTA, antibiotics, and protease inhibitors are materials for the prevention of protein degradation. Samples undergo sequential density-based ultracentrifugation (10,000 at 4 °C for 2 h; d = 1.004, 45,000 rpm at 4 °C for 24 h; d = 1.019, 45,000 rpm at 4 °C for 24 h; d = 1.063, 45,000 rpm at 4 °C for 48 h), and after that, LDL (d = 1.019-1.063) can be purified. Additional three times dialyzed against TRIS / EDTA buffer at pH 8.0 and later sterilized by 0.22 pm filter, the LDL sample can be further isolated into five subfractions by a fast-protein liquid chromatography (FPLC) system equipped with an UnoQ12 column. L5 LDL is the most electronegative subfraction. VLDL: very- low-density lipoprotein; IDL: intermediate-density lipoprotein; LDL: low-density lipoprotein; HDL: high-density lipoprotein; L5: electronegative LDL; FPLC: fast-protein liquid chromatography; rpm: revolutions per minute; 1 x Protease Inhibitor: cOmplete. (Roche Diagnostics, Basel, Switzerland).Assessments

[0080] In another aspect, the invention provides methods for analyzing the size distribution of LDL(-) by differential charged-particle mobility analysis. In someembodiments, one or more LDL(-) and / or LDL(-) biomarkers are obtained from a body fluid such as a plasma specimen from an individual. In some embodiments, the method further includes the step of using the determined LDL(-) and / or LDL(-) biomarkers size distribution to conduct an assessment of the individual, the assessment selected from the group consisting of lipid-related health risk, cardiovascular condition, risk of cardiovascular disease, and responsiveness to a therapeutic intervention.

[0081] "Assessment" in the context of lipid-related health risk, cardiovascular condition, and risk of cardiovascular disease, refers to a statistical correlation of the resulting LDL(-) and / or LDL(-) biomarkers size distribution with population mortality and risk factors, as well known in the art. Assessment in the context of responsiveness to a therapeutic intervention refers to comparison of the LDL(-) biomarker concentrations before and after a therapeutic intervention is conducted. Exemplary therapeutic interventions include, without limitation, the administration of drugs to an individual for the purpose of lowering serum cholesterol, lowering LDL, IDL, and VLDL, Lp(a) and / or raising HDL, as known in the art.

[0082] In some embodiments, the results of LDL(-) and / or LDL(-) biomarker analyses are reported in an analysis report. "Analysis report" refers in the context of LDL(-) and / or LDL(-) biomarker and other lipid analyses contemplated by the invention to a report provided, for example to a clinician, other health care provider, epidemiologist, and the like, which report includes the results of analysis of a biological specimen, for example a plasma specimen, from an individual. Analysis reports can be presented in printed or electronic form, or in any form convenient for analysis, review and / or archiving of the data therein, as known in the art. An analysis report may include identifying information about the individual subject of the report, including without limitation name, address, gender, identification information (e.g., social security number, insurance numbers), and the like. An analysis report may include biochemical characterization of the lipids in the sample, for example without limitation triglycerides, total cholesterol, LDL cholesterol, and / or HDL cholesterol, and the like, as known in the art and / or described herein. An analysis report may further include characterization of LDL(-) and / or LDL(-) biomarkers, and references ranges therefore, conducted on samples prepared by the methods provided herein. The term "reference range" and like terms refer to concentrations of componentsof biological samples known in the art to reflect typical normal observed ranges in a population of individuals. Exemplary characterization of lipoproteins in an analysis report may include the concentrations of LDL(-) and / or LDL(-) biomarkers determined by differential charged-particle mobility. Further exemplary characterization of lipoproteins, determined for example by differential charged-particle mobility analyses conducted on samples prepared by methods of the invention, include the concentration and reference range for VLDL, IDL, LDL(-), Lp(a), LDL and HDL, and subclasses thereof. An analysis report may further include lipoprotein size distribution, obtaining for example by differential charged-particle mobility analysis, of a sample prepared by methods of the invention.

