The HS-Omega-3 Index Methodological Considerations

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2 The HS-Omega-3 Index Methodological Considerations William S. Harris, PhD The HS-Omega-3 Index is the EPA+DHA content of red blood cells (RBCs) expressed as a percent of total identified RBC fatty acids. It is measured using a proprietary methodology developed over several years of research. OmegaQuant, LLC is a CLIA certified lab (#43D ). Fatty acids (n=24) are quantitatively identified using capillary column gas chromatography with an internal-standard-based, three-point calibration curve approach based loosely on the classic method of Morrison and Smith 1. This method is precise and accurate because it allows for adjustment of day-to-day and instrument-to-instrument variations in FID response factors. This keeps analytical variability very low. Observed Index(%) Predicted Index (%) Figure 1. The correlation between the expected Figure and 1. The observed correlation values between for the the HS-Omega-3 expected and Index observed This values shows that for the across HS-Omega-3 the clinically Index. significant This shows range that across of values, the the clinically test gives significant accurate range results. of values, the test gives accurate results Linearity: The observed HS-Omega-3 Index value matches precisely the expected value when mixtures of RBCs with different amounts of EPA+DHA are analyzed (Figure 1). In addition, the response is linear between 2.5 and 30 ul of RBCs (Figure 2). Precision: Current coefficients of variation (CVs) were determined over 200 runs across 4 instruments. At a low HS-Omega-3 Index (1.7%) the CV is 6.8%, and at a high HS-Omega-3 Index (11.0%) the CV is 2.5%. Sensitivity: The method can reliably detect very low levels of FAs (i.e., 0.1% abundance). GC Area Counts Analytical Linearity RBC volume (ul) Figure Linearity of response of response of the of HS-Omega-3 the HS-Omega-3 Index to Index increasing to increasing amount amount of RBC sample of RBC analyzed. sample analyzed. The test is routinely The test done is routinely with 30 done ul of with packed 30 ul RBCs. of packed R 2 =0.97 RBCs. R=0.97

3 Biological Stability: In a study of 60 placebotreated patients on a stable diet over 10 weeks, the mean HS-Omega-3 Index did not change (4.6±1.% vs. 4.6±1.2%) 2. In another study 3 with 20 healthy volunteers studied 7 times over 6 weeks, the within-subject coefficient of variation (CV) for EPA+DHA in RBCs and whole plasma were 4.1%±1.9% and 15.9%±6.4%, respectively 3 (Figure 3). (The CV for whole blood EPA+DHA was 6.7%±4.0%). Thus, the RBC test had the lowest biological variability which means that a single measurement (not several) is all that is needed to determine the true level. Fasting vs. Fed: The effect of consuming a large meal on the EPA+DHA content of RBCs, whole blood, and plasma was examined in the same 20 subjects as described above3. The EPA+DHA level in RBCs did not differ by fasting-fed status (-1%), but it was slightly lower in whole blood (-5%, p=0.03), and much lower in plasma (-11%, p<0.05) due to the dilution of plasma EPA+DHA with non-omega-3 FAs contained in the test meal. Plasma percent of total FAs RBC percent of total FAs 10% 9% 8% 7% 6% 5% 4% 3% 2% 1% 0% 12% 10% 8% 6% 4% 2% 0% Figure 3. Biologic variability: RBC vs Plasma. EPA+DHA content of both sample types was measured weekly in 20 volunteers for 6 weeks. Determination of the HS-Omega-3 Index from a Dried Blood Spot (DBS): The DBS method for determining the HS-Omega-3 Index was validated in a study including 106 subjects from whom DBS cards and blood tubes were collected. The latter were immediately processed for RBCs which were frozen at -80 C; the former were express shipped to various cities around the country, and from there, returned by regular US mail back to OmegaQuant for routine analysis. The correlation between the EPA+DHA content of the two samples was very high (r=0.96; p<0.0001), and the 95% confidence interval for the DBS-estimated value was ± 1% (Figure 4). RBC EPA+DHA DBS EPA+DHA Figure 4. Correlation between the EPA+DHA content of dried blood spots (DBS) and the (RBCbased) HS-Omega-3 Index. DBS samples were collected from 106 subjects, shipped via standard US mail, and compared to RBC samples frozen immediately after collection. R=0.96, p<

