WO2004087737A2 - Omega-3 fatty acid assays for disease risk assessment - Google Patents

Abstract

Methods for determining an individual’s overall risk of death from coronary heart disease are provided. The methods utilize measurements of the individual’s levels of omega-3 polyunsaturated fatty acids in order to stratify them according to their risk of death from CHD. The risk-stratification can be used to classify the individual’s risk of death from CHD as low, intermediate, or high based on levels of omega-3 fatty acids of 8% or higher, between 4% and 8%, and 4% or below, respectively. The present invention then provides methods of monitoring an individual’s progress toward lowering their risk by periodically measuring their omega-3 levels and instructing the individuals to alter their intake of omega-3 fatty acid-containing foods or supplements.

Description

OMEGA-3 FATTY ACID ASSAYS FOR DISEASE RISK ASSESSMENT

RELATED APPLICATIONS The present application claims the benefit of Provisional Patent Application Serial No. 60/_ filed on March 26, 2003, entitled OMEGA-3 FATTY ACID ASSAYS FOR

DISEASE RISK ASSESSMENT, the content and teachings of which are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention generally pertains to methods of determining an individual's risk of death from coronary heart disease. More particularly, the present invention is concerned with methods of determining an individual's risk of death from coronary heart disease (CHD) by assaying a biological sample for the individual's level of omega-3 polyunsaturated fatty acids. The individual's level of omega-3 polyunsaturated fatty acids are then compared to a standard indicating the individual's risk of death from CHD relative to the population as a whole. Still more particularly, the present invention is concerned with risk-stratifying individuals based on the levels of omega-3 polyunsaturated fats in a biological sample taken from that individual. These omega-3 levels are compared to a standard wherein levels less than or equal to 4% indicate a relatively high risk, levels between 4% and 8% indicate an intermediate risk, and levels of 8% and greater indicate a relatively low risk of death from CHD.

Description ofthe Prior Art

Approximately 50 % of cardiovascular disease fatalities occur suddenly. A number of risk factors for sudden cardiac death such as serum lipids, blood pressure, diabetes, NYHA functional class, ejection fraction, and syndromes such as Bragada or Wolfe-Parkinson- White have been identified. However, these risk factors suffer from low predictive power, low specificity, or only apply to minute fractions ofthe population. Therefore, the vast majority of sudden cardiac deaths occur in persons not readily identifiable by current approaches. The beneficial effects of diets incorporating omega-3 polyunsaturated fatty acids (omega-

3's) have been speculated about for over 25 years. It was not until November of 2002 that the evidence had become sufficiently strong to allow the American Heart Association (AHA) to officially recommend the use of omega-3 polyunsaturated fatty acids for risk reduction. Their recommendation is about 1-g per day of eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) (the principal omega-3 polyunsaturated fatty acids in fish oils) for reducing risk for death from CHD in patients with established disease. In addition, for individuals without known disease, the AHA recommendation is to consume at least two, preferably oily, fish meals per week. Although these nutritional recommendations are an important step forward, measuring a blood biomarker of omega-3 FA intake, and establishing a target level of that biomarker would help physicians assess individual risk and compliance with specific recommendations. Such a biomarker would then become a modifiable risk factor for CHD death.

Previous investigations of the relationship between omega-3's and CHD have been structured to show first that a relationship existed between omega-3 (actually fish) intakes and the risk for death due to CHD. A few more recent studies correlated risk of death from CHD with specific blood levels of omega 3's. No studies have attempted to quantify risk of death from CHD according to blood levels of omega-3's, nor has there been an attempt to stratify risk into categories (low, intermediate, or high). Instead, the prior art has focused on determining whether or not a relationship actually exists between omega-3 levels and risk of death from CHD. These studies have not recommended specific target blood levels for risk prediction, although they have shown that the inverse association between risk and blood levels is stronger than and independent of traditional risk factors.

One such study (Siscovick et al., Dietary Intake and Cell Membrane Levels of Long- Chain Omega-3 Polyunsaturated Fatty Acids and the Risk of Primary Cardiac Arrest, 274:17, JAMA, pages 1363-1367 (1995) the teachings and content of which are hereby incorporated by reference) found a relationship between either the estimated dietary intake or the blood levels of omega-3 polyunsaturated fatty acids and a reduced risk of primary cardiac arrest. Similarly, the recent (non-prior art) study by Albert, et al., (Blood Levels of Long-Chain Omega-3 Fatty Acids and the Risk of Sudden Death, 346:15 N. Engl. J. Med., 1113-1118 (2002) the teachings and content of which are hereby incorporated by reference) suggested that blood omega-3 levels were associated with reduced risk of sudden cardiac death. This study measured whole blood long- chain omega-3's which include EPA, DHA, and docosapentaenoic acid (DP A). However, neither of these studies quantified the levels of omega-3's that would result in a low, intermediate, or high risk of death from CHD, nor did they recommend that blood omega-3 fatty acid levels be considered a risk factor for CHD.

Accordingly, what is needed in the art is a method of quantifying an individual's risk of death from CHD based on their levels of omega-3 's. What is further needed is a standard reference that could be used to compare levels of omega-3's with the risk of death from CHD. What is still further needed is a method of measuring the effects of different intake levels of omega-3 's on the levels of omega-3 's from biological samples . Such measurements could be used to determine individual differences in metabolism of omega-3 's as well as track patient compliance with and effectiveness of increasing the intake amounts omega-3's in different individuals in order to decrease their risk of death from CHD. What is even further needed is a method of increasing the accuracy of determining an individual ' s overall risk of death from CHD by using omega-3 levels as a risk factor that must be taken into account when determining the overall risk. Such a risk factor could be used alone or in combination with other known risk factors.

SUMMARY OF THE INVENTION The present invention overcomes the problems inherent in the prior art and provides quantified levels of omega-3 's from which individual's can be stratified according to their risk of death from CHD. This quantification is termed the Omega-3 Index and it is defined as the eicosapentaenoic (EPA; C20:5) and docosahexaenoic acid (DHA: C22:6) content of red blood cells (RBC) expressed as a percent of total red blood cell (RBC) fatty acids (FA). DPA can also be measured as a part ofthe Omega-3 Index, however, it is preferable to exclude this omega-3 polyunsaturated acid from the Omega-3 Index since it does not add predictive value. As used herein, when the terms omega-3 s or Omega-3 Index are used, these terms refer to EPA and DHA and exclude DPA unless specifically noted.

The risk levels of the Omega-3 Index can be classified as low, intermediate, or high, depending upon the precise levels of omega-3's measured in a biological sample from the individual. As used herein, the term "low" with respect to the risk of death from CHD refers to a risk that is lesser, reduced, or otherwise below average. The term "intermediate" as used herein with respect to the risk of death from CHD refers to a risk that is medium, average, or between low and high. The term high as used herein with respect to the risk of death from CHD refers to risk that is greater, increased, or otherwise above average.

DHA and, to a lesser extent, EPA are found in most biological membranes due to direct consumption of these fatty acids or to bioconversion from alpha-linolenic acid. Accordingly, omega-3 levels from biological samples such as tissues including adipose, muscle, skin, and cheek, whole blood, whole serum (or plasma), serum lipid fractions including phospholipids, cholesterol esters, triglycerides, and non esterified fatty acids, lipoprotein fractions including chylomicrons, very low density lipoproteins, low density lipoproteins, and high density lipoproteins, lipoprotein lipid fractions, erythrocytes, platelets, leukocytes and red blood cells can be used for purposes ofthe present invention. Of these potential sources for biological samples, it is preferred to use RBCs. RBCs have many advantages over the other potential sources including 1 ) being a lipid bilayer that is more reflective of tissue fatty acid levels than serum fatty acids, 2) RBC omega-3 half-life is 4-6 times longer in RBCs than serum omega-3 half-life which better reflects long-term exposure, 3) the omega-3 levels in RBCs are not influenced by fed or fasting states, 4) the omega-3 levels in RBCs are responsive to increasing intakes, 5) their composition is less influenced by dyslipidemias than is the composition of serum fatty acids, 6) the omega-3 levels in RBCs are less variable than their levels in serum, 7) laboratory assessment of omega-3 levels in RBCs is simpler than in lipoprotein or lipid fraction fatty acids, and 8) the RBCs are resilient to variations in pre-analytical storage conditions. The Omega-3 Index cut points were determined based on data from previous studies as well as studies described herein. The target value for the Omega-3 Index was the level associated with the lowest risk for death from CHD. As described above, one ofthe key studies examining the relationship between a biomarker of omega-3 fatty acid intake and risk for sudden cardiac death utilized, not the Omega-3 Index, but whole blood long-chain omega-3 fatty acid (EPA+DHA plus docosapentaenoic acid, (DPA) C22:5 omega-3) composition. The percent of whole blood fatty acid comprised by the omega-3 fatty acids is quite different from the Omega-3 Index owing to 1) the inclusion of DPA, and 2) the presence of other non-omega-3 fatty acids carried in lipoproteins, leukocytes and platelets. Thus, the Omega-3 Index must be estimated from the whole blood parameter. This estimation was accomplished in two steps: first, by subtracting the contribution of DPA to the whole blood omega-3 fatty acid content, and then by converting whole blood EPA+DHA into the Omega-3 Index by application of an experimentally- determined equation relating one with the other. The latter was established by comparing these two parameters in a random set of 40 fasting blood samples.

Another key study reported the effects of EPA+DHA supplementation on CHD events in a randomized, prospective trial. These authors have recently published the plasma phospholipid EPA+DHA levels, both at baseline and after treatment. In order to estimate the Omega-3 Index in this study, both of these parameters were measured in another set of 65 samples drawn in a variety of past studies from our laboratory. The equation describing this relationship was then determined and applied it to the values reported by Nilsen et al. (Effects of a High-dose Concentrate of Omega-3 Fatty Acids or Corn Oil Introduced Early After Acute Myocardial Infarction on Serum Triacylglycerol and HDL Cholesterol, 1A Am. J. Clin. Nutr., 50-56 (2001) the teachings and content of which are hereby incorporated by reference) to produce an estimate ofthe Omega-3 Index from the data obtained in their study.

The results from a third study were also used to determine the effects of relatively small, defined intakes of EPA+DHA on the Omega-3 Index. Healthy adult subjects provided written, informed consent for this study which had been approved by the Saint Luke's Hospital Institutional Review Board. Subjects for this study had to be free of any diseases and not taking any drugs known to affect lipid metabolism, digestion or absorption, and had to have serum triglycerides between 100 and 300 mg/dL, serum LDL-cholesterol < 130 mg/dL and serum HDL- cholesterol > 40 mg/dL. Subjects were queried regarding the previous month's oily fish intake and were excluded if it exceeded one serving. Subjects on stable background diets were randomized to 0 (placebo), 0.5, 1.0 and 2.0 g of EPA+DHA per day for five months following a one-month placebo run-in period. They were given blinded bottles containing 1 -g capsules and instructed to take seven per day for the entire six-month study. The placebo capsules contained corn oil, and the EPA+DHA capsules contained ROPUFA '30' omega-3 Food Oil (Roche Vitamins, Parsippany, NJ). ROPUFA was given either full-strength (2 g group), pre-blended with corn oil 50:50 (1 g group) or 25:75 (0.5 g group). ROPUFA contained 11% EPA and 18% DHA. Since rather small intakes of omega-3 FA were being studied, subjects were instructed to completely avoid consumption of any oily fish (e.g., salmon, sardines, albacore tuna, mackerel) for the duration of the study but to otherwise make no changes in their diets. Pre-study interviews regarding oily fish intake suggested that this restriction would not significantly impact the normal dietary patterns of individuals living in the central US. Compliance was assessed by pill counts. The 5-month treatment period was sufficiently long to allow RBC FA composition to stabilize.