[0083] The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.EXAMPLES

[0084] The following Examples illustrate the synthesis of representative compounds used in the invention and the following Reference Examples illustrate the synthesis of intermediates in their preparation. These examples are not intended, nor are they to be construed, as limiting the scope of the invention. It will be clear that the invention may be practiced otherwise than as particularly described herein. Numerous modifications and variations of the invention are possible in view of the teachings herein and, therefore, are within the scope of the invention.EXAMPLE 1LDL isolation and analysis:

[0085] Narrow density subfractions of LDL were isolated from plasma of a single individual by density gradient ultracentrifugation using the method described in: Shen, et al. (1981). Heterogeneity of serum low-density lipoproteins in normal human subjects. J. Lipid Res., 22, 236-244. PMID: 7240955. Eleven subfractions of 0.5 ml each spanning the range of 1.019 g / mL and 1.063 g / mL were isolated by this procedure for the analyses shown here. Ion mobility (also referred to herein as “differential charged-particle mobility analysis”) was used to measure concentrations of lipoprotein particles (y axis, arbitrary units) within individual particle diameter intervals (x-axis, nm). In FIG 7 isshown the data for a single representative subfraction of narrow density (~1 .04 g / ml) in the middle of the LDL density range). Surprisingly, both midzone and IDL-sized particles are found in narrow LDL subfractions isolated by ultracentrifugation. In addition to the major LDL species (~21 nm) there are two additional peaks, the smaller corresponding in size to the midzone and the larger having a size range comparable to IDL (intermediate density lipoproteins). However, these are not true IDL since their density is much less than that of LDL (<1.019 g / ml) and as shown below (FIG 8) they appear instead to be dimers of a subset of LDL particles in the major peak. Among 11 narrow ultracentrifugally isolated LDL density subfractions, molecular mass of IDL sized particles is exactly two-fold that of LDL in these fractions. Consistent with IDL-sized particles within the LDL density range (i.e., not true IDL) formed by dimerization of LDL.Midzone peak assessment:

[0086] For each of the 11 narrow density LDL subfractions mentioned above, the peak LDL species diameter and peak midzone species diameter were measured. The results were plotted in a chart in FIG 9 where peak LDL diameter (nm) is on the x axis and peak midzone diameter (nm) is on the y axis. A high correlation (r=0.98) exists between the peak LDL species diameter and peak midzone species diameter. Among 11 narrow ultracentrifugally isolated LDL subfractions, both midzone diameter and concentration are highly correlated with the respective values for LDL (r=0.98). Thus the midzone is a biomarker for properties of subsets of plasma LDL particles throughout their density range.

[0087] For each of the 11 narrow density LDL subfractions mentioned above, the peak LDL concentration and peak midzone concentration were measured. The results were plotted in a second chart in FIG 9 where peak LDL concentration (nm / L) is on the x axis and peak midzone concentration (nm / L) is on the y axis. A high correlation (r=0.98) exists between the peak LDL concentration and peak midzone concentration.

[0088] The striking correlations between midzone and LDL values for both measures strongly suggest that these particles are related metabolically. The midzone particles are too small to consist of LDL (the smallest of which are >18-19 nm), effectively ruling out the possibility that the midzone particles can be converted to LDL. However thecorrelations are consistent with the possibility that midzone particles are produced in conjunction with LDL metabolism as described further in FIG 3 (Musliner, Michenfelder, Krauss, J. Lipid Res. 29: 349, 1988; Musliner et al., J.Lipid Res. 32:903, 1991). The presence of midzone particles in the same density fraction as LDL indicates that they are likely weakly associated with LDL and then released due to the very low ionic strength of the buffer used for the ion mobility procedure.IDL peak assessment:

[0089] The molecular volumes, and hence masses, of particles at the peak diameters of the LDL and IDL sized species were determined for each of the 11 narrow density LDL subfractions mentioned above using the formula for a sphere: / Sttr3. The results were plotted in FIG 8 where the molecular volume of the LDL-sized particles (arbitrary units) is on the x axis and the molecular volume of the IDL-sized particles (arbitrary units) is on the y axis.

