METHOD FOR DETERMINING CARDIOVASCULAR RISK BY POSTPRANDIAL CHANGES IN HDL3

Abstract

Embodiments of the present invention include a method (10) for assessing a patient for cardiovascular risk. The method includes the steps of: drawing a first blood sample from a patient in a fasting condition; administering a liquid based protein meal to the patient; drawing a second blood sample from the patient after a time period of 60-120 minutes after the step of administering the meal; measuring HDL3-P of the first blood sample so as to determine a fasting HDL3-P amount (20); measuring HDL3-P of the second blood sample so as to determine a postprandial HDL3-P amount (30); comparing the fasting HDL3-P amount and the postprandial HDL3-P amount; and detecting cardiovascular risk based on the comparison.

Claims

1. A method (10) for assessing a patient, the method comprising the steps of: drawing a first blood sample from a patient in a fasting condition; administering a liquid based protein meal to the patient; drawing a second blood sample from the patient after a time period of 60-120 minutes after the step of administering the meal; measuring HDL3-P of the first blood sample so as to determine a fasting HDL3-P amount (20); measuring HDL3-P of the second blood sample so as to determine a postprandial HDL3-P amount (30); comparing said fasting HDL3-P amount and said postprandial HDL3-P amount; and detecting cardiovascular risk based on step of comparing.

2. The method for assessing, according to claim 1, wherein said fasting condition is comprised of at least eight hours of fasting completed by said patient before the step of drawing said first blood sample.

3. The method for assessing, according to claim 1, wherein said liquid base protein meal is comprised of one half of a recommended dietary allowance of protein.

4. The method for assessing, according to claim 3, wherein said recommended dietary allowance is 0.8 g of protein per kg of body weight per day.

5. The method for assessing, according to claim 3, wherein said recommended dietary allowance is for a person with moderate physical activity.

6. The method for assessing, according to claim 5, wherein said recommended dietary allowance for said person with moderate physical activity is 0.6 g of protein per kg of body weight per day

7. The method for assessing, according to claim 6, wherein said liquid protein meal is comprised of 0.3 g of protein per kg of body weight of said patient.

8. The method for assessing, according to claim 1, wherein said liquid base protein meal is high protein, low carbohydrate, low fat with known grams of protein per ounce.

9. The method for assessing, according to claim 1, wherein said time period is 90 minutes.

10. The method for assessing, according to claim 1, wherein said time period is equal to a postprandial time period corresponding to a statistically significant increase in HDL particles from a protein meal over a mono unsaturated fat meal.

11. The method for assessing, according to claim 10, wherein said statistically significant increase in HDL particles is determined by protein.

12. The method for assessing, according to claim 10, wherein said statistically significant increase in HDL particles is determined by an increase in HDL3 particles.

13. The method for assessing, according to claim 1, wherein the step of comparing is comprised of: subtracting said fasting HDL3-P amount from said postprandial HDL3-P amount so as to determine an HDL3-P activation index; and wherein said step of detecting is comprised of: determining a health profile corresponding to cardiovascular fitness; and matching said HDL3-P activation index to said health profile.

14. The method for assessing, according to claim 13, wherein said health profile is comprised of an increase in said HDL3-P activation index, equivalent HDL2b-P particle amounts in said fasting condition and in said postprandial condition, and equivalent HDL-C cholesterol amounts in said fasting condition and in said postprandial condition.

15. The method for assessing, according to claim 14, wherein said increase in said HDL3-P activation index is between 0-100%.

16. The method for assessing, according to claim 14, further comprising the step of: making a treatment recommendation according to the step of matching.

17. The method for assessing, according to claim 1, wherein said step of detecting is comprised of: determining an overall cardiovascular risk score for said patient; and correlating said HDL3-P activation index to said overall cardiovascular risk score.

18. The method for assessing, according to claim 17, wherein said HDL3-P activation index is inversely related to said overall cardiovascular risk score.

19. The method for assessing, according to claim 17, further comprising the step of: making a treatment recommendation according to the step of correlating.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0045] FIG. 1 is a schematic view of a table showing HDL subgroups.

[0046] FIG. 2 is a schematic view of a table showing results of healthy individuals and individuals with a metabolic syndrome.

[0047] FIG. 3 is a schematic view of a graph illustration for determining the HDL3-P Activation Index, according to an embodiment of the method of the present invention.