Considering all ofthe data and analysis herein, the present invention proposes Omega-3 Index outpoints that correlate with low, intermediate and high risk of death from CHD and methods of determining an individual's risk of death from CHD based on their Omega-3 Index. Based on the evidence presented, considering both measured and mtake-estimated Omega-3 Indices from epidemiological and prospective intervention trials, and considering the likely upper limit of benefit, the proposed target Omega-3 Index would be 8-10%. This range would appear to be both necessary and sufficient to provide significant protection against sudden cardiac death. In addition, an Omega-3 Index of 8-10% is readily achievable by following the current AHA dietary guidelines, both for primary as well as secondary prevention. This target range is conservative (i.e., potentially higher than absolutely necessary), however, when dealing with a risk factor that presents no safety concerns if exceeded, aiming too high is better than aiming too low. It is also proposed that an Omega-3 Index of ≤ 4% be considered high risk, whereas an Index between 4% and 8% be considered an intermediate risk.

As noted above, omega-3 FA are found in virtually all biological membranes. These come from either from direct consumption or from bioconversion from alpha-linolenic acid (which is rapid in utero and in the early postnatal period but exceedingly inefficient in adulthood). Since most adult tissues (except neurons) respond with increased EPA+DHA levels when the intake of these FA is increased, there are many candidate tissues that could serve as biomarkers. Although a case can be made for each of these in context, perhaps the strongest case - both from physiology and laboratory practicality - can be made for RBCs. Moreover, much of the rationale presented for choosing specific Omega-3 Index cut-points is based upon epidemiological data in which either RBC or whole blood omega-3 FA levels were related to risk for sudden cardiac death. Thus, there is perhaps no better marker than the Omega-3 Index for estimating risk for CHD death.

The present invention also uses the Omega-3 Index as a risk factor for CHD death. In order to be considered a risk factor, there are several requirements that any putative risk factor/marker must meet in order to be clinically useful. These requirements include: l)the consistency of epidemiological data between populations, withm populations, and prospective cohorts; 2) a strong association between the biomarker and disease; 3) independence from other risk factors; 4) biological plausibility; 5) safely, quickly, and cheaply modifiable; 6) modification of the factor reduces risk; 7) standardized measure; 8) low biological variability; 9) high analytical reproducibility; and 10) low cost. As shown herein, the Omega-3 Index already fulfills many of the most critical components. The epidemiological data, both between and within populations, as well as from prospective cohort studies, is summarized in the 2002 AHA Scientific Statement (Kris-Etherton et al., Fish Consumption, Fish Oil, Omega-3 Fatty Acids, and Cardiovascular Disease, 106 Circulation, 2747-2757 (2002) the content and teachings of which are hereby incorporated by reference). Since that time, other studies have appeared that continue to support the rationale for measuring blood omega-3 FA to estimate risk for death from CHD (see Lemaitre et al., -3 Polyunsaturated Fatty Acids, Fatal Ischemic Heart Disease and Non- fatal Myocardial Infarction in Older Adults. The Cardiovascular Health Study, 76 Am. J. Clin. Nutr. 319-325 (2002); Mozaffarian et al., Cardiac Benefits of Fish Consumption May Depend on the Type of Fish Meal Consumed: the Cardiovascular Health Study, 107 Circulation JTD - 0147763 1372-1377 (2003); Hu et al., Fish and Long-chain Omega-3 Fatty Acid Intake and Risk of Coronary Heart Disease and Total Mortality in Diabetic Women, 107 Circulation, 1852-1857 (2003); and Paganelli et al., Altered Erythrocyte -3 Fatty Acids in Mediterranean Patients with Coronary Artery Disease, 78 hit. J. Cardiol., 27-32 (2001) the content and teachings of which are hereby incorporated by reference). Importantly, the associations between the biomarker and disease were independent of all known CHD risk factors including serum lipids, blood pressure, BMI, age, and gender. There is biological plausibility for a relationship between membrane omega-3 FA levels and sudden cardiac death. Currently, the most likely mechanism by which they appear to operate is via reducing the susceptibility ofthe myocardium to developing lethal arrhythmias. EPA and DHA can inhibit the fast, voltage-dependent sodium current and the L- type calcium currents. Their antithrombotic properties may also contribute to this stabilization. In addition, omega-3 FA contribute towards plaque stabilization, and may be antiatherosclerotic via a variety of other mechanisms.

The Omega-3 Index is independent of other known CHD risk factors as a predictor of risk for sudden cardiac death. In the studies by Albert et al. and Siscovick, et al., the Index remained a statistically significant risk predictor in multivariate models which included other known risk factors for CHD. In the latter, these included adjustment for age, smoking status, assignment to aspirin/beta-carotene/placebo; body mass index; history of diabetes, hypertension, or hypercholesterolemia; alcohol consumption; exercise frequency; parental history of MI before age 60 yrs; and trans and monounsaturated FA intake. With risk reductions of approximately 90% in the highest quartiles, the Omega-3 Index is both a strong and an independent predictor of risk for sudden cardiac death. With respect to the modifiability of the Omega-3 Index as a risk factor, such a characteristic is perhaps the most important because this property requires that a change in the risk factor alter the disease outcomes. To date, only two interventions have been shown, in randomized controlled prospective trials, to have the potential to reduce risk for sudden cardiac death: implanted cardiac defibrillators and supplementation with omega-3 FAs. Although more studies are clearly needed to refine and extend the findings with omega-3 FA, this perhaps most critical criterion for evaluating the utility of a risk factor in clinical medicine has been met by omega-3 FA.

Finally, one requirement remaining to be satisfactorily addressed for a blood-based risk factor is the establishment of standardized laboratory methods to measure the Omega-3 Index. The assay is relatively labor intensive, and has been approached with multiple extraction techniques, varying derivatization methods, and differing gas chromatographic conditions. This has resulted in widely divergent values being reported in the literature for RBC EPA+DHA, even among individuals presumably eating similar diets. For example, the Omega-3 Index for individuals consuming typical Western diets has ranged from less than 1.5% to over 7% or more. Thus, before the Omega-3 Index can be applied widely, uniform methods and widely available quality control materials will be needed.

The clinical utility of any proposed risk factor is greatly enhanced if it can be modified. Clearly, there are useful unmodifiable risk markers for CHD (age, gender, family history), but modifiable ones (cholesterol, blood pressure, body weight, smoking, etc.) offer the patient an opportunity to change the natural history of his/her disease, hi order to raise the Omega-3 Index, additional omega-3 FA must be consumed. Accordingly, safety, efficacy and cost issues associated with this treatment must be considered.

With respect to safety, in 1997, the US Food and Drag Administration (FDA) granted GRAS (Generally Recognized As Safe) status to refined menhaden fish oil. In so doing, the agency indicated that the consumption of up to 3 g per day of EPA+DHA from all sources (fish, fish oils and potentially fortified foods) would be considered safe for American adults. Thus up to this dose, there does not appear to be any significant safety concern. Thus, the most extreme recommendation ofthe AHA (for patients with known CHD) of about one gram of EPA+DHA per day, presents no significant safety concerns. In populations chronically consuming AHA- levels and higher, no safety concerns have been noted with the possible exception of anecdotal reports bleeding tendencies, but these have not been seen in controlled trials. The best example of the lack of effect on clinical bleeding was the study by Leaf et al. (Do Fish Oils Prevent Restenosis after Coronaiy Angioplasty? , 90 Circulation, 2248-2257 (1994) the content and teachings of which are hereby incorporated by reference herein) wherein 275 patients undergoing percutaneous transluminal coronary angioplasty (PTC A) were randomized to 7 g of EPA+DHA per day (or corn oil placebo) for 6 months. Even with the daily administration of 325 mg of aspirin in addition to the consumption of this high amount of omega-3 FA, no bleeding problems were reported, even among those patients who eventually went on to coronary artery bypass surgery. There is currently no evidence that omega-3 FA interact adversely with other drugs used to treat CHD (including, as noted above, anti-platelet agents). Thus, in keeping with the regulatory authorities, it is concluded that from a safety point of view, no argument can be made against consuming up to 3 g/day EPA+DHA (although, as noted earlier, such high intakes are usually not necessary to achieve target Omega-3 Index levels).

It was reported that high mercury levels in fish may diminish the cardioprotective effects of the omega-3 FA they contain. Although recent data have suggested that the concern with children and mercury toxicity from fish consumption may be overstated, current FDA Advisories continue to advise young children, nursing mothers and women who either are or are trying to become pregnant to avoid certain high-mercury fish (shark, swordfish, tile fish and King mackerel); the rest ofthe population is advised to limit their weekly intake of these species to 12 oz. The AHA has recommended that individuals eat a wide variety offish to keep the potential mercury intake low. Fish oil capsules, on the other hand, do not contain mercury and do not pose a threat. A recent independent analysis of 16 fish oil capsules sold in the US by the Consumer's Union found no significant contamination with either metals or chlorinated hydrocarbons, and all ofthe labels reflected the EPA and DHA content relatively accurately. Accordingly, the use of nearly all species of oily fish and of fish oil capsules to increase the Omega-3 Index may be done safely. With respect to efficacy, the Omega-3 Index is easily modified. The dose ranging study described herein as well as hundreds of previous trials in which blood (serum) levels of omega-3 FA have been followed as a measure of compliance to a fish oil intervention testify to the ease with which the Index may be modified. As noted herein, the Omega-3 Index in the SCIMO trial rose from 3.4% at baseline to 7.9 % after 18 months of 1.5 g/day. In other words, the baseline Omega-3 Index in Munich (CHD patients) was about 1 percentage point lower at baseline than the Index in Kansas City (healthy subjects) suggesting that there may be differences in background intakes of these FA. Furthermore, in Munich, 1.5 g/d of EPA+DHA for 18 months raised the Index to about 8% whereas in Kansas City, this level was reached with a dose of 500 mg/d in only four months. These observations, although not made contemporaneously within the same laboratory or patient population, suggest that 1) the Omega-3 Index may be a useful reflection of background omega-3 FA intakes, and 2) that recommended omega-3 FA doses may need to be tailored individually based upon baseline levels. In other words, the Index may find utility in both assessing initial risk and in monitoring treatment success. With respect to cost, obtaining 500-1 ,000 mg of EPA+DHA per day can be achieved with either oily fish consumption, fish oil capsules or cod liver oil. The costs associated with these strategies can vary widely, from $20-$40 per day if a salmon entree is ordered at a fine restaurant, to $0.07 per day if fish oil capsules are purchased at wholesale buying clubs. Using other capsules to obtain about one gram per day can cost up to about $0.60 per day. Needless to say, the costs required to correct a deficient Omega-3 Index are minimal.