[0090] A high correlation (r=0.98) exists between the molecular volume of the LDL- sized particles and the molecular volume of the IDL-sized particles. This high correlation, with a slope corresponding to a 2-fold higher value for the IDL-sized species, strongly suggests that it is a dimer formed from particles within the major LDL peak.The dimer would have the same composition and thus the same density as the monomer.EXAMPLE 2Data utilized for correlation analyses:

[0091] Plasma samples from a subset of 452 healthy individuals were subjected to ion mobility analysis. This subset is from a previously characterized study population described in Simon et al (2006). Phenotypic predictors of response to simvastatin therapy among African Americans and Caucasians: the Cholesterol and Pharmacogenetics (CAP) Study. Am. I. Cardiology, 97, 843-850. PMID: 16516587.Individual particle diameter interval vs. mean concentration of lipoprotein particles:

[0092] Ion mobility was used to measure mean concentrations of lipoprotein particles within 550 individual particle diameter intervals designated by their ion mobility particle bin numbers.

[0093] The relationship between the concentration of midzone particles and other plasma lipoproteins was assessed by correlating values for the 15.1 nm peak with those for individual particle size intervals across the full particle diameter spectrum using ion mobility. Note that in addition to the expected midzone correlation surrounding the 15.1 nm peak, there are correlations with distinct peaks within the HDL, LDL, and IDL size ranges, indicating that the midzone may be a biomarker for inter-related levels of these other particle species. Results are shown in FIG 6.EXAMPLE 3A description for the metabolic pathway proposed for LDL(-) production

[0094] The mechanisms responsible for the formation of LDL(-) and the marked differences in its composition compared with LDL of normal charge have not been previously determined. This slide presents a scheme that proposes a metabolic pathway responsible for LDL(-) production and provides a basis for linking levels of both midzone particles and IDL-sized LDL dimers to LDL(-) levels. It is based on in vitro studies described in Musliner et al, Krauss, J. Lipid Res. 29: 349, 1988 & Musliner et al., J.Lipid Res. 32:903, 1991. The steps in this pathway are shown in FIG 3.1) Physical interaction of an LDL particle with a triglyceride-rich very low density lipoprotein (VLDL) in plasma.2) Free Fatty Acids (FFAs) on the VLDL surface derived from lipolysis of VLDL triglyceride by lipoprotein lipase promote stabilization of a VLDL-LDL complex, with consequent transfer of various VLDL constituents to LDL: e.g., triglycerides, FFAs, and apolipoproteins, most notably apolipoprotein C-III (apoCIII) and apolipoprotein A-I (apo Al).3) Interaction of this complex with small lipid-poor HDL particles releases the modified LDL from the complex in conjunction with transfer to this LDL of apoAI, the major HDL protein constituent. Note that the increased content of both apoAI and apoCIII in the modified LDL is likely to impart an increased negative charge to the LDL based on the charge properties of these proteins.4) The modified LDL then form dimers, consistent with increased potential for aggregation of LDL(-) that has been reported by others.5) In conjunction with release of modified LDL from the VLDL-complex promoted by interaction with HDL, a “dissociation complex” is formed consisting of small discoidal particles composed of ~4 molecules of HDL-derived apoAI and surface lipids derived from LDL, as illustrated in FIG 11.

[0095] Electron micrographs of dissociation complexes derived from interaction of HDL and FFA with EDE show them to consist of stacks of discoidal particles with long axes of ~15 nm (from Musliner et al., J.Lipid Res. 32:903, 1991). It is therefore reasonable to surmise that they correspond to the midzone particle of this size seen in the LDL density fractions by ion mobility.