[0048] FIG. 4 is a schematic view of another graph illustration for determining the HDL3-P Activation Index, according to an embodiment of the method of the present invention.

[0049] FIG. 5 is a schematic view of a table showing Postprandial HDL3-P Activation, according to an embodiment of the method of the present invention.

[0050] FIG. 6 is a schematic view of a graph illustration for a health profile of a healthy patient for the method according to the present invention.

[0051] FIG. 7 is a schematic view of a graph illustration for a health profile of a metabolic syndrome patient for the method according to the present invention.

[0052] FIG. 8 is a schematic view of a graph illustration for a health profile of a low fasting HDL3-P patient for the method according to the present invention.

[0053] FIG. 9 is a schematic view of a table showing results of the HDL3 Study, according to an embodiment of the method of the present invention.

[0054] FIG. 10 is a schematic view of Table 2, showing Pearson Correlation Coefficients.

DETAILED DESCRIPTION OF THE INVENTION

[0055] Studies have shown that HDL2a particles and especially HDL2b particles are fully loaded with cholesterol and are near the end of the reverse cholesterol transport cycle for cholesterol delivered to the liver.

[0056] The HDL2b particle was traditionally thought to be the main indicator of cardiovascular risk since the HDL2b particle is the end result of HDL cholesterol loading. A low risk patient or healthy individual should have approximately 25 percent or more HDL particles as HDL2b particles. There is no doubt that low HDL2b particles, as in metabolic syndrome patients, indicates reduced functionality of HDL.

[0057] The cardiovascular risk assessment associated with HDL has recently added another dimension, HDL3 particles. There must be a balance between HDL2b and HDL3, since HDL3 is required as the precursor of HDL2b. Also, the ability of the body to produce more HDL3 particles for a different pathway of efficient reverse cholesterol transport is critical as determined in a number of studies. There are different functionalities of HDL3 that require consideration before any single measurement of HDL3 can be interpreted or relied upon as a risk factor.

[0058] For example, HDL3 can affect the understanding of HDL2b along the reverse cholesterol transport process. Low amounts of HDL2b particles, as in metabolic syndrome patients, indicate reduced functionality of HDL, but the reverse proposition that very high amounts of HDL2b particles indicate health and good HDL functionality may not necessarily be true. Instead, very high amounts of HDL2b may indicate an inability to complete the cycle of reverse cholesterol transport, that is, an inability to unload stored cholesterol to the liver. In that example, measurement of HDL3 would be a better indicator of cardiovascular risk and the potential of HDL to complete the reverse cholesterol transport process. HDL3 particles start the reverse cholesterol process from HDL3 to HDL2 to the liver and the direct reverse cholesterol process from HDL3 to the liver. Previous measurements of HDL2 as indicators of cardiovascular health did not account for HDL3, and the previous measurements of HDL3 were not interpreted correctly or ignored. In the prior art, a higher amount of fasting HDL3 was interpreted to indicate the inability to complete the reverse cholesterol transport process through HDL2. The prior art considered the result to be a less favorable medium that too much HDL3 meant that HDL2 was not being formed to complete the reverse cholesterol transport process.

[0059] Simply measuring HDL3 does not allow a reliable determination of cardiovascular risk. There are also existing methods to simply measure the amount of lipoproteins in a blood sample. The Lipoprotein Particle Profile (LPP) method for lipoprotein particle number measurement has been described in detail in the prior art, including U.S. Pat. No. 7,856,323, issued on Dec. 21, 2010 and WIPO publication WO 2016/049528 A1, published on Mar. 31, 2016. Other known measurements of HDL3 particle types may also be incorporated into the present invention. Known lipoprotein detection methods can already measure the subgroups of all lipoproteins and especially the HDL subgroups needed in the present invention which is aimed at the dietary functionality of HDL3.

[0060] The present invention addresses the need for a method to apply the step of measuring HDL3 particles in order to reliably determine cardiovascular risk.