The Omega-3 Index also compares favorably with other CHD risk factors. Using data from the Physicians' Health Study, Albert et al. have published the relative risk for sudden cardiac death across the four quartiles of several risk factors. The three risk factors that show statistically significant trends are C-reactive protein, the total/HDL cholesterol ratio and the Omega-3 Index. The factor that shows the steepest gradient in risk (reaching 90% reduction at the highest quartile) is blood omega-3 FA. As noted above, the Omega-3 Index is easily, quickly, safely and cheaply brought into a healthy range. This is not true for the other, traditional risk factors for CHD. Serum lipoproteins and blood pressure can be modified by diet, but very commonly, a pharmaceutical approach is needed, and omega-3 FA are clearly more benign than any drug, including aspirin. Perhaps the risk factor most like the Omega-3 Index is homocysteine since its levels are modifiable by nutritional supplementation with folate, pyridoxine and cobalamin. The connection between homocysteine and CHD has yet to be demonstrated in randomized, prospective trials with clinically-important endpoints, and the first reported study was negative. Therefore, the Omega-3 Index is at least as useful as the other known risk factors for death from CHD. Although the case is quite strong for simply recommending that all adults consume approximately 500-1,000 mg of EPA+DHA per day (depending on their CHD risk status), the present invention demonstrates that individuals should measure omega-3 FA levels for a variety of reasons. First, it is difficult to know how much EPA+DHA one is actually consuming. The omega-3 FA content of a serving of any given fish is unknown. Tables of omega-3 FA content by species present average levels, but the ranges can vary markedly depending on season, maturity, the fish's diet, post-catch processing and cooking methods. In addition to uncertainties in omega-3 intake, certain fish are contaminated with small amounts of a variety of industrial chemicals (e.g., methylmercury, pesticides, herbicides, etc.). Thus, the consumer may be unsure of both the omega-3 FA content and presence of undesirable pollutants in the fish on his/her plate. In addition, the omega-3 FA content of encapsulated oils is not Federally regulated. Therefore, the consumer cannot necessarily trust that the label claim of potency is true. While reputable manufacturers are careful to list these values accurately (as recently reported), the consumer cannot know for sure who is and who is not reputable. (This may change as national testing organizations begin to institute certification programs for nutritional supplements). Even if one could know for certain how much omega-3 FA they were consuming, this would still not guarantee a particular tissue level would be achieved. Each individual is metabolically unique, with idiosyncrasies in digestion, absorption, tissue distribution, and cellular metabolism conspiring together to produce different levels in different people all consuming the same amount of omega-3 FA. In the dose ranging study described herein, the Omega-3 Index varied from 3% to 7% in the placebo group, from 4% to 10% in the 500 mg/d group, and from 5% to 13% in the 1 g/d group. Individual variations in the in vivo conversion of cc-linolenic acid (the plant-derived omega-3 FA) into EPA and DHA, as well as other dietary variables (e.g., kcalories, omega-6 FA) can also influence tissue omega-3 FA levels. Consequently, as an indicator of risk for sudden cardiac death, an estimated intake cannot adequately substitute for knowledge of tissue levels. Algorithms including other risk factors in conjunction with the Omega-3 Index should be developed to improve the ability to predict (and thus prevent or greatly reduce) sudden cardiac death. As noted above, although one is proposed herein, no standardized method for measuring the Omega-3 Index is yet available, and developing such an assay is ofthe highest priority so that the results of future studies may be compared and contrasted. In addition, other blood-based markers of omega-3 FA intake should be evaluated, and the specific omega-3 FA included in the Index (whatever the tissue) may need to be re-considered. The effects offish vs. capsules on the Index will need to be clarified, and precisely how rapidly steady state levels can be achieved is not known with certainty. The impact upon the Omega-3 Index of different ratios of dietary EPA and DHA (not to mention α-linolenic acid) will need to be studied. How the Omega-3 Index correlates with risk in other disease states is not known, nor is it clear that patients with CHD, when fed the same amounts of EPA+DHA as healthy volunteers, actually achieve the same blood levels.

In summary, the present invention presents a case, not only for measuring a biomarker of omega-3 FA intake for CHD risk stratification, but specifically for utilizing the Omega-3 Index as that biomarker. In addition, the present invention suggests that the Omega-3 Index be used to segregate three levels of risk for death from CHD - high, intermediate and low - defined as Omega-3 Indices of <4%, 4%-8%, and 8-10%). These values are derived from prospective epidemiological investigations and randomized controlled trials, and are conservative. The FDA has approved the safety up to 3 g of EPA+DHA for inclusion in the US food supply, and such intakes may be necessary in certain individuals persons with documented CHD and low baseline Omega-3 Indices. The Omega-3 Index could thus be an important step in reducing risk for sudden cardiac death.

In one aspect of the present invention, an improved method for determining an individual's overall risk of death from coronary heart disease is provided. The method generally comprises the known step of assaying a biological sample for at least one risk factor associated with CHD and the improvement comprises assaying the biological sample for the level of omega- 3 polyunsaturated fatty acids therein. This basic method may further include the step of using the assayed level of omega-3 polyunsaturated fatty acids as one component ofthe individual's overall risk of death from CHD. Thus, the method provides yet another factor which could be used in combination with the other known risk factors to assess and predict an individual's risk of death by CHD. Alternatively, the assayed levels of omega-3 s could be used alone as a predictor of an individual's risk of death from CHD. Preferably, the level of omega-3 polyunsaturated fatty acids is assayed in red blood cells. It is also preferred for this method to measure omega-3 polyunsaturated fatty acids that are selected from the group consisting of EPA, DHA, and combinations thereof as these two omega-3 s are more indicative of actual levels of omega-3 s in the biological membranes of individuals. The method may further comprise the step of separately risk-stratifying the individual's risk of death from CHD based on the level of the omega-3 polyunsaturated fatty acids. When this additional step is used, the risk-stratifying may classify the individual as having a low, intermediate, or high, risk of death from CHD. Such designations are commonly understood by the general public and will result in a better understanding by the individual of what their risk of death from CHD is, relative to the general public. The high risk classification preferably indicates an omega-3 level of less than or equal to 4%, the intermediate risk classification preferably indicates an omega-3 level between 4% and 8%, and the low risk classification preferably indicates an omega-3 level of 8% or greater. In another aspect of the present invention, a method of stratifying an individual ' s risk of death from CHD is provided. The method generally comprises the steps of determining the individual' s omega-3 fatty acid level and comparing the determined omega-3 fatty acid level with an index related to the risk of death from CHD and omega-3 fatty acid levels. As with other methods of the present invention, the index preferably stratifies the risk of death from CHD based on the omega-3 fatty acid levels as high, intermediate or low. The method may further include the step of using the determined level of omega-3 polyunsaturated fatty acids as one component of the individual's overall risk of death from CHD. That is to say that the individual's omega-3 level maybe used in combination with other know risk factors for death from CHD. Alternatively, the individual's omega-3 levels maybe used as a separate, solitary risk factor assessment. Preferably, the level of omega-3 polyunsaturated fatty acids is determined in red blood cells and the omega-3 polyunsaturated fatty acids are selected from the group consisting of EPA, DHA and combinations thereof. Preferably, stratifying individuals into low, intermediate, or high risk categories for death from CHD is based on the determined levels of omega-3s from a biological sample taken from the individual. Cut-points at 4% and 8% are preferably used to distinguish between individuals at low, intermediate, or high risk of death from CHD with individuals with levels less than or equal to 4% indicating a high risk, between 4% and 8%> indicating an intermediate risk, and 8% or above indicating a low risk.

In another aspect ofthe present invention, an index for classifying an individual's risk of death from CHD is provided. The index distinguishes between high, intermediate, or low, levels ofthe risk based on cut-off points related to levels of omega-3 polyunsaturated fatty acids in biological samples. Each of these cut-off points provides an endpoint for one of the risk classifications. In practice, a biological sample is obtained from an individual and the sample is assayed for omega-3 levels therein. The measured omega-3 levels are then compared to the index which categorizes the individual's risk of death from CHD as low, intermediate, or high, based upon the measured level of omega-3 s . Preferably, the index has a cut-off points at 4% and 8% wherein individuals having an omega-3 level of less than or equal to 4% are classified as having a high risk of death from CHD, individuals having an omega-3 level of between 4% and 8% are classified as having an intermediate risk of death from CHD, and individuals having an omega-3 level of 8% or greater are classified as having a low risk of death from CHD. Preferably, the assayed or measured level of omega-3 polyunsaturated fatty acids is determined in red blood cells. It is also preferable to measure omega-3 polyunsaturated fatty acids selected from the group consisting of EPA, DHA, and combinations thereof. Such an index couldbe used as one component or as a separate, individual factor ofthe individual's overall risk of death from CHD. The index may be used as a reference standard for determining an individual's risk of death from CHD based on the level of omega-3 polyunsaturated fatty acids in a biological sample from the individual.

In another aspect ofthe present invention, a method of decreasing an individual's risk of death from CHD is provided. Preferably, the individual has a first level of omega-3 polyunsaturated fatty acids of less than 8% as determined by an assay of the individual's biological fluid prior to performing the steps of this method. The method generally comprises the step of causing the individual to ingest omega-3 polyunsaturated fatty acids in an amount effective for increasing the individual's level of omega-3 polyunsaturated fatty acids to a second level, wherein the second level is 8% or greater as determined by an assay of the individual's biological fluid that is performed after the ingestion ofthe omega-3s. In practice, an individual has an assay performed in order to assess their omega-3 level. If the level of omega-3s in the sample taken from the individual falls below 8%, the individual is instructed to ingest an amount of food or supplement containing omega-3s over a selected period of time with the amount of food or supplement being in addition to any foods or supplements containing omega-3 s that the individual currently ingests. After a period of time, a second biological sample is then obtained from the individual and this second sample is also assayed for the levels of omega-3 s therein. Thus, this method can be used to determine how the omega-3 levels were affected by the increased intake of food or supplement which contained omega-3s. It may also be used in order to lower an individual's risk of death from CHD and to track progress toward that goal. This method is important and significant because different foods contain varying amounts of omega-3s and supplements may have more or less omega-3s than is indicated on their label. Additionally, different individuals may metabolize omega 3's differently and such individuals may also be of different size than others and therefore, the amount of omega-3s necessary for any individual to raise their omega-3 levels to 8% or greater will vary. Such a method overcomes such variability problems. Preferably, the individual has a first level of omega-3 polyunsaturated fatty acids of less than or equal to 4% and is at high risk for death from CHD, and the method results in a second level of omega-3s that is above 8% and therefore reduces the individual's risk to where they are at a low risk of death from CHD. In some cases, the effective amount will comprise an average of up to 7 grams of omega-3 polyunsaturated fatty acid per day. More preferably, the amount will comprise between 0.1 and5 grams, still more preferably between 0.25 and2.5, even more preferably between 0.5 and 1.5 grams, and even more preferably, between about 0.5 and 1.0 grams of omega-3 polyunsaturated fatty acid per day. Preferably, the additional source of ingested omega-3 polyunsaturated fatty acids are derived from a source selected from the group consisting offish, fish oil supplements, and combinations thereof. Additionally, it is preferred that the omega-3 polyunsaturated fatty acids measured and ingested are selected from the group consisting of EPA, DHA, and combinations thereof. It is preferred that the levels of omega-3 polyunsaturated fatty acids are assayed in red blood cells.