[0096] In FIG 15 we demonstrate that the metabolic pathway described in Example 3 and FIG 3 yields LDL(-), which is here identified as peak L5 using the existing procedure for separating and measuring LDL(-) by ion exchange chromatography as described in “Charged LDL(-) biomarkers reference concentration” herein. Notably, the production of LDL(-) is shown to be dependent on lipolysis of VLDE triglyceride by lipoprotein lipase, consistent with the proposed mechanism for generation of LDL(-).Summary

[0097] The pathway of FIG 3 supports the use of ion mobility-determined concentrations of midzone and / or IDL-sized particles within LDL to provide an assay of LDL(-).

[0098] In one example, most straightforward ion mobility-based clinical assay for assessing LDL(-) would be the measurement of midzone lipoprotein particles as an LDL(-) biomarker. This assay could be performed in plasma that has been depleted of HDL and non-lipoprotein proteins that overlap the midzone particle size region. An example for achieving this in a high throughput manner would be a precipitation method employing blue dextran sulfate coated beads. Alternatively, the assay could be performed using LDL isolated from plasma by ultracentrifugation, though this is less amenable for clinical laboratory application.EXAMPLE 4Differential PrecipitationMethod A

[0099] Whole plasma samples are pre-treated with blue dextran reagent (3.75mg / mL Blue Dextran, 2.5mg / mL Dextran Sulfate, 0.5mg / mL disodium EDTA) in a 1:4 plasma: reagent ratio for 15 minutes on ice. After pretreatment, 22 uL of the mixture is layered on top of 132 uL of deuterium oxide into ultracentrifuge tubes (17x20 mm) and centrifuged in a Beckman 42.2Ti rotor at 42,000 rpm for 2 hours and 15 minutes. Aftercentrifugation, the top 90 uL (“top fraction”) is collected from each centrifuge tube and mixed to homogenize. The top fraction is diluted 205-fold with dilution buffer (25mM Ammonium Acetate, 0.5mM Ammonium Hydroxide. 30pg / mL Dextran Sulfate) and analyzed by ion mobility.Method B

[0100] Plasma is treated with 17% ethanol which removes >97% of fibrinogen, and lipoproteins are then precipitated with dextran sulfate (2 mg / mL) and calcium (0.15 M). Precipitated lipoproteins are harvested on paramagnetic particles, washed to remove free salt and proteins and then resuspended in 25 mM ammonium acetate for analysis by ion mobility. This method recovers all measurable apoB (105%), apoA-I (96%) and total cholesterol (103%). Removal of plasma proteins by this method was assessed by the following proteins (final concentration remaining after extraction compared with original serum concentration): IgG (3%), albumin (<4%), transferrin (0%). This method is described in the Supplementary Material of: Atherogenic Lipoprotein Subfractions Determined by Ion Mobility and First Cardiovascular Events After Random Allocation to High-Intensity Statin or Placebo: The Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER) Trial. Mora S, Caulfield MP, Wohlgemuth J, Chen Z, Superko HR, Rowland CM, Glynn RJ, Ridker PM, Krauss RM. Circulation. 2015 Dec 8; 132(23):2220-9. PMID: 26408274.EXAMPLE 5

[0101] Electronegative LDL: LDL(-): -LDL subspecies defined by high negative charge; —Heterogeneous size; does not correspond to LDL-C, LDL-P or LDL subclasses; -Is not equivalent to oxidized LDL; -Normal level < ~5 % of total LDL-P (Note high- risk level of Lp(a): >7 % of total LDL-P); —Composition differs markedly from LDL of normal charge (Higher TG (1.5-fold), FFA (2-fold), multiple apoproteins); -Challenging to measure - currently requires ion exchange chromatography. Bancellos et al„ J.Lipid.Res. 51: 3508, 2010; Akyo et al., Current Atherosclerosis Reports 26:317, 2024.EXAMPLE 6