[0061] The conventional step of measuring particles is in the fasting condition. Most lipids are measured in the fasting state to get a more accurate measure of triglycerides; however, the fasting state is not representative of the daily lipoprotein activity. People are in a non-fasting state most of the day, so the levels of lipoproteins and especially HDL subgroups, such as HDL3, are not the same as in the fasting patient. Therefore, the prior art measurements of HDL activity are biased views of the HDL particles for reverse cholesterol transport. Further complicating the assessment of lipoprotein activity is the fact that lipoprotein particles numbers can change in different ways from their cholesterol content. HDL-P for particles and HDL-C for cholesterol available to the HDL-P particles are different measurements. Generally, it has been observed that the lipoprotein particle numbers (HDL-P) in non-fasting patients are very similar to lipoprotein particle numbers (HDL-P) in fasting patients, except for Very Low Density Lipoproteins (VLDL) lipoproteins, where much of the triglycerides are stored. Non-fasting VLDL particles numbers show a shift from the smaller VLDL subgroup of VLDL3 to the larger particles of VLDL1 & 2. Other lipoprotein subgroups were not known to make much of an adjustment.

[0062] The prior art has previously addressed the steps of measuring HDL particles in both the fasting condition and the non-fasting condition. The results showed that a high protein (HP) meal compared to a high mono unsaturated fat (HMF) meal had no significant change in the cholesterol (HDL-C), but total particles (HDL-P) and the medium-small particles (HDL3+HDL2a particles of the HDL-P total) showed a significant increase (p=0.01). The prior art determined that the HP meal was a less favorable medium compared to the HMF meal, since the HDL-C cholesterol was unchanged and the larger HDL2b particles did not increase.

[0063] The significance of the increase in HDL-P particles overall due to the medium-small particles (HDL3+HDL2a) is largely unexplained in the prior art. As a side note in the prior art studies, the overall conclusion still supported established guidelines for increasing HDL-C cholesterol and the larger HDL2b particles. Having the increase in the HDL3 did not contradict the findings to increase HDL-C cholesterol and the larger HDL2b particles. There was no suggestion, motivation, or teaching that the prior art steps of measuring HDL particles in a fasting condition and measuring HDL particles in the postprandial condition can provide any other information, other than confirmation of known conclusions for dietary recommendations and cardiovascular health.

[0064] The present invention addresses the need for a method to apply the step of measuring HDL3 particles in a fasting condition and the step of measuring HDL3 particles in a postprandial condition in order to reliably determine cardiovascular risk.

[0065] Embodiments of the present invention include a method for assessing a patient for cardiovascular risk. The method comprises the steps of: drawing a first blood sample from a patient in a fasting condition; administering a liquid based protein meal to the patient; drawing a second blood sample from the patient after a time period of 60-120 minutes after the step of administering the meal; measuring HDL3-P of the first blood sample so as to determine a fasting HDL3-P amount; measuring HDL3-P of the second blood sample so as to determine a postprandial HDL3-P amount; comparing the fasting HDL3-P amount and the postprandial HDL3-P amount; and detecting cardiovascular risk based on the comparison.

[0066] According to embodiments of the step of drawing the first blood sample, the fasting condition is comprised of at least eight hours of fasting completed by the patient before the step of drawing the first blood sample. The eight hours without food or beverage intake, other than water, is a conventional standard for blood tests.

[0067] According to embodiments of the step of administering the liquid based protein meal, a specific dietary supplement is needed, similar to a suitable supplement for an oral glucose tolerance test. The meal of the present invention requires a high protein content and enough calories to generate an appropriate dietary response. Since the prior art determined the HDL3-P particle increases after the high protein meal, it was necessary to determine whether the HDL3-P particle increases related to the high protein content or the low fat content. In limited testing, it appeared that the addition of either fat or carbohydrate calories had little effect on the HDL3-P particle increases or HDL3-P activation. Furthermore, it was reasonable to select a high protein meal because the first step in the production of HDL3 is the formation of Apo A-1, a lipoprotein formed from the amino acid nutrients in a high protein drink. The Apo A-1 is combined with phospholipids to form a nacent pre-Beta HDL. Thus, embodiments of the present invention disclose a high protein drink as the meal to separate the fasting condition and the postprandial condition.

[0068] The relative high protein meal or drink required further standardization. In the present invention, the liquid base protein meal is comprised of one half of a recommended dietary allowance of protein. The Recommended Dietary Allowance (RDA) for a healthy adult is 0.8 grams of protein per kg of body weight per day. See Wu, G., Food Function, 2016 March; 7(3):1251-65. A person with moderate physical activity needs 1.3 grams of protein per kg of body weight per day or 0.6 grams per pound per day so a good estimate of a person's meal requirement would be one half of this dietary allowance or 0.3 grams of protein per pound of body weight. A high amount of protein is half of the total amount of protein recommended for the entire day in a single meal. Generally, the liquid base protein meal is high protein, low carbohydrate, and low fat with known grams of protein per ounce. A number of commercially available high protein liquid based meals are also considered for the liquid based protein meal of the present invention. Desirable liquid based meals are ones that have at least 40 grams of protein and minimal fat and carbohydrate calories.