In yet another aspect ofthe present invention, a method of measuring patient compliance with a diet designed to increase the level of omega-3 polyunsaturated fatty acids in a biological sample taken from the patient is provided. The method generally comprises the steps of taking a first biological sample from the patient, assaying the level of omega-3 polyunsaturated fatty acids in the first sample to provide a first omega-3 level, taking a second biological sample from the patient, assaying the level of omega-3 polyunsaturated fatty acids in the second sample to provide a second omega-3 level, and comparing the first omega-3 level with the second omega-3 level. Preferably, the first biological and the second biological sample being taken from the patient at least 7, more preferably between 7 days and 10 years apart, still more preferably between 30 days and 1 year apart, still more preferably between 120 days and 180 days apart. The method may also comprise the step of instructing the patient to increase their consumption of omega-3 polyunsaturated fatty acids a specific amount based on the first omega-3 level. Preferably, the specific amount is an amount effective for raising the second omega-3 level to at least 8%. As with the other methods ofthe invention, the omega-3 polyunsaturated fatty acids measured and ingested are preferably selected from the group consisting of EPA, DHA, and combinations thereof and the first and said second omega-3 levels are assayed in red blood cells.

In yet another aspect ofthe present invention, a kit for determining the presence of fatty acids in a biological sample is provided. Generally, the kit comprises a container for holding the biological sample, a set of instructions for using the kit, and at least one standardized fatty acid sample. Preferably, the kit is designed to determine the presence and/or the quantity or level of omega-3 fatty acids in the biological sample. Still more preferably, the fatty acids are selected from the group consisting of EPA, DHA, and combinations thereof. The kit may further include a second standardized fatty acid sample. In some forms, one fatty acid sample will be a sample having 8% omega-3 fatty acids therein and the other fatty acid sample will be a sample having 4% omega-3 fatty acids therein. The instructions included with the kit preferably include instructions on how to determine omega-3 presence or levels in the biological sample. In some preferred forms, the kit will also include all necessary reagents for determining the presence of omega-3 fatty acids and/or all necessary equipment for determining the presence of omega-3 fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph illustrating the relationship between the Omega-3 Index as measured in whole blood and in RBCs;

Fig.2a is a graph illustrating the stability ofthe Omega-3 Index in samples stored at room temperature for up to five days; Fig. 2b is a graph illustrating the stability ofthe Omega-3 Index in samples refrigerated for up to five days;

Fig. 3 is a graph comparing the Omega-3 Index for ten individuals from Kansas City and ten Alaska natives living in Nome, Alaska; Fig. 4 is a graph illustrating the sensitivity and responsivity ofthe Omega-3 Index;

Fig. 5 is a graph illustrating the baseline Omega-3 Indices from numerous studies for various countries;

Fig. 6 is a graph illustrating the effects of increasing intakes of EPA and DHA on RBC EPA + DHA levels pre- and post- 20 weeks of supplementation of healthy subjects; Fig. 7 is a graph illustrating the effects of EPA and DHA supplementation in heart transplant patients; and

Fig. 8 is a graph illustrating the relationship between the Omega-3 Index as measured in plasma phospholipids and RBCs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following examples set forth preferred embodiments ofthe present invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope ofthe invention.

EXAMPLE 1

This example determined the relationship between whole blood omega-3 levels found in the Albert et al. study and the Omega-3 Index. Materials and Methods:

To establish the relationship between the Omega-3 Index derived from whole blood and the Omega-3 Index derived from RBCs, the whole blood omega-3 content was compared with RBC omega-3 content in a random set of 40 blood samples. 40 samples of blood were obtained and then each sample was divided into two separate portions for analysis using the procedures described below in Example 2. One portion was used to measure EPA and DHA from whole blood and the second was used to measure EPA and DHA in RBCs. The next step was to subtract the whole blood DPA levels [which were not different between cases and controls (0.98% and 1.01%, p=0.25)] from the reported whole blood EPA+DHA+DPA values from Albert et al. to arrive at the average whole blood EPA+DHA. Average DPA levels were determined by reviewing the data from the cases and controls ofthe Albert et al. study. After subtracting 1% for DPA which represents the average amount of DPA that was included in the Albert et al. data, the equation derived from the data shown in Figure 1 was applied to estimate the Omega-3 Index associated with each level of risk in Albert et al.

Results and Discussion:

The relationship between the Omega-3 Index derived from whole blood and the Omega-3 Index derived from RBCs is demonstrated in Fig. 1. This Fig. shows that the two values are closely related. Such a relationship then permitted the extrapolation of the data from the Albert et al. study which utilized data from the Physicians Health Study, h this study, 14,916 healthy male physicians were screened for a wide variety of risk factors and provided baseline blood samples between 1982 and 1984. Over the next 17 years, 94 men experienced sudden cardiac death. The study then compared whole blood long-chain omega-3 FA (i.e., EPA+DHA+DPA) in these cases to that of 184 age and smoking-status-matched controls. Those subjects with the highest blood omega-3 FA levels had approximately 10% of the risk of those in the lowest category. Utilizing the approach described in this example to estimate the Omega-3 Index in Albert et al. resulted in an average Index of 7.3% for the highest quartile with a range of 6.5% to 10.8%. Levels in the lowest quintile were estimated to be 3.9% with a range of 2.3% to 4.6%. The mean Omega-3 Index in the highest quartile was 7.3% of total RBC FA with a range of 6.5% to 10.8%). Levels in the lowest quintile averaged 3.9% with a range of 2.3% to 4.6%. The Albert et al., study thus provided an initial window of possible target Omega-3 Indexes.

EXAMPLE 2 This example describes the procedures used to determine the Omega-3 Index from a biological sample comprising blood.

Materials and Methods:

A blood sample is drawn into a 5 mL EDTA purple top tube. This tube is centrifuged for 15 minutes at 3000 rpm, at 4°C, in the TJ-6 centrifuge to separate the cells from the plasma.

Next, the plasma and buffy coat were removed using a plastic transfer pipette and discarded, leaving only packed RBCs in the bottom ofthe tube. The RBCs were washed three times with cold saline and then frozen at -80"C. To extract total lipids from the RBC membranes, the RBC pellet is thawed at room temperature. Using the pipettor bottle of isopropanol (IP A), add 2.8 mL of containing 50 mg/Lbutylatedhydroxytoluene(BHT; Sigma)to a 16 X 125mm screw-cap tube. Next, pipette 0.125 mL ofthe washed RBCs into the tube with the IPA. The RBC stroma will begin to clump upon the addition of the IPA. Shake well by hand for 30 seconds. Next, using the pipettor bottle of hexane, slowly add 1.8 mL of hexane to the tube and shake well by hand for 30 seconds. Centrifuge the tube for 15 minutes, at 3000 rpm, at 4°C in the TJ-6 clinical centrifuge. After centrifugation, most ofthe RBC stroma will be tightly pelleted at the bottom ofthe tube. A small percentage ofthe stroma will remain 'floating' in the solvent layer. The lipids will also be in the solvent layer. Transfer the solvent layer into a 16 x 125, screw-capped, glass tube using a plastic transfer pipette taking care to leave the stroma behind. Dry down the lipid extract under N₂, using the N-evap or Reacti-vap (N₂ must be used for all dry downs in this procedure to prevent oxidation of fatty acids). The tubes are heated in the water bath (or heating block) to 45 "C to accelerate solvent evaporation. After the extract is completely dried down, proceed to the methylation step. If the sample is to be saved for later methylation, it should be filled with nitrogen and covered with parafilm and stored in the refrigerator.

For the methylation and extraction steps, turn on the heating block to 100°C in preparation for the methylation procedure. To the dried fatty acid methyl esters (FAME), add 1 mL (14%>) boron trifluoride (BF₃) in methanol, cap and vortex for 5-10 seconds. Incubate for lOminutes at 100° C in a heating block. Let cool and add 2 mL saturated sodium chloride solution and 2 mL of hexane before shaking vigorously for 20-30 seconds. Next, spin for 3 minutes at 3,000 rpm at room temperature. Remove the top (hexane) layer using a plastic transfer pipette and place the extract into a 12 X 75 mm glass tube, dry under N₂ and bring up to 50 uL with hexane before vortexing briefly. Transfer to a glass GC vial with a silicone septum covered cap.

For the next step, gas chromatography is used with the following specifications: Column: Supelcowax 10 fused silica capillary column (Supelco, Bellefonte, PA) 30 m, .32mm ID, .25um film thickness; cat.# 2-4080 GC Gas Chromatograph: Shimadzu GC14A

Split 1:25 Purge 15-20 ml/min

Carrier Gas: Hydrogen

Make Up Gas: Nitrogen

FID (hydrogen and oxygen generated flame) GC Pressure Gauge readings (in kg/cm²)

Oxygen 0.3

Hydrogen 0.5

Nitrogen 0.3

Hydrogen (carrier) 1.0 Temperature Program:

Initial temperature 120°C for 1 min

Ramp up to 160 C at 4°C/min (10 min ramp)

Hold at l60°C for 2 min

Ramp up to 200°C at 4 C/min (10 min ramp) Hold at 200°C for 23 min

(Display on GC computer only for 35 min)

Burn off and cool down program after each sample:

Ramp up from 200°C to 240°C at 20°C/min (2 min ramp)

Hold at 240°C for 4 min Ramp down to 120° C at 40° C/min (3 min ramp)

Hold at 120°C for 5 min

Ready for the next sample

To interpret the gas chromatograph data, both high and low controls (frozen RBC samples) should be run each day. Both should generate Omega-3 Indexes within 2 standard deviations of normal for the runs to be accepted. If either is not, the problem should be determined and the samples re-run once control has been re-established. To begin, the integrator is set to begin integrating after the BHT peak elutes. The run finishes after DHA (C22:6ω3) elutes. The computer then integrates the area under each peak. The operator should inspect each chromatogram to determine that the integrator set the 'start' and 'stop' integration markers properly for the major peaks of interest. The area percent for EPA (C20:5 ω3) and DHA is summed and reported out as the Omega-3 Index. EXAMPLE 3 This example established controls for the Omega-3 Index.

Materials and Methods:

In order to create these controls for the Omega-3 Index, 200 mL (about 72 unit) of blood was drawn from each of eight individuals. Four of these individuals had been taking omega-3 FA supplements regularly and their samples constituted the high controls. The other four ate virtually no fish and were not taking omega-3 supplements; their samples constituted the low control. The blood was drawn into 10-cc EDTA tubes, spun as usual, and the plasma and buffy coat removed. The RBCs from each set of four were pooled, and aliquotted into about 700, 0.3 mL aliquots, capped, and frozen at -70° C. These controls were first assayed for 24 runs in a row to determine mean and SD values. Once determined, these values were assigned, and thereafter, whenever a batch of unknowns was run, the controls with known values were also run. If the control values fall within 2 SD ofthe established mean values, then the run will be accepted as valid. If either ofthe controls falls outside ofthe 2 SD range, the run is rejected.