[0102] Some Pathologic Properties of L5 LDL: -Increased inflammatory molecules: ceramide, lysoPC, LpPLA2; —Not recognized by LDL receptor -> prolonged clearance;-Signals through the lectin-like oxidized LDL receptor (LOX-1 ), promoting endothelial cell cytokine release and dysfunction; -Attracts monocytes and lymphocytes to endothelial cells, promotes apoptosis; -Enhances ADP signaling in platelets via LOX-1 and PAF receptor; -Prone to aggregation and generation of circulating immune complexes. Akyo et al., Current Atherosclerosis Reports 26:317, 2024.EXAMPLE 7

[0103] Origin and factors determining levels of LDL(-) are unknown: -LDL(-) is enriched in TG, FFA, multiple apoproteins, and inflammatory components; -How does LDL acquire these properties? -There is a metabolic model that can account for them and thus for the generation of LDL(-).EXAMPLE 8

[0104] The metabolic model is based on in vitro studies. Is there evidence that this pathway operates in vivo? We turn to studies of plasma LDL using ion mobility methodology.EXAMPLE 9

[0105] Conclusions and clinical implications: -LDL(-) is an important subfraction of LDL that is associated with increased risk for MI and stroke via pro-inflammatory and pro thrombotic properties; -It is not related to other standard LDL measures; -Existing methodology is prohibitive for routine clinical measurement; -Midzone lipoprotein particle concentration can be a biomarker for determining level of LDL(-); - Development of a clinical assay for LDL(-) could significantly augment existing measures of assessing CVD risk and treatment effects.

[0106] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0107] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present invention is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, if such features, systems, articles, materials, and / or methods are not mutually inconsistent, is included within the scope of the present invention.

[0108] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0109] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e.. the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of." will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0110] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0111] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio.Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide. 2-hydroxy-ethanesulfonate, lactobionate. lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(CI-4 alkyiy salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0112] Optionally substituted refers to a group which may be substituted or unsubstituted. In general, the term "substituted" means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. Theterm "substituted" is contemplated to include substitution with all permissible substituents of organic compounds, and includes any of the substituents described herein that results in the formation of a stable compound. The present invention contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.

[0113] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

WHAT IS CLAIMED IS:

1. A method for determining a cardiovascular condition, or a predisposition to develop the cardiovascular condition, of a patient, the method comprising:(a) separating lipoprotein particles having an average density of from about 1.020 g / mL to about 1.060 g / mL from the blood of said patient, wherein the product of step (a) comprises LDL(-) biomarkers;(b) converting the LDL(-) biomarkers into charged LDL(-) biomarkers;(c) determining the charged LDL(-) biomarkers concentration; and(d) determining said cardiovascular disease, or a predisposition to develop said cardiovascular condition, of the patient.

2. The method of claim 1, further comprising prescribing and / or administering a cardiovascular condition therapy to the subject when the cardiovascular condition is determined by the product of step (d).

3. The method of claim 1, further comprising prescribing and / or administering a cardiovascular condition prophylaxis to the subject when the predisposition to develop said cardiovascular condition is determined by the product of step (d).

4. The method of a preceding claim, wherein the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”).

5. The method of claim 4, wherein the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent.

6. The method of claims 1-3, wherein the LDL(-) biomarkers have a diameter from about 28.0 nm to about 32.0 nm as measured by ion mobility (“IDL-sized”).

7. The method of claim 1, wherein the charged LDL(-) biomarkers from the patient are classified on the basis of mobility by a differential mobility analyzer, and the mobility classification is scanned to create the charged LDL(-) biomarkers concentration.

8. The method of a preceding claim, wherein said charged LDL(-) biomarkers concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition.

9. A method for determining a level of therapeutic responsiveness to a cardiovascular drug or other therapeutic intervention directed at a cardiovascular condition of a patient, comprising:(a) administering said cardiovascular drug or other therapeutic intervention to said patient;(b) obtaining lipoprotein particles from said patient;(c) separating lipoprotein particles having an average density of from about 1.020 g / mL to about 1 .060 g / mL from the product of step (b) wherein the product of step (c) comprises LDL(-) biomarkers;(d) converting the LDL(-) biomarkers into charged LDL(-) biomarkers;(e) determining the charged LDL(-) biomarkers concentration; and(f) determining the level of therapeutic responsiveness to a cardiovascular drug or other therapeutic intervention directed at a cardiovascular condition of a patient.