[0069] According to embodiments of the step of drawing the second blood sample, the time period is equal to a postprandial time period corresponding to a statistically significant increase in HDL particles of a protein meal over a mono unsaturated fat meal. The prior art studies showed that between 120 minutes and 180 minutes, there was not a significant change in medium and small HDL-P, the HDL3-P particles. Thus, shorter postprandial times of 75, 90, and 100 minutes were tested, and the responses of the HDL3-P particles were not significantly different within the range of 60-120 minute time period. Since the present invention utilizes the high protein meal, the statistically significant increase in HDL particles should be determined by protein, not fat as previously discussed. Furthermore, the statistically significant increase in HDL particles is determined by an increase in HDL3 particles, not any HDL2 particles. In particular embodiments, the time period is 90 minutes, wherein the current assay protocol is to draw blood on a fasting patient and then have the patient drink a liquid based high protein meal and wait for 90 minutes before making a second blood draw. The timing of the second draw is flexible for at least 15 minutes after the 90 minutes waiting time since it was shown that there was no significant change in HDL3-P from 75 to 120 minutes in the prior art studies.

[0070] According to embodiments for the steps of measuring HDL3 of the first blood sample and measuring HDL3 of the second blood sample, the present invention relies on known particle measurement methods, including Ultracentrifugation as in the Lipoprotein Particle Profile (LPP (registered trademark)), Ion Mobility, 2-Dimensional electrophoresis, and NMR technology.

[0071] According to embodiments for the step of comparing, the present invention includes subtracting the fasting HDL3-P amount from the postprandial HDL3-P amount so as to determine an HDL3-P activation index, while the step of detecting is comprised of determining a health profile corresponding to cardiovascular fitness and matching the HDL3-P activation index to the health profile. FIG. 3 shows the determination of the HDL3-P activation index. FIGS. 4 and 5 show the determination of the health profile with factors besides the HDL3-P activation index, specifically, the modest HDL2a-P increase, the lack of HDL2b-P increase, and the equivalent HDL-C cholesterol amounts in the fasting condition and in the postprandial condition. The health profile can be comprised of an increase in the HDL3-P activation index, equivalent HDL2b-P particle amounts in the fasting condition and in the postprandial condition, and the equivalent HDL-C cholesterol amounts in the fasting condition and in the postprandial condition. The health profile is not just the HDL3-P activation index alone. In some embodiments, the increase in the HDL3-P activation index is between 0-100%. Based on the test results of FIG. 9, the increase in total HDL3-P corresponds to an increase in Total HDL-P of over 30%, and the lowest quartile for HDL3 activation index is less than 600 nmol/L, setting less than 600 nmol/L as a low HDL3 activation index. The HDL3-P is considered to be a primary lipoprotein increased for improved macrophage and cellular free cholesterol efflux.

[0072] Embodiments of the method further comprise making a treatment recommendation according to the step of matching. When a patient matches a health profile of an individual with a metabolic syndrome with high cardiovascular risk factors, the patient can be prescribed medications and activities to improve cardiovascular health. The patient can also be warned to avoid certain activities. FIG. 6 shows a health profile of a healthy individual. FIG. 7 shows a health profile of a metabolic syndrome patient. FIG. 8 shows how the prior art can be reconciled with the present invention, wherein low amounts of fasting HDL3-P had previously different interpretations. In FIG. 8, the low fasting HDL3-P is not necessarily determinative of cardiovascular risk itself. The HDL3-P activation index remains high, so this individual is still able to react with HDL3-P pathways for direct reverse cholesterol transport.

[0073] According to alternate embodiments for the step of detecting is comprised of: determining an overall cardiovascular risk score for the patient; and correlating the HDL3-P activation index to the overall cardiovascular risk score. FIG. 9 shows determination of the overall cardiovascular risk score and the comparison to the HDL3-P activation index. The HDL3-P activation index can be inversely related to the overall cardiovascular risk score. Similar to other embodiments, the method can further comprise making a treatment recommendation according to the step of correlating. The patient with a low HDL3-P activation and a high cardiovascular risk score can be prescribed medications and activities to improve cardiovascular health. The patient can also be warned to avoid certain activities.