Results and Discussion:

The values assigned to the high and low controls currently in use in the laboratory are: 9.0 ± 0.36% and 3.2 ± 0.22%. The coefficients of variation for these two controls are 4% and 1%, respectively. It is essential to have controls (RBC samples of known EPA+DHA content) that span the expected normal range that can be run with each batch of unknowns in order to confirm that the analytical method is performing properly start to finish. This approach tests not only the performance ofthe GC equipment, but also the extractions (are the solvent still good?), the derivatizing reagents (is the methylation procedure still working?) and the GC analysis itself.

EXAMPLE 5 This example tested whether the Omega-3 Index was influenced by specific anticoagulants. Materials and Methods:

Two individuals volunteered to give blood samples for this study: One individual had high blood omega-3 levels and one had low/normal levels. Four separate blood tubes, each containing a different anticoagulant, were drawn from each individual: EDTA, sodium citrate, heparin, and sodium fluoride. The Omega-3 Index was measured in triplicate for each of these samples. Results and Discussion:

The anticoagulant did not affect the Omega-3 Index. Mean values for the anticoagulants were: 8.7%. 8.5%, 8.8% and 8.9%>, respectively. Nevertheless, because of the potential antioxidant properties of EDTA (binds Calcium and free Iron), it is recommended that the Omega-3 Index be run on EDTA samples.

EXAMPLE 6 This example tested the effect of 0, 1 and 2 freeze-thaw cycles on the Omega-3 Index.

Materials and Methods:

The same blood samples as drawn for the anticoagulant test were used to determine the effects of freeze-thaw cycles. After removing the plasma and white blood cells, each sample was assayed "fresh" and then frozen at -20°C for at least 24 hours. These samples were then thawed, a sample ofthe RBCs removed for the Omega-3 Index analysis, and the remainder was re-frozen.

At least 24 hours later, the sample was again thawed and the Omega-3 Index measured.

Results and Discussion:

It was found that the Omega-3 Index was reduced (compared to fresh) by one cycle (6.7+0.12%) vs.5.8+0.14%), but there was no further decrease after the second cycle. Results for this are provided below in Table 1. Accordingly, the Omega-3 Index assay routinely includes one freezing as the first step in the assay. This is important because virtually all ofthe studies that have been done looking at blood or RBC omega-3 FA and risk for death from CHD have used samples that have undergone at least one freeze-thaw cycle. EXAMPLE 7 This example tested wether the RBCs needed to be washed prior to analysis. Materials and Methods:

Five random blood sample were each analyzed without washing (simply as packed RBCs) and after three washes with cold saline. Results and Discussion:

Washing had no impact on the Omega-3 Index. These results are also included in Table

1. Historically, RBC membranes have been isolated by first removing the plasma and white cells followed by up to three washes with cold saline to remove all remnants of plasma. Such a step is very time consuming and therefore, determining whether it effects obtaining an accurate

Omega-3 Index is important for future use.

EXAMPLE 8 This example tested whether the Omega-3 Index was affected by feeding.

Materials and Methods:

For this study, ten healthy adults had fasting blood drawn in the morning, and were then given a meal pass to the hospital cafeteria and asked to eat whatever they wanted for breakfast. Four hours after eating (the peak of postprandial lipemia), they gave a second blood sample. The Omega-3 Index was measured in each.

Results and Discussion:

There was no difference between fasting and the fed Omega-3 Indexes. Again, these results are provided below in Table 1. Accordingly, the Omega-3 Index can be determined in blood drawn at any time ofthe day. Because the Omega-3 Index is the amount of EPA+DHA expressed as a percent of total fatty acids, it could theoretically be influenced if the RBC fatty acid composition changes with eating. Plasma fatty acid composition definitely changes with food consumption owing to the influx of triglyceride fatty acids in chylomicrons. This experiment was of tremendous practical significance because, if there is no difference between the fasting and fed RBC FA composition, then the Omega-3 Index could be measured in casual blood samples taken at any time ofthe day. This would be much more convenient for the patient and physician because having to fast overnight is often challenging.

EXAMPLE 9

This example tested to determine whether there were differences in the Omega-3 Index in men versus women or across the age spectrum in the population. Materials and Methods:

The Omega-3 Index was analyzed as described above in 125 men and women not taking fish oil supplements who were living in either the Kansas City area or Charlotte, North Carolina. Results and Discussion:

There was no difference between men and women in the mean Omega-3 Index, nor was there any significant difference by decade of life. These results are provided below in Table 1.

Table 1. Omega-3 Index: Effects of Age, Gender and Laboratory Manipulation

EXAMPLE 10 This example tested the repeatability of one sample analyzed multiple times in one run. Materials and Methods:

To test how reproducible the gas chromatographic analysis ofthe Omega-3 Index is, the high omega-3 sample used in Example 5 was analyzed nine consecutive times.

Results and Discussion:

The mean value was 12.0%> and the standard deviation 0.39%. This calculated into a coefficient of variation of 3%.

EXAMPLE 11

This example compared the short vs long GC analytical program. Materials and Methods:

In this experiment, the Shimadzu GC- 14A was used and compared an isothermal program (210°C for 12 minutes) vs. a ramping temperature program (120 °C held for 1 minute; ramp at 4°C/min to 160°C and hold for 2 min; ramp again at °C/min to 200° C and hold for 23 min). The same two samples as used in Example 5 were analyzed in triplicate under each condition. Each GC was equipped with a Supelcowax 10, 30 m column.

Results and Discussion: It was found that the short program, although desirable for higher throughputs, produced higher Omega-3 Indexes than the longer program. This appeared to be due to the inability ofthe shorter program to separate DHA (C22:6ω3) from a small peak appearing just after it. In other words, in the short program, the DHA peak contained a contaminant which added to its peak area and falsely elevated the Omega-3 Index. Accordingly, it was concluded that the longer program was more accurate.

There are an almost infinite number of ways in which the GC can be temperature programmed. A temperature program is the set of temperatures through which the GC oven (and thus the column) progresses as the sample is being analyzed. In an "isothermal" program, the oven temperature does not change throughout the run whereas in a temperature-ramping program, the temperature typically goes from a relatively low to a relatively high temperature. This is done to allow both the slow- and the fast-eluting peaks to be detected in as short a time as possible while still maintaining adequate peak resolution.

EXAMPLE 12

This example compared results from a high resolution GC column with results from a typical GC column in order to determine whether or not there are contaminant compounds coeluting with EPA and/or DHA under the standard analysis conditions. Materials and Methods: To test whether or not there are contaminant compounds coeluting with EPA and/or DHA under the standard analysis conditions, the typical Supelcowax 10, 30-m column was compared to a Supelco SP2560, 100-m column which is over 3 times longer (much greater resolving power) and which required over twice as much time to perform the analysis. With this column, any "hidden" peaks contaminating the omega-3 FA peaks would very likely be flushed out. For the longer column, the temperature program was : 190 ° C for 50 minutes; ramp at 2.5 ° per minute to 240°C and hold for 47 minutes. For the shorter (typical) column, the 35-minute temperature program described above was used. The question was whether the EPA+DHA content would be equally well-determined with each column. The Omega-3 Index in 10 samples from Eskimos in Nome, Alaska was measured in both instruments.

Results and Discussion:

In this experiment the answer to the question, are there 'contaminant' compounds coeluting with EPA and or DHA under our typical analysis conditions was sought. Such a contaminant would, as occurred above with the short temperature program, falsely report the Omega-3 Index. The Omega-3 Index was determined to be virtually identical in both instruments (5.4+4.0 and 5.4+4.4). Because the 100-m column and longer run time can potentially resolve many more peaks, it was comforting to find that there were apparently no contaminants in the EPA or DHA peaks from the 30-m column that were resolved in the 100-m column. This suggests that the EPA and DHA peaks are "pure" and that the Omega-3 Index is actually what it claims to be, the sum of EPA+DHA in RBC membranes. EXAMPLE 13 This example tested whether methylene chloride and hexane (other solvents than are frequently used in the initial RBC extraction step) function as well as chloroform. Materials and Methods:

Two samples for each solvent condition were analyzed in triplicate.

Results and Discussion:

There was no difference in the Omega-3 Index with any solvent used. Accordingly, it is recommended to use hexane because it is more volatile (evaporates faster), it is used later in the procedure, and it is less hepatotoxic than either ofthe chlorinated compounds.

EXAMPLE 14 This example tested the effects of time and temperature of storage on the Omega-3 Index of blood samples.

Materials and Methods:

Four EDTA tubes were drawn from four individuals supplementing their diets with omega-3 FA. Each sample was processed with one freeze-thaw step as part of the routine procedure. The control sample was aliquotted and frozen on day 0; the other three tubes were left out at room temperature for 1, 2 and 5 days. On each of these days, the sample was processed (centrifuged, RBC isolated, an aliquot taken and frozen). The four frozen aliquots were then thawed and analyzed on the same day. This would simulate the real-life scenario where blood might be drawn in an office and sent by express mail in an unrefrigerated container to the lab for analysis. Results and Discussion:

Although there was a trend towards a decrease in the Omega-3 Index over time (see Fig. 2a for room temperature and 2b for refrigerated sample results), there was no significant difference among the samples by ANOVA. Nevertheless, no more than 1 day of room temperature storage is optimal. The same study was done with the same samples stored at 4°C, and in this setting, the samples showed a higher degree of stability. Thus, samples should be refrigerated if they will be in transit for more than 1 day. EXAMPLE 15 This example compared the Omega-3 Index from an sample of individuals from the Eskimo population with a sample of individuals from the middle portion ofthe continental U.S. Materials and Methods:

A preliminary study often random RBC samples from Eskimos in Nome, Alaska was taken and compared to that of ten individuals in Kansas City with respect to their respective Omega-3 Indexes. In addition to the 10 Kansas Citians, the Omega-3 Index was measured in four Kansas City area doctors who had all been taking omega-3 supplements. Results and Discussion:

Theoretically, the Eskimo group should have a higher Omega-3 Index because their traditional diets contain seal, whale, and fish whereas the diets of Americans in the "lower 48 States" do not, especially in the Midwest. It was found that, although the mean Omega-3 Index was indeed higher with the Eskimos (5.7+2.3% vs. 3.6+0.7%, p=0.009), there were several individuals from the Eskimo group whose Omega-3 Index was not materially different from those in Kansas City. These results are provided in Figure 3. This confirmed the hypothesis that some Alaska Natives had moved largely away from the traditional diets to typical western diets, and that this might explain their increasing rates of coronary heart disease. The results ofthe testing performed on the doctors who were taking omega-3 supplements are also presented in Fig. 3. As shown by these results, it is clear that "Eskimo" levels ofthe Omega-3 Index could be achieved by consuming concentrated fish oil supplements.