4 Preventing Loss of EPA and DHA on Dried Blood Spots with OxyStop : The long-chain omega-3 fatty acids that comprise the HS-Omega-3 Index (EPA and DHA) are highly polyunsaturated and therefore very susceptible to oxidative degradation. At OmegaQuant, we use our proprietary antioxidant treatment called OxyStop to retard this natural process in samples collected as dried blood spots on filter paper (QuantCards ). In the experiment illustrated in Figure 5, one blood sample was applied to 3 different types of card/antioxidant systems: QuantCards with OxyStop, Fluka cards without BHT and Fluka cards with BHT. (Fluka cards and BTH are available from Sigma- Aldrich). Cards were prepared in an identical manner and stored in plastic bags in the dark at room temperature for up to 2 weeks. On the indicated day, one of each type of card was removed, 3 punches of dried blood were taken from each, and then all were analyzed for the HS-Omega-3 Index. The average of each triplicate analysis is plotted. There was no loss of EPA and DHA in the OxyStop-treated QuantCard whereas there was loss in the Fluka cards, less with BHT than without. HS-Omega-3 Index 9% 8% 7% 6% 5% 4% 3% 2% 1% OxyStop Fluka with BHT Fluka without BHT 0% DayDays Figure 5. Comparison of QuantCards with OxyStop v Figure Fluka 5. cards Comparison with and of without QuantCards BHT. The with HS-Omega-3 OxyStop vs Index Fluka was cards measured with and in without triplicate BHT. at The the HS-Omega-3 Index was measured in triplicate on the indi- indicated day cated day. Recovery of Fatty Acids from Spiked Samples: Experiments were conducted to determine the a- ccuracy of both RBC and plasma fatty acid analytical methods. Accuracy was defined as the percent recovery of 6 major fatty acids, all esterified in phosphatidylcholine (their primary chemical form in plasma and RBCs), added to native RBC and plasma samples. The analyses were performed in triplicate using three levels of added fatty acids analyzed with the internal-standard, 3-point calibration curve method. Table 1 shows the fatty a- cids assessed and the percent recoveries. Clearly, the method quantitatively determines the fatty acid content of both sample types. TABLE 1: Fatty Acid Recovery % Recovery Fatty Acid RBC Plasma C16:0 98% 98% C18:0 98% 98% C18:1n9 101% 102% C18:2n6 101% 98% C20:4n6 100% 94% C22:6n3 104% 98%

5 EDTA Blood Samples Analyzed Wet Vs Dry on QuantCards: Some researches prefer to send a spot of blood rather than a tube of blood to OmegaQuant. In order to document comparability of these two sample types, EDTA (lavender top) tubes were drawn from 20 subjects. The tubes were opened, a drop of blood was placed on a QuantCard and allowed to dry. The card and the liquid blood sample were then analyzed. The mean ±SD HS-Omega-3 Index was 5.08±1.9% for the EDTA-blood and 5.05±1.9% for blood card. The correlation was 0.97 (Figure 6). Comparison with Other Laboratories: International, cross validation studies were conducted with two other laboratories that are using the HS-Omega-3 Index technology [OmegaQuant Asia (Seoul, Korea) and OmegaMetrix (Munich, Germany)]. The HS-Omega-3 Index measured in these labs varied by less than 0.1 percentage points from the value obtained at OmegaQuant. This confirms that the test is transferrable to other labs and that accuracy and precision can be achieved when identical methodologies are employed. Different methodologies, however, can generate different results. To illustrate, a comparison was made using 366 RBC samples. In this study, the samples were analyzed at OmegaQuant using the HS-Omega-3 technology, and in Lab X (in Scandinavia) using their own methodology. Values are significantly higher for nearly every fatty acid in the OmegaQuant analysis (see Table 2 showing selected fatty acids). The reason for this difference has more to do with the denominator than the numerator. In other words, since fatty acid levels are expressed as a percent of total, deciding which fatty acids are included in the denominator (the total ) can influence the results. At OmegaQuant we focus our analysis on the fatty acids esterified in a particular, physiologically-active subset of the membrane glycerophospholipids. Lab X uses another method which measures these plus other types of fatty acids (italics in the Table 2) predominantly carried in sphingolipids. The latter fatty acids add about 11% to the denominator. In addition, Lab X includes the area under ALL peaks on the chromatogram (whether positively identified as fatty acids or not, unknown ), whereas we include only a suite of the most important, clearly identified fatty acids in our calculations. Fingerstick (%) Fatty Acid EDTA blood (%) Figure Comparison of the HS-Omega-3 Index Index measured on dried blood spots spots using using blood blood collected collected either from either a fingerstick from a or fingerstick from a EDTA or tube from (n=20). a EDTA tube (n=20) TABLE 2: Inter-lab Comparison Lab X % of fatty acids OMQ C16: C16:1n C18: C18: 1 trans C18: C18:2n C20:4n C20:5n C22: C24: C24:1n C22:4n C22:5n C22-6n Omega-3 Index Unknown