[0074] The present invention discloses a method to measure the full potential of the HDL3-P particles in a non-fasting environment, when there is a maximum effect on reverse cholesterol transport. The present invention more closely resembles the non-fasting state of daily life. A number of variables exist that need to be determined, such as composition of the dietary supplement and the optimal length of postprandial time for the measurement. However, the general prior art disclosures of postprandial effects determined by meal selection and time period tested after the meal fail to disclose the method of the present invention.

[0075] It is known that patients can vary dramatically in the HDL3-P increase due to dietary supplementation and with the length of postprandial time. Specifically, patients with high very buoyant HDL2b-P often have low fasting levels of HDL3-P that increases with a meal. Also, patients with low HDL2b-P often had high HDL3-P levels that did not change with a meal. FIG. 9 shows that some patients had a modest or no improvement in HDL3-P, where other patients showed a significant increase. Additional work is necessary to further all results of the present invention, but the evidence clearly supports a relationship between HDL3-P and cardiovascular risk. For example, it is not yet been proven that the percent (30%) or quantity (less than 600 nmol/L) as measured in nmol/L of HDL3 particle numbers is directly and causally related to cardiovascular health or if only the final response is more predictive of reverse cholesterol transport and risk. The HDL3 levels measured in fasting patients in the prior art were inconclusive regarding cardiovascular risk with little guidance to resolve the discrepancies between studies. The present invention presents a method to reconcile the current state of the prior art.

[0076] An in-house correlation study of 600 random patients in FIG. 10 indicated that HDL3-P is truly an independent marker which agrees with previous work. The largest lipoprotein correlation was with Total HDL-P which is understandable since HDL3-P it is a component of HDL-P. HDL3-P was poorly or negatively correlated with HDL2a-P and HDL2b-P which emphasizes the importance of measuring only HDL-3-P and not including HDL2a-P or medium size HDL. Interestingly, negative weak correlations exist between high sensitivity C-reactive protein (CRP-hs), an inflammation marker and fat metabolism markers of Leptin and Adiponectin since these are all associated with cardiovascular risk. Also, Apo A-1 correlations were examined since Apo A-1 is the indicator in 2-D electrophoresis measurements and the main protein on HDL. A high correlation of Apo A-1 with HDL2b of r=0.86 was found and a very low correlation of r=0.26 for HDL2a. For HDL3 a correlation of r=0.11 was determined which is probably due to the fact that one or two Apo A-1 molecules are present on HDL3. This observation makes the quantitative measurements of HDL subgroups, using Apo A-1 as the indicator, subject to large errors.

[0077] The measurement of the dietary response of lipoproteins and more specifically HDL3-P can be a useful metric, not only to determine cardiovascular risk but also in the testing of various therapies to stimulate HDL production. A number of studies have been done using Cholesterol Ester Transfer Protein (CETP) inhibitors to improve HDL for risk reduction. These approaches have failed in clinical trials but this may be due to targeting the production of HDL2b-C and not the production of HDL3-P. Until now a method to study HDL3-P without interference from HDL2a-P has not been developed. Additionally, most studies looked at HDL cholesterol rather than particle numbers so the benefit of increasing HDL3-P was not noticed. The dietary influence on HDL3-P was not studied in the CETP trials so the true level of HDL activation was unknown.

[0078] The HDL Activation Assay:

[0079] This purpose of the present invention is as an assay to measure the postprandial levels of HDL3-P to determine cardiovascular risk and HDL3-P activation. Some prior art studies have shown that fasting low levels of HDL3-C or HDL3-P are associated with higher risk and some studies show that high levels of HDL3-C or HDL3-P are associated with cardiovascular disease. Since HDL3 is needed for indirect reverse cholesterol transport, low levels of HDL3 could impair this process. Low levels of HDL3 in the fasting condition is not necessarily bad and associated with cardiovascular disease (See FIG. 8). Most of the day a person is in a non-fasting state, so it is logical that the postprandial values of HDL3-P are important. Since total HDL-C does not increase in the postprandial state it cannot be used to measure this potential. From previous work it was shown that a meal, especially a high protein meal will stimulate the body to produce HDL3-P particles that are nearly free of cholesterol. This is the activation of HDL3-P and believed to be important in cardiovascular risk.