EXAMPLE 16

This example compared omega-3 levels that were measured using the protocol presented herein with a reference protocol. Materials and Methods:

Blood samples were drawn from seven individuals and analyzed by both methods. The first method consisted of a consensus fatty acid analysis protocol and the second is the method described in Example 2. The consensus or reference protocol is used for quantitative determination of EPA and DHA content in omega-3 products and is applicable to triglyceride and ethyl ester product forms with results expressed as mg DHA/g and mg EPA/g after correction to free fatty acid equivalents. All operations were carried out as rapidly as possible, avoiding exposure to actinic light, oxidizing agents, oxidation catalysts (for example, copper and iron) and air.

Gas chromatography. The assay is carried out on the methyl or ethyl esters of all-cis- eicosa-5,8,ll,14,17-pentaenoic acid (EPA; 20:5 -3) and all-cw-docosa-4,7,10,13,16,19- hexaenoic acid (DHA; 22:6 -3) in the sample to be examined.

Internal standard. Methyl tricosanoate R. Test solution (a). Prepare 3 solutions for each sample.

1. Dissolve the sample to be examined according to the table below and about 70.0 mg ofthe internal standard in a 0.05 g/1 solution of butylhydroxytolueneR in trimethylpentane R and dilute to 10.0 ml with the same solution.

Approx. sum EPA + DHA Amount sample to be weighed 30 - 50 % 0.4 - 0.5 g

50 - 70 % 0.3 g

70 - 80 % 0.25 g

Ethyl esters are now ready for analysis. For triglycerides continue as described in 2.

2. Introduce 2.0 ml of the solution obtained from step 1 into a quartz tube and evaporate the solvent at 40-50 °C with a gentle current of nitrogen R. Add 1.5 ml of a 20 g/1 solution of sodium hydroxide R in methanol R, cover with nitrogen R , cap tightly with a polytefrafluoroethylene-lined cap, mix and heat on a steaming water-bath for 7 min. Allow to cool to 40-50°C.

3. Add 2 ml of boron trichloride-methanol solution R, cover with nitrogen R, cap tightly, mix and heat on a water-bath for 30 min. Cool to 40-50°C, add 1 ml of trimethylpentane R, cap and shake vigorously for at least 30 s. Immediately add 5 ml of a saturated solution of sodium chloride R, cover with nitrogen R, cap and shake vigorously for at least 15 s. Transfer the upper layer to a separate tube. Shake the methanol layer once more with 1 ml of trimethylpentane R . Wash the combined trimethylpentane extracts with 2 quantities, each of 1 ml, of water R and dry over anhydrous sodium sulphate R.

Test solution (b). (to be prepared at the same time as test solution (a))

Dissolve 0.300 g of the sample to be examined in a 0.05 g/1 solution of butylhydroxytoluene R in trimethylpentane R and dilute to 10.0 ml with the same solution. Proceed as described for test solution (a).

Reference solution (a). Prepare 3 individual solutions (to be prepared at the same time as test solution (a))

Dissolve 60.0 mg of docosahexaenoic acid ethyl ester CRS, about 70.0 mg ofthe internal standard and 90.0 mg of eicosapentaenoic acid ethyl ester CRS in a 0.05 g/1 solution of butylhydroxytoluene R in trimethylpentane R and dilute to 10.0 ml with the same solution. For analysis of ethyl esters the solutions are now ready for analysis. For analysis of triglycerides continue as described in step 2 for preparation of test solution (a).

Reference solution (b) (for system suitability of recovery vs. the theoretical response of the Flame Ionisation Detector (FID). Introduce 0.3 g of methyl palmitate R, 0.3 g of methyl stearate R, 0.3 g of methyl arachidate R and 0.3 g of methyl behenate R into a 10 ml volumetric flask, dissolve in a 0.05 g/1 solution of butylhydroxytoluene R in trimethylpentane R and dilute to 10.0 ml with the same solution.

Reference solution ©) (for system suitability of chromatographic resolution). Introduce a sample containing about 55.0 mg docosahexaenoic acid methyl ester CRS and about 5.0 mg of 15-tetracosenoic acid methyl ester CRS diluted to 10.0 ml of a 0.05 g/1 solution of butylhydroxytoluene R in trimethylpentane R.

Column: CP-Wax 52CB, 25 m x 0.25 mm LD. 0.2 μm film thickness, Chrompack cat. no. 7713 or equivalent will be suitable. Material: fused silica

Dimensions: / = at least 25 m, 0 = 0.25 mm

Stationary phase: bonded polyethylene glycol polymer (film thickness 0.2 μm)

Carrier gas: hydrogen for chromatography R or helium for chromatography. Split: 1 :200, alternatively splitless with temperature control (samples need to be diluted 1:200 with a 0.05 g/1 solution of butylhydroxytoluene R in trimethylpentane R before injection)

Temperature:

Time (min) Tern perature

CG)

Column 0 - 2 170

2 -25.7 170 - 240

25.7 - 28 240

Injection port 250

Detector 270

Detection: flame ionisation.

Injection: twice 1 μl of each solution.

The assay is not valid unless: 1) the chromatogram obtained with reference solution (b) gives area per cent compositions increasing in the following order: methyl palmitate, methyl stearate, methyl arachidate, methyl behenate; the difference between the percentage area of methyl palmitate and that of methyl behenate is less than 2 area per cent units; 2) the chromatogram obtained with reference solution ©) shows 2 resolved peaks corresponding to docosahexaenoic acid methyl ester CRS and 15-tetracosenoic acid methyl ester CRS, giving a chromatographic resolution of minimum 1.2; 3) the chromatogram obtained with test solution (a) shows a resolution of methyl tricosanoate R and any heneicosenoic acid methyl ester present when compared with the chromatogram obtained with test solution (b) (if not, a correction term has to be used); and experiments using the method of standard additions to test solution (a) show more than 95 per cent recovery of the added eicosapentaenoic acid ethyl ester CRS and docosahexaenoic acid ethyl ester CRS, when due consideration has been given to the correction by the internal standard.

Calculate the content of EPA and DHA as mg fatty acid/g oil using the following expression and taking into account the certified value ofthe reference substances. The results should be rounded to the nearest 10 mg/g based on the method's precision.

m₁ = mass ofthe internal standard in test solution (a), in milligrams, m₂ = mass ofthe sample in test solution (a), in milligrams, m₃ = mass ofthe internal standard in reference solution (a), in milligrams, m^. = mass of eicosapentaenoic acid ethyl ester CRS or docosahexaenoic acid ethyl ester CRS in reference solution (a), in milligrams, A_x = area of the peak corresponding to eicosapentaenoic acid ester or docosahexaenoic acid ester in the chromatogram obtained with test solution (a), A_xr = area of the peak corresponding to eicosapentaenoic acid ester or docosahexaenoic acid ester in the chromatogram obtained with reference solution (a), A₁ = area of the peak corresponding to the internal standard in the chromatogram obtained with test solution (a), A₃ = area ofthe peak corresponding to the internal standard in the chromatogram obtained with reference solution (a), C = a conversion factor to fatty acids based on the difference in molecular weight of ethyl esters in the standard and fatty acid CEP A = 0.915, CDHA = 0.921. Total omega-3-acids.

From the assay for EPA and DHA, calculate the content ofthe total omega-3 -acids using the following expression and identifying the peaks from the chromatograms:

A_n . (EPA + DHA) EPA + DHA+ A_EPA + A_DHA

EPA = content of EPA obtained from the assay for EPA and DHA,

DHA = content of DHA obtained from the assay for EPA and DHA,

A„_.3 = sum of the areas of the peaks corresponding to C18:3 -3, C18:4 -3, C20:4 -3,

C21 :5 -3 and C22:5 -3 methyl esters in the chromatogram obtained with test solution (b), A_EPA = area ofthe peak corresponding to EPA methyl ester in the chromatogram obtained with test solution (b),

A_DHA = area of the peak corresponding to DHA methyl ester in the chromatogram obtained with test solution (b).

Results and Discussion :

A Consensus FA analysis protocol was adopted by the Omega-3 Manufacturers and the Council for Responsible Nutrition (CRN). The purpose ofthe CRN consensus method was to create a standardized approach for analysis of omega-3 rich oils to which all manufacturers could agree and which would thus allow for comparisons of label claims for EPA and DHA among products. The consensus method is much more involved and lengthy than the protocol ofthe present invention. The Omega-3 Index was not different between methods. The method ofthe present invention resulted in fatty acid measurements of 4.4 ±3.5 while the reference method resulted in fatty acid measurements of 4.6 ±3.7 (p=0.67). Thus, the approach of the present invention is comparable to what is currently the "gold standard" method.

EXAMPLE 17 This example prepared and tested quality control mixes of pure fatty acids in order to determine the accuracy of the gas chromatographs used in the present invention. Materials and Methods:

Standardized mixes of pure fatty acids of known composition available commercially were run in order to determine whether the GCs used in the present experiments were able to separate and accurately determine the mass of FAME in a known sample. To test this, 4 mixes were obtained from Supelco: NLH-D and F, and GLC-10 and 50. Each of these mixes contains a different assortment of FA. The mixes were analyzed by the GCs under standard operating protocol disclosed above.

Results and Discussion: It was determined that the GC worked well, producing values very close to expected.

Results of this experiment are given below in Table 2.

Table 2: Performance of Gas Chromatograph (with SupelcowaxlO column) with samples of known composition and concentration Mix FA* observed expected Mix FA* observed expected

GLC10 16-0 19.4 20.0 NIH-D 14-0 9.8 11.8

18-0 21.2 20.0 16-0 23.0 23.6

18-1 19.2 20.0 16-1 6.5 6.9

18-2 20.2 20.0 18-0 16.1 13.1

18-3 20.0 20.0 18-1 44.7 44.6

GLC50 16-1 22.8 25.0 NIH-F 14-0 2.2 2.5

18-1 24.9 25.0 16-0 4.1 4.2

20-1 26.4 25.0 18-0 7.4 7.3

22-1 26.0 25.0 20-0 13.8 13.6 22-0 25.6 25.4 *Fatty Acid nomenclature: for example, 16-0 means the fatty acid is 16 carbons long and contains 0 double bonds (i.e., palmitic acid).

EXAMPLE 18

This example tested the responsiveness ofthe assay ofthe present invention to increasing amounts of omega-3's in RBC membranes. Materials and Methods:

Increasing amounts of a high-Omega-3 Index sample were blended with a low-Omega-3 Index sample and the resulting mixture was assayed the mixture. The high Index sample contained 9.6% omega-3 FA and the low Index sample contained 2.8% omega-3FA according to the Index. Five mixtures were prepared that had ratios of high Index sample: low Index sample of 0: 100, 25:75, 50:50, 25:75, and 100:0. These mixtures were then tested according to the procedure of Example 2 to determine the responsiveness ofthe assay ofthe present invention.

Results and Discussion:

As shown by Fig.4, the assay ofthe present invention accurately detected the increasing amounts of omega-3 fatty acids.

EXAMPLE 19 This example demonstrated the need for standardization between omega-3 measurements. Materials and Methods:

This "experiment" consisted of a search of the medical literature to find studies that reported the Omega-3 Index for populations of subjects in a variety of countries consuming various amounts of omega-3 FA. Results and Discussion:

Table 3 provides the results of this study.