10. The method of claim 9, further comprising:(g) reducing the amount of said cardiovascular drug or other therapeutic intervention to said patient when the product of step (f) is above the desired therapeutic responsiveness; or increasing the amount of said cardiovascular drug or other therapeutic intervention to said patient when the product of step (f) is below the desired therapeutic responsiveness.

11. The method of claims 9-10, wherein the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”).

12. The method of claim 11, wherein the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent.

13. The method of claims 9-10, wherein the LDL(-) biomarkers have a diameter from about 28.0 nm to about 32.0 nm as measured by ion mobility (“IDL-sized”).

14. The method of claim 9, wherein the charged LDL(-) biomarkers are classified on the basis of mobility by a differential mobility analyzer, and the mobility classification is scanned to create the charged LDL(-) biomarkers concentration.

15. The method of claims 9-14, wherein said charged LDL(-) biomarkers concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition.

16. A method for determining a cardiovascular condition, or a predisposition to develop the cardiovascular condition, of a patient, the method comprising:(a) obtaining a charged LDL(-) biomarkers concentration from the patient; and(b) determining the cardiovascular condition or the predisposition to develop the cardiovascular condition, of the patient.

17. The method of claim 16, further comprising prescribing and / or administering a cardiovascular condition therapy to the subject when the cardiovascular condition is determined by the product of step (a).

18. The method of claim 16, further comprising prescribing and / or administering a cardiovascular condition prophylaxis to the subject when the predisposition to develop said cardiovascular condition is determined by the product of step (a).

19. The method of claims 16-18, wherein the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”).

20. The method of claim 19, wherein the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent.

21. The method of claims 16-18, wherein the LDL(-) biomarkers have a diameter from about 28.0 nm to about 32.0 nm as measured by ion mobility (“IDL-sized”).

22. The method of claim 16, wherein at least one of the steps of (a) and (b) is carried out by a deterministic algorithm.

23. The method of claims 16-22, wherein said charged LDL(-) biomarkers concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition.

24. A method for assessing lipid-related health risk comprising the steps of:(a) separating lipoprotein particles having an average density of from about 1.020 g / mL to about 1.060 g / mL from the blood of said patient, wherein the product of step (a) comprises LDL(-) biomarkers;(b) adding to the product of step (a) one or more diluents, thereby forming a diluted sample;(c) electrospraying said diluted sample;(d) reducing the electrosprayed sample to a uniform charge;(e) passing said uniformly charged diluted sample to a volatilizing chamber to evaporate said diluents, forming individual uniformly charged LDL(-) biomarker ions;(f) transporting said individual uniformly charged LDL(-) biomarker ions into a size- selectable differential mobility analyzer;(g) counting the number of individual uniformly charged LDL(-) biomarker ions, dn+, in a defined sampling time, dt, at each size selection, s, of the selectable differential mobilityanalyzer, *' resulting in a size output;(h) scanning the size output over a range of size selections, resulting in an output histogram of charged LDL(-) biomarkers counted in the defined sampling time versus size; and(i) assessing the lipid-related health risk.

25. The method of claim 24, further comprising prescribing and / or administering a lipid-related health risk therapy to the subject when the lipid-related health risk therapy is assessed by the product of step (i).

26. The method of claim 24 or 25, wherein the range of size selections is from about 13.0 nm to about 18.0 nm.

27. The method of claim 26, wherein the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent.

28. The method of claim 24 or 27, wherein the range of size selections is from about 28.0 nm to about 32.0 nm.

29. The method of claims 24-28, wherein said output histogram of charged LDL(-) biomarkers correlates with the lipid-related health risk.