6 So the fact that Lab X includes more/different fatty acid species in the denominator reduces the apparent levels of the Omega-3 Index as well as all other fatty acids. Not only are there are no standardized conventions defining which fatty acids should be included in the denominator, there is no consensus regarding how the fatty acids are chemically liberated from the RBC samples, nor for how baselines are defined for each type of peak. Therefore, differences in methods can result in very different fatty acid compositions. As another example, the same blood sample was sent to 4 commercial US labs for analysis of the omega-3 index (Figure 7). Compared to the OmegaQuant value (10.4%), the other labs returned values varying from 3.81% to 13.74%. This underscores the wide variability in results that can be obtained commercially. Omega-3 Index (%) OMQ LABG LABM LABB LABN Figure 7. The same blood sample was sent to 4 commercial labs in the US for measurement Figure of omega-3 7. The index. same The results blood confirm sample lab to lab was s to differences. 4 commercial labs in the US for measurement of the omega-3 index. Th results confirm that lab to lab differenc OmegaQuant The Gold Standard: We consider the OmegaQuant results to be the gold standard because our unique HS-Omega-3 Index technology has been and continues to be used in several major epidemiologic 4-13 and intervention studies. These studies are, for the first time, linking specific fatty acid values with risk for important clinical outcomes (death, heart attacks, depression, dementia, etc). Thus, investigators and clinicians wanting to take an evidence-based approach to monitoring and titrating their patients omega-3 status must use the test that was originally used to derive the evidence. For example, if an HS-Omega-3 Index of 8% or greater is considered cardioprotective based on several published studies using OmegaQuant, then the healthcare provider who used Lab M or Lab B (Figure 7) will mistakenly conclude that his/her patient needs to increase their omega-3 intake. In like manner, a sample sent to Lab G, which overestimated the Omega-3 Index by 3.4%, could be falsely reported as being in the target zone when an increased omega-3 intake should have been prescribed. With the HS-Omega-3 Index clinicians can be confident that they are using the most researched omega-3 status test available.

7 Reference List (1) Morrison WR, Smith LM. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol. J Lipid Res 1964;5: (2) Carney RM, Freedland KE, Rubin EH et al. Omega-3 augmentation of sertraline in treatment of depression in patients with coronary heart disease: a randomized controlled trial. JAMA ;302: (3) Harris WS, Thomas RM. Biological variability of blood omega-3 biomarkers. Clin Biochem ;43: (4) Block RC, Harris WS, Reid KJ et al. EPA and DHA in Blood Cell Membranes from Acute Coro nary Syndrome Patients and Controls. Atherosclerosis 2007;197: (5) Block RC, Harris WS, Reid KJ et al. Omega-6 and trans fatty acids in blood cell membranes: a risk factor for acute coronary syndromes? Am Heart J 2008;156: (6) Cohen BE, Garg SK, Ali S et al. Red blood cell docosahexaenoic acid and eicosapentaenoicacid concentrations are positively associated with socioeconomic status in patients with established coronary artery disease: data from the heart and soul study. J Nutr 2008;138: (7) Farzaneh-Far R, Lin J, Epel ES et al. Association of marine omega-3 fatty acid levels with telo meric aging in patients with coronary heart disease. JAMA 2010;303: (8) Farzaneh-Far R, Harris WS, Garg S et al. Inverse association of erythrocyte n-3 fatty acid levels with inflammatory biomarkers in patients with stable coronary artery disease: The Heart and Soul Study. Atherosclerosis 2009;205: (9) Harris WS, Assaad B, Poston WC. Tissue omega-6/omega-3 fatty acid ratio and risk for coro nary artery disease. Am J Cardiol 2006;98:19i-26i. (10) Moyers B, Farzaneh-Far R, Harris WS et al. Relation of Whole Blood n-3 Fatty Acid Levels to Exercise Parameters in Patients With Stable Coronary Artery Disease (from the Heart and Soul Study). Am J Cardiol :1/49-54 (11) Shearer GC, Pottala JV, Spertus JA et al. Red blood cell fatty acid patterns and acute coronary syndrome. PLoS ONE 2009;4:e5444. (12) Baghai TC, Varallo-Bedarida G, Born C et al. Major depressive disorder is associated with car diovascular risk factors and low Omega-3 index. J Clin Psychiatry 2010 (epub). (13) Harris WS, Sands SA, Windsor SL et al. Omega-3 Fatty Acids in Cardiac Biopsies from Heart Transplant Patients: Correlation with Erythrocytes and Response to Supplementation. Circula tion 2004;110: (14) Harris WS, Gonzales M, Laney N et al. Effects of omega-3 fatty acids on heart rate in cardiac transplant recipients. Am J Cardiol 2006;98: (15) Harris WS, Pottala JV, Sands SA et al. Comparison of the effects of fish and fish-oil capsules on the n 3 fatty acid content of blood cells and plasma phospholipids. Am J Clin Nutr 2007;86: (16) Harris WS, Lemke SL, Hansen SN et al. Stearidonic acid-enriched soybean oil increased the omega-3 index, an emerging cardiovascular risk marker. Lipids 2008;43: (17) Larson MK, Ashmore JH, Harris KA et al. Effects of omega-3 acid ethyl esters and aspirin, alone and in combination, on platelet function in healthy subjects. Thromb Haemost 2008;100: OmegaQuant, OxyStop and HS-Omega-3 Index are registered marks of Harris Scientific, Inc All rights reserved.

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