Table 3. Omega-3 Index at Baseline (before any intervention) in 60 Data Sets

Author (ref) Subject Type n Background Diet θ3I

Berlin Normals 40 US diet w/placebo 1.0

Santos Normals 20 Spanish diet 1.4

Nelson Normals 4 US diet 1.4

Santos CHD 20 Spanish diet 2.4

Peterson T2DM 36 Vegetarians S. Asia 2.5

Palozza Normals 40 Italian diet 3.1 Lund Normals 17 UK diet 3.2

Belluzzi Crohn's 33 Italian diet 3.3 von Schacky CHD 223 German diet 3.4 van den Ham Normals 129 Dutch diet 3.5

PRIME Study Group Normals 2S3 Pan European diet 3.6

Peet Normals 15 UK diet 3.6

Belluzzi Crohn's 50 Italian diet 3.7

Peterson T2DM 24 UK diet 3.7

Tynan Normals 12 Irish diet 3.9

Warren Normals 25 UK diet 3.9

Vidgren Normals 39 Finnish diet 4.0

Dodge Normals 10 US diet 4.1

Francois Normals 8 US diet 4.1

Mills Normals 18 Canadian diet 4.2

Assies Normals 19 Dutch diet 4.3

Siscovic SCD 334 Seattle diet 4.3

Leaf CHD 84 US diet 4.6

Marangoni Normals 8 Italian diet 4.8

Giltay Normals 18 UK diet 4.8

Manku normals 40 Nova Scotian diet 4.9

Siscovick controls 493 Seattle diet 4.9

Cartwright normals 5 UK diet 5.0

Sanders normals 26 UK diet 5.0

Stanford normals 15 US diet 5.0

Katan normals 14 Dutch diet 5.2

Tynan hyperlipidemia 31 Irish diet 5.3

Ruiz normals 9 Spanish diet 5.3

Brown normals 11 Australian diet 5.4 von Schacky normals 6 German diet 5.5

Hamazaki normals 42 Japanese diet 5.5

Alexander normals 24 normal 5.5

Paganelli CHD 26 French diet 5.6

Albert * SCD cases 94 US diet 5.6

Albert * SCD controls 184 US diet 5.6

Di Marino normals 16 Italian diet 5.7

Brown and Morrice normals 30 Scottish diet 5.7

Rivellese HTG-T2DM 16 Italian diet 5.8

Haines T1DM 41 UK diet 6.0

Conquer normals 19 Canadian diet 6.1

Luo T2DM 6 US diet w/placebo 6.3

Brown normals 12 Australian diet 6.9

Paganelli normals 24 French diet 7.3

Kark CHD 14 Israeli diet 7.3

Heude normal elderly 246 French diet 7.4

Bjerve normals 9 Norwegian diet 8.2

Terano normals 8 Japanese diet 8.9

Agren normals 29 Finnish diet 9.1

Katan normals 15 Dutch diet 9.3

Hessel normals 8 Finnish diet 9.3

Sarkkinen normals 157 low fat 9.5

Kamada T2DM 12 Japanese diet 9.8

Flaten normals 64 Norwegian diet 9.8

Kamada normals 11 Japanese diet 11.1 Average 5.35

* estimated from whole blood EPA+DHA+DPA Standard Deviation 2.22

If all ofthe traditionally high-fish eating populations are removed (Norwegians, Japanese,

Finns) and all patients (heart disease, type 2 diabetes mellitus, sudden cardiac death, hypertriglyceridemia, etc.) are removed, then the distribution of reported Omega-3 Index in populations with relatively uniform fish intake can be compared. Such a comparison is shown in Fig. 5. The variability in reported population averages (not individual values) suggests that assay variation may be great. A standardized method is essential for clinical utility or cross- population research studies.

EXAMPLE 20

This example determined the responsiveness ofthe Omega-3 Index to dietary Omega-3 fatty acids. Materials and Methods:

The purpose of this study was to determine the effects of relatively small, defined intakes of EPA+DHA on the Omega-3 Index. Healthy adult subj ects (n=65) provided written, informed consented for this study which had been approved by the Saint Luke's Hospital IRB. Besides being free of any diseases and not taking any drugs known to affect lipid metabolism, digestion or absorption, subjects had to have serum triglycerides betweenlOO and 300 mg/dL, serum LDL- cholesterol ©) < 130 mg/dL and serum HDL-c > 40 mg/dL. Subjects were queried regarding the previous month' s oily fish intake and were excluded if it exceeded one serving. A randomized, prospective, placebo-controlled, dose-ranging design was then employed. Subjects on stable background diets were randomized to 0 (placebo), 0.5, 1.0 and 2.0 g of EPA+DHA per day for five months following a one-month placebo run-in period. They took seven, 1 -g capsules per day for the entire six-month study. The placebo capsules contained corn oil, and the EPA+DHA capsules contained ROPUFA '30' omega-3 Food Oil (Roche Vitamins, Parsippany, NJ).

ROPUFA was given either full-strength (2 g group), pre-blended with corn oil 50:50 (1 g group) or 25:75 (0.5 g group). ROPUFA contained 11% EPA and 18% DHA. Since rather small intakes of omega-3 FA were being studied, subjects were instructed to completely avoid consumption of any oily fish (e.g., salmon, sardines, albacore tuna, mackerel) for the duration ofthe study but otherwise to make no changes in their diets. Pre-study interviews regarding oily fish intake suggested that this restriction would not significantly impact the normal dietary patterns of individuals living in the central US. Compliance was assessed by pill counts. The 5-month treatment period was sufficiently long to allow RBC FA composition to stabilize. The Omega-3 Index was then measured as described in Example 2. Whole blood FA patterns were also determined using the same methods as described above for RBCs. For comparison ofthe plasma phospholipid EPA+DHA content with the Omega-3 Index, plasma lipids were extracted with methylene chloride:methanol as previously described. The lipid extract was subj ected to thin layer chromatography on silica gel G with hexane/diethyl ether/formic acid (70/30/1) as the mobile phase in order to isolate the phospholipid band. This was methylated and analyzed by gas chromatography as described above.

Results and Discussion:

The effects ofthe various intakes of omega-3 FA on the Omega-3 Index are illustrated in Fig. 6. For the placebo group (n=22), the Index decreased slightly but significantly during the study, most likely because the subjects were instructed to avoid all oily fish during the study period. The Index rose from baseline values of 4.7±0.9% to 7.91=1.7% in the 0.5 g/d group, to 9^2.9% with 1 g/day, and 11.6+2.4% with 2 g/d. Each change (pre- to post-supplementation) was statistically significant within groups, and the changes from baseline between groups were significantly different for all but the 1 vs.2 g groups. Although a small study, these findings suggest that, in Kansas City, the AHA's current recommendation of "about 1 g/d" of EPA+DHA for CHD patients produces average Omega-3 Indices of about 10% (in Kansas City) whereas an intake of 500 mg/d results in an Omega-3 Index of about 8%. Thus, an intake of 800-900 mg/day might be expected to produce an Omega-3 Index of about 9%. Using these results, it was possible to estimate what Omega-3 Indexes were likely to have been in studies from which only intake data are available.

Evidence is considered here derived from prospective, randomized, controlled trials testing the effects of omega-3 FA (from capsules or oily fish) on risk for death from CHD. Neither the Omega-3 Index nor any parameter reducible to it was reported in these studies, therefore, how the omega-3 FA dose provided would likely have impacted the Omega-3 Index will be discussed. Four such studies have been reported. The first of these studies was by Nilsen and colleagues (incorporated by reference above) who enrolled 300 patients in Stavanger, Norway who had recently survived a myocardial infarction. They were randomized to four capsules per day of either Omacor (Pronova Biocare, Oslo, Norway; 85% ethyl esters of EPA+DHA) or a corn oil placebo. The former provided 3.4 g of EPA+DHA per day. After 18 months of follow-up, relative risks were not different from 1.0 for X, Y and Z. Thus, no evidence of CHD benefit was found. The authors hypothesized that the high fish intake typical of the Norwegian diet may have produced sufficiently high background omega-3 FA levels such that no further increase would be beneficial. An analysis of the plasma phospholipid EPA+DHA levels from a subset of patients from this study has recently been published and is provided herein as Table 4.

Table 4. Markers of Omega-3 FA Exposure in the Stavanger Study*

*adapted from Nilsen et al. **As described in Example 1

The average weekly consumption of three fish meals and the pre-study use of fish oils produced a baseline Omega-3 Index of over 9%, and increasing it to over 13% did not afford any clinically-significant CHD benefit. Thus an Index of greater than 9%, although apparently not harrtrful, may not be necessary. A second study (RB. Singh et al., Randomized, double-blind, Placebo-Controlled Trial of Fish Oil and Mustard oil in patients With Suspected Acute Myocardial Infarction: The Indian Experitnent of InfarctSurvival-4, 11 Cardiovasc. Drugs Ther., 485-491 (1997) the content and teachings of which are hereby incorporated by reference) was too small and short (despite extremely high 1-year event rates) to generate data useful for this analysis. The two remaining studies were conducted in patients who had survived a myocardial infarction.

The first was the Diet and Reinfarction Trial (DART) (Burr et al., Effects of Changes in Fat, Fish, and Fibre Intakes on Death and Myocardial Reinfarction: Diet and Reinfarction Trial (DART), 2 Lancet, 757-761 (1989) the content and teachings of which are hereby incorporated by reference herein). In this study, 2033 men were randomized to either receive (or not receive) advice to increase their oily fish intake to about 300 g/week. After two years of followup, those receiving the fish advice experienced a 29% reduction in all cause mortality and a 32%> decrease in ischemic heart disease mortality compared to controls . Dietary surveys indicated an average, achieved intake of oily fish was 292 g per week. The average EPA+DHA content of twenty ofthe most commonly eaten, "oily" fish is 1,221 mg per 85 g (3 oz.) based on the US Department of Agriculture Foods database. Based on this calculation, 292 g of oily fish would provide about 600 mg of EPA+DHA per day. The authors estimated an intake of 2.5 g of EPA per week or 357 mg d. Using the same database, the average contribution of EPA to the total EPA+DHA content of oily fish is about 40%>. Using this figure, the estimated intake of EPA+DHA in DART was about 900 mg/d. It can therefore be safely concluded that the EPA+DHA intake in the DART was somewhere between these two values and will be assumed to have been about 750 mg/d.

The second and largest study testing the effects of omega-3 FA supplementation on death from CHD was the GISSI-Prevention Study (GISSI-Prevenzione Investigators, Dietary Supplementation with N-3 Polyunsaturated Fatty Acids and Vitamin E in 11,324 Patients with Myocardial Infarction: Results of the GISSI-prevenzione Trial, 354 Lancet, 447-455 (1999) the content and teachings of which are hereby incorporated by reference). In this trial, 11,324 patients receiving modern cardiac pharmacotherapy (including statins, beta-blockers, anti-platelet drugs, and angiotensin converting enzyme inhibitors) were randomized to either 850 mg/d of EPA+DHA, 300 mg/d of vitamin E or to the unsupplemented control group. After 3.5 years of follow up, the group given the omega-3 fattyacidsexperienceda20%reductioninall-causemortality(p=0.01), anda45%> reduction in sudden death (p<0.001) compared to the control group. These effects became statistically significant within 3-4 months of randomization. Thus, similar intakes of EPA+DHA in the DART and the GISSI studies resulted in similar death benefits.