30. A method for determining a cardiovascular condition, or a predisposition to develop the cardiovascular condition, of a patient, the method comprising:(a) precipitating non-lipoprotein proteins from the blood of said patient, wherein the product of step (a) comprises a supernatant fraction and the supernatant fraction comprises LDL(-) biomarkers;(b) converting the LDL(-) biomarkers into charged LDL(-) biomarkers;(c) determining the charged LDL(-) biomarkers concentration; and(d) determining said cardiovascular disease, or a predisposition to develop said cardiovascular condition, of the patient wherein the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”).

31. The method of claim 30, further comprising prescribing and / or administering a cardiovascular condition therapy to the subject when the cardiovascular condition is determined by the product of step (d).

32. The method of claim 30, further comprising prescribing and / or administering a cardiovascular condition prophylaxis to the subject when the predisposition to develop said cardiovascular condition is determined by the product of step (d).

33. The method of claim 30, wherein the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent.

34. The method of claims 30-33, wherein said charged LDL(-) biomarkers concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition.

35. A method for determining a level of therapeutic responsiveness to a cardiovascular drug or other therapeutic intervention directed at a cardiovascular condition of a patient, comprising:(a) administering said cardiovascular drug or other therapeutic intervention to said patient;(b) precipitating non-lipoprotein proteins from the blood of said patient, wherein the product of step (a) comprises a supernatant fraction and the supernatant fraction comprises LDL(-) biomarkers;(c) converting the LDL(-) biomarkers into charged LDL(-) biomarkers;(d) determining the charged LDL(-) biomarkers concentration; and(e) determining the level of therapeutic responsiveness to a cardiovascular drug or other therapeutic intervention directed at a cardiovascular condition of a patient wherein the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”).

36. The method of claim 35, further comprising:(f) reducing the amount of said cardiovascular drug or other therapeutic intervention to said patient when the product of step (e) is above the desired therapeutic responsiveness; or increasing the amount of said cardiovascular drug or other therapeutic intervention to said patient when the product of step (e) is below the desired therapeutic responsiveness.

37. The method of claim 35 or 36, wherein the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent.

38. The method of claims 35-37, wherein said charged LDL(-) biomarkers concentration correlates with the cardiovascular condition, or the predisposition to develop said cardiovascular condition.

39. A method for assessing lipid-related health risk comprising the steps of:(a) precipitating non-lipoprotein proteins from the blood of said patient, wherein the product of step (a) comprises a supernatant fraction and the supernatant fraction comprises LDL(-) biomarkers;(b) adding to the product of step (a) one or more diluents, thereby forming a diluted sample;(c) electrospraying said diluted sample;(d) reducing the electrosprayed sample to a uniform charge;(e) passing said uniformly charged diluted sample to a volatilizing chamber to evaporate said diluents, forming individual uniformly charged LDL(-) biomarker ions;(f) transporting said individual uniformly charged LDL(-) biomarker ions into a size- selectable differential mobility analyzer;(g) counting the number of individual uniformly charged LDL(-) biomarker ions, dn+, in a defined sampling time, dt, at each size selection, s, of the selectable differential mobilityanalyzer, resulting in a size output;(h) scanning the size output over a range of size selections, resulting in an output histogram of charged LDL(-) biomarkers counted in the defined sampling time versus size; and(i) assessing the lipid-related health risk wherein the LDL(-) biomarkers have a diameter from about 13.0 nm to about 18.0 nm as measured by ion mobility (“midzone”).

40. The method of claim 39, further comprising prescribing and / or administering a lipid-related health risk therapy to the subject when the lipid-related health risk therapy is assessed by the product of step (i).

41. The method of claim 39, wherein the LDL(-) biomarkers comprise a particle comprising apolipoprotein Al as the main protein constituent.

42. The method of claims 39-41, wherein said output histogram of charged LDL(-) biomarkers correlates with the lipid-related health risk.

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