[0080] The results of a fasting patient of the present invention show a significant postprandial increase in HDL3-P, that the HDL3-P particles must have performed their function by being converted to HDL2b-P or returning to the liver as HDL3 particles to complete the reverse cholesterol transport cycle. This direct reverse cholesterol transport cycle is also an example of healthy functioning HDL. In metabolic syndrome patients, where small postprandial increases in HDL3-P are observed and low fasting HDL2b-P is present that doesn't change with a meal, the reverse cholesterol transport cycle is dysfunctional. The small postprandial increases represent a disease state that can be detected by the assay of the present invention and is not accurately measured by a fasting HDL3 assay alone.

[0081] Metabolic syndrome patients usually have high fasting HDL3-P but this is different type of HDL3-P that has been formed by the action of CETP and hepatic lipases in the presence of elevated triacylglycerols. This enzymatic activity reduces the size of HDL2's to that of an HDL3. For clarity, this type of HDL3 is HDL3X-P. It appears that HDL3X-P is not recognized by hepatic SR-B1 (scavenger receptor B1) for functional reverse cholesterol transport so in spite of a reasonable total HDL particle number, metabolic syndrome patients actually have very little HDL2b-P and non-functional HDL3X-P.

[0082] The methods of measuring HDL3 are known. The profiles of HDL particles are composed from the integrated intensity of 20 Gaussian profiles at specific densities that define each lipoprotein group and subgroup. HDL-P is composed of two Gaussians for HDL2b-P positioned at a density of 1.063-1.100 g/ml, one for HDL2a-P positioned at a density of 1.100-1.125 g/ml and two for HDL3-P positioned at a density of 1.125-1.200 g/ml. The result for HDL3-P was not previously reported separately but rather as a component of total HDL-P since there was no clinical guidance for this result.

[0083] Accurate individual measurement of small, HDL3-P particle numbers has not been reported previously without the influence of mid-sized HDL-P or HDL2a-P. As shown in the lipoprotein particle profile result (FIG. 4), there is a clear separation of HDL2a-P and HDL3-P and these results have a very low Pearson Correlation Coefficient of HDL3-P to HDL2a-P of r=0.15 (FIG. 10, Table 2). The current Lipoprotein Particle Profile now reports HDL3-P as a separate result.

[0084] The present invention is a method to measure the difference between fasting and postprandial lipoprotein particle number values to determine cardiovascular risk. On the lipoprotein particle profile report up to four measurements can be presented to compare results and perform calculations related to those results. The present invention is focused on the cardiovascular risk association from the activation of lipoprotein subgroup HDL3-P. In FIG. 8, patients with average to higher fasting HDL3-P values often had modest percentage increases in HDL3-P and this appeared to be associated with metabolic syndrome type profiles or higher cardiovascular risk. In one embodiment, a reference value was determined for a small number of activated specimens representing patients with various levels of cardiovascular disease. This value was determined to be approximately 600 nmol/L.

[0085] The Cardiovascular Risk and HDL3-P Activation of the assay from the method of the present invention can be evaluated based on results from:

[0086] 1. The final postprandial increased HDL3-P value.

[0087] 2. The increase in HDL3-P between the fasting and postprandial results.

[0088] 3. The changes that result between fasting and postprandial results for all lipoprotein subgroups.

[0089] FIG. 9 shows cardiovascular and health risk scoring from assay results for a study from a variant population. HDL3-P activation was shown to change from 0% to 100% depending on the patient. In other words, the up to a 100% increase in previously unknown reverse cholesterol transport potential. The increase also corresponds to an increase in total HDL-P of over 30%. More importantly, the study shows an indication of direct correlation with both apparent health and apparent cardiovascular risk of the patients. Poor HDL3-P activation appears to be associated pre-diabetic or diabetic patients, existing atherosclerosis, obesity and poor physical condition and strong family history of atherosclerosis. These cardiovascular risk conditions can be treated if identified early. Since HDL3-P appears to be the primary component in reverse cholesterol transport and the delivery of anti-oxidants it may very well be the single most important lipoprotein yet in the fasting state its actual potential in unknown. The HDL3-P activation assay has the ability to reveal the true patient cardiovascular risk.

[0090] More detailed studies based on the best correlation between the above three metrics and other lipid and advanced lipoprotein results, other analytesmany of which are listed in FIG. 10, additional medical diagnostics, height, weight, blood pressure, exercise, diet, possible disease states, medication, vitamins and supplements.

[0091] The foregoing disclosure and description of the invention is illustrative and explanatory thereof. Various changes in the details of the described method can be made without departing from the true spirit of the invention.