The results from the dose ranging study would suggest that 750 mg/d of EPA+DHA in the DART would produce an Omega-3 Index of about 9%, whereas the 850 mg/d in the GISSI-P study might have led to an Omega-3 Index of about 9.5%.

EXAMPLE 21 This example demonstrated the impact of 1.1 g of EPA + DPA per day on the Omega-3 Index in heart transplant patients. Materials and Methods

Twenty-five cardiac transplant patients who had been medically stable for at least 6 months were recruited. After obtaining informed consent, each subject gave a blood sample, and in addition, a sample of cheek (buccal) cells and cardiac tissue. The latter was obtained as part ofthe routine clinical evaluation of these patients. An assay to determine the Omega-3 Index in accordance with Example 2 was performed on the blood sample. The subjects were then given 1.1 g of EPA+DHA per day for the next 6 months. At that time they returned and blood and tissue samples were again obtained. A second assay was then performed to measure each subject's Omega-3 Index.

The current hypothesis regarding how omega-3 FA protect the heart is that these FA get incorporated into the tissues ofthe heart itself and thereby (in an as yet unknown manner) increase the ability ofthe muscle to withstand the ill-effects of a heart attack. Therefore, the purpose of this study was to determine the extent to which typical, cardioprotective intakes of omega-3 FA would alter the tissue FA composition of a variety of human tissues, most especially, the heart. Fig. 7 illustrates how each individual responded to the same intake. This figure clearly shows that 1) supplementation increased the Omega-3 Index form the low level of 4.7% to a 9.4%, well within the cardioprotective zone, and 2) that differences in background or baseline Omega-3 Indexes remained the same after supplementation. In other words, patients with low baseline values went up as much as those with higher values, but all subjects did not achieve the same target values. Hence, intakes must be individualized to achieve the target of 8%

EXAMPLE 22

This example analyzed the relationship between plasma phospholipid EPA + DHA and the Omega-3 Index. Materials and Methods:

Plasma Phospholipid EPA + DHA and the Omega-3 Index were compared by obtaining samples from 65 individuals, dividing these samples into 2 portions and comparing the two measurements.

Results:

Results for this example are provided in Fig. 8 which shows that the two measurements are closely related. The evidence of such a relationship was reduced to an equation and then the equation was applied to the values reported by Nilsen et al., and another study which examined the relationship between a blood measure of omega-3 FA and risk for CHD death (Lemaitre et al., Omega-3 Polyunsaturated Fatty Acids, Fatal Ischemic Heart Disease and Non-fatal Myocardial Infarction in Older Adults. The Cardiovascular Health Study, 76 Am. J. Clin. Nutr., 319-325 (2002) the content and teachings of which are hereby incorporated by reference herein). The Lemaitre investigators found a strong, protective relationship between (in this case) serum phospholipid EPA+DHA andrisk for fatal ischemic heart disease. The odds ratio associated with a one-standard deviation increase in this biomarker was 0.3. Converting the reported phospholipid EPA+DHA values to the Omega-3 Index by the equation derived above (Figure 8) revealed that those subj ects (approximately the upper fertile) with an Omega-3 Index above 9.5% [one standard deviation (2.4%) above the mean (7.1%)] were at 70%> lower risk for fatal ischemic disease than those at the mean. This value corresponds well with the results from Siscovick and Albert.

The teachings and content of all references disclosed above are expressly incorporated by reference. Additionally, the content and teachings ofthe following references are also incorporated by reference herein.

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Claims

I claim:

1. In a method of deteimining an individual's overall risk of death from coronary heart disease, said method including the steps of assaying a biological sample for at least one risk factor associated with coronary heart disease, wherein the improvement comprises assaying said sample for the level of omega-3 polyunsaturated fatty acids therein.

2. The method of claim 1 , said method further including the step of using said assayed level of omega-3 polyunsaturated fatty acids as one component ofthe individual's overall risk of death from coronary heart disease.

3. The method of claim 1, said level of omega-3 polyunsaturated fatty acids being assayed in red blood cells.

4. The method of claim 1 , said omega-3 polyunsaturated fatty acids being selected from the group consisting of EPA, DHA, and combinations thereof.

5. The method of claim 1 , further comprising the step of separately risk-stratifying the individual's risk of death from coronary heart disease based on the level of the omega-3 polyunsaturated fatty acids.

6. The method of claim 5, said risk-stratifying including classifying the individual as having a low, intermediate, or high, risk of death from coronary heart disease.

7. The method of claim 6, said high risk classification indicating an omega-3 level of less than or equal to 4%.

8. The method of claim 6, said intermediate risk classification mdicating an omega-3 level between 4% and 8%.

9. The method of claim 6, said low risk classification indicating an omega-3 level of 8% or greater.

10. A method of stratifying an individual's risk of death from coronaiy heart disease comprising the steps of: determining the mdividual's omega-3 fatty acid level; and comparing said detennined omega-3 fatty acid level with an index related to the risk of death from coronary heart disease and omega-3 fatty acid levels.

11. The method of claim 10, said index stratifying the risk of death from coronary heart disease based on the omega-3 fatty acid levels as high, intermediate or low.

12. The method of claim 10, said method further including the step of using said deteirnined level of omega-3 polyunsaturated fatty acids as one component ofthe mdividual's overall risk of death from coronaiy heart disease.

13. The method of claim 10, said level of omega-3 polyunsaturated fatty acids being determined in red blood cells.

14. The method of claim 10, said omega-3 polyunsaturated fatty acids being selected from the group consisting of EPA, DHA and combinations thereof.

15. The method of claim 10, said stratifying including classifying the individual as having a low, intennediate, or high, risk of deatli from coronaiy heart disease, based on said detennined level of omega-3 polyunsaturated acids.

16. The method of claim 15, said high risk classification mdicating an omega-3 level of less than or equal to 4%.

17. The method of claim 15, said intermediate risk classification indicating an omega-3 level between 4% and 8%.

18. The method of claim 15, said low risk classification indicating an omega-3 level of

8% or greater.

19. An index for classifying an individual's risk of death from coronary heart disease as high, intermediate, or low, based on the individual's level of omega-3 polyunsaturated fatty acids as detemiined from an assay of a biological sample from the individual, said index comprising cut-off points related to levels of omega-3 polyunsaturated fatty acids in biological samples wherein each said cut-off point provides an endpoint for one of said risk classifications.

20. The index of claim 19, said index having a cut-off point at 4% wherein individuals having an omega-3 level of less than or equal to 4% are classified as having a high risk of death from coronary heart disease.

21. The index of claim 20, said index having a second cut-off point at 8% wherein individuals having an omega-3 level of between 4% and 8% are classified as having an intermediate risk of death from coronary heart disease.

22. The index of claim 20, said index having a second cut-off point at 8% wherein individuals having an omega-3 level of 8% or greater are classified as having a low risk of death from coronary heart disease.

23. The index of claim 19, said level of omega-3 polyunsaturated fatty acids being determined in red blood cells.

24. The index of claim 19, said omega-3 polyunsaturated fatty acids being selected from the group consisting of EPA, DHA, and combinations thereof.

25. The index of claim 19, said index being used as one component ofthe individual's overall risk of death from coronary heart disease.

26. The index of claim 19, said index being used as a reference standard for determining an individual's risk of death from coronary heart disease based on the level of omega-3 polyunsaturated fatty acids in a biological sample from the individual.

27. A method of decreasing an individual's risk of death from coronary heart disease, said individual having a first level of omega-3 polyunsaturated fatty acids of less than 8%_> as determined by an assay ofthe individual's biological fluid, said method comprising the step of: causing said individual to ingest omega-3 polyunsaturated fatty acids in an amount effective for increasing the individual's level of omega-3 polyunsaturated fatty acids to second level, said second level being 8% or greater as determined by an assay ofthe individual's biological fluid.

28. The method of claim 27, said individual having a first level of omega-3 polyunsaturated fatty acids of less than or equal to 4%.

29. The method of claim 27, said effective amount comprising an average of at least 0.5 grams of omega-3 polyunsaturated fatty acid per day.

30. The method of claim 29, said effective amount comprising an average of at least 1.0 grams of omega-3 polyunsaturated fatty acid per day.

31. The method of claim 27, said ingested omega-3 polyunsaturated fatty acids being derived from a source selected from the group consisting of fish, fish oil supplements, and combinations thereof.

32. The method of claim 27, said omega-3 polyunsaturated fatty acids being selected from the group consisting of EPA, DHA, and combinations thereof.

33. The method of claim 27, said levels of omega-3 polyunsaturated fatty acids being assayed in red blood cells.

34. A method of measuring patient compliance with a diet designed to increase the level of omega-3 polyunsaturated fatty acids in a biological sample taken from the patient comprising the steps of; taking a first biological sample from the patient; assaying the level of omega-3 polyunsaturated fatty acids in said first sample to provide a first level; taking a second biological sample from the patient; assaying the level of omega-3 polyunsaturated fatty acids in said second sample to provide a second level; and comparing said first level with said second level.

35. The method of claim 34, said firstbiological and said second biological sample being taken from the patient at least 7 days apart.

36. The method of claim 34, said first biological sample and said second biological sample being taken from the patient at least 30 days apart.

37. The method of claim 34, said first biological sample and said second biological sample being taken from the patient at least 120 days apart.

38. The method of claim 34, further comprising the step of instructing the patient to increase their consumption of omega-3 polyunsaturated fatty acids a specific amount based on said first level.

39. The method of claim 38, said specific amount being an amount effective for raising the level of omega-3 polyunsaturated fatty acids to at least 8%>.

40. The method of claim 34, said omega-3 polyunsaturated fatty acids being selected from the group consisting of EPA, DHA, and combinations thereof.

41. The method of claim 34, said first and said second levels being assayed in red blood cells.

42. A kit for determining the presence of fatty acids in a biological sample, said kit comprising: a container for holding the biological sample; a set of instructions for using said kit; and a first standardized fatty acid sample.

43. The kit of claim 42, said fatty acids being omega-3 fatty acids.

44. The kit of claim 43, said fatty acids being selected from the group consisting of EPA, DHA, and combinations thereof.

45. The kit of claim 42, said kit further comprising a second standardized fatty acid sample.

46. The kit of claim 42, said first fatty acid sample comprising a sample having 8% omega-3 fatty acids therein.

47. The kit of claim 45, said second standardized fatty acid sample comprising a sample having 4% omega-3 fatty acids therein.

48. The kit of claim 42, said instructions including instructions on how to determine omega-3 levels in said biological sample.

49. The kit of claim 42, said kit further comprising all necessary reagents for determining the presence of omega-3 fatty acids.

50. The kit of claim 42, said kit further comprising all necessary equipment for determining the presence of omega-3 fatty acids.