Scholarly article on topic 'Comparative effects of an acute dose of fish oil on omega-3 fatty acid levels in red blood cells versus plasma: Implications for clinical utility'

Comparative effects of an acute dose of fish oil on omega-3 fatty acid levels in red blood cells versus plasma: Implications for clinical utility Academic research paper on "Health sciences"

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{"n-3 Fatty acids" / Erythrocytes / Plasma / Biomarker / Biovariability}

Abstract of research paper on Health sciences, author of scientific article — William S. Harris, Stephen A. Varvel, James V. Pottala, G. Russell Warnick, Joseph P. McConnell

Background Omega-3 fatty acid (n-3 FA) biostatus can be estimated with red blood cell (RBC) membranes or plasma. The matrix that exhibits the lower within-person variability and is less affected by an acute dose of n-3 FA is preferred in clinical practice. Objective We compared the acute effects of a large dose of n-3 FA on RBC and plasma levels of eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA). Methods Healthy volunteers (n = 20) were given 4 capsules containing 3.6 g of n-3 FA with a standardized breakfast. Blood samples were drawn at 0, 2, 4, 6, 8, and 24 hours. The EPA + DHA content of RBC membranes and plasma (the latter expressed as a percentage of total FA and as a concentration) were determined. General linear mixed models were used to analyze the mean response profiles in FA changes over time for plasma and RBCs. Results At 6 hours after load, the plasma concentration of EPA + DHA had increased by 47% (95% confidence interval [CI], 24% to 73%) and the plasma EPA + DHA percentage of total FA by 19% (95% CI, 4.7% to 36%). The RBC EPA + DHA percentage of composition was unchanged [–0.6% (95% CI, −2.6% to 1.5%)]. At 24 hours, the change in both of the plasma EPA + DHA markers was 10-fold greater than that in RBCs. Conclusions An acute dose of n-3 FA (eg, a meal of oily fish or fish oil supplements) taken within a day before a doctor’s visit can elevate levels of EPA + DHA in plasma, whether expressed as a percentage or a concentration, but not in RBC membranes. Similar to hemoglobin A1c, which is not affected by an acute glycemic deviation, RBCs provide a more reliable estimate of a patient’s chronic EPA + DHA status than does plasma.

Academic research paper on topic "Comparative effects of an acute dose of fish oil on omega-3 fatty acid levels in red blood cells versus plasma: Implications for clinical utility"

Journal of Clinical Lipidology (2013) 7, 433-440

Journal of

Clinical Lipidology

Comparative effects of an acute dose of fish oil on omega-3 fatty acid levels in red blood cells versus plasma: Implications for clinical utility*

William S. Harris, PhD*, Stephen A. Varvel, PhD, James V. Pottala, PhD, G. Russell Warnick, MS, MBA, Joseph P. McConnell, PhD

Health Diagnostic Laboratory, Inc, 737 N 5th Street, Suite 103, Richmond, VA 23219, USA (Drs. Harris, Varvel, Pottala, and McConnell; and Mr. Warnick); OmegaQuant Analytics, LLC, Sioux Falls, SD, USA (Dr. Harris); and Department of Internal Medicine, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD, USA (Drs. Harris and Pottala)

BACKGROUND: Omega-3 fatty acid (n-3 FA) biostatus can be estimated with red blood cell (RBC) membranes or plasma. The matrix that exhibits the lower within-person variability and is less affected by an acute dose of n-3 FA is preferred in clinical practice.

OBJECTIVE: We compared the acute effects of a large dose of n-3 FA on RBC and plasma levels of eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA).

METHODS: Healthy volunteers (n = 20) were given 4 capsules containing 3.6 g of n-3 FA with a standardized breakfast. Blood samples were drawn at 0, 2, 4, 6, 8, and 24 hours. The EPA 1 DHA content of RBC membranes and plasma (the latter expressed as a percentage of total FA and as a concentration) were determined. General linear mixed models were used to analyze the mean response profiles in FA changes over time for plasma and RBCs.

RESULTS: At 6 hours after load, the plasma concentration of EPA 1 DHA had increased by 47% (95% confidence interval [CI], 24% to 73%) and the plasma EPA 1 DHA percentage of total FA by 19% (95% CI, 4.7% to 36%). The RBC EPA 1 DHA percentage of composition was unchanged [-0.6% (95% CI, -2.6% to 1.5%)]. At 24 hours, the change in both of the plasma EPA 1 DHA markers was 10-fold greater than that in RBCs.

CONCLUSIONS: An acute dose of n-3 FA (eg, a meal of oily fish or fish oil supplements) taken within a day before a doctor's visit can elevate levels of EPA 1 DHA in plasma, whether expressed as a percentage or a concentration, but not in RBC membranes. Similar to hemoglobin A1c, which is not affected by an acute glycemic deviation, RBCs provide a more reliable estimate of a patient's chronic EPA 1 DHA status than does plasma. © 2013 National Lipid Association. All rights reserved.

Potential conflicts of interest. All authors are employed by either Health Diagnostic Laboratory, Inc or OmegaQuant Analytics, LLC or both. Both of these companies offer red blood cell fatty acid testing, and the latter (primarily a research laboratory) also offers plasma assays. W.S.H. has received consulting fees and honoraria from GlaxoSmithKline and Amarin Corporation (companies with interests in pharmaceutical n-3 fatty acids). He is also on the scientific advisory boards of Omthera and Aker Biomarine Antarctic.

* This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Corresponding author.

E-mail address: Bharris@hdlabinc.com

Submitted December 26, 2012. Accepted for publication May 1, 2013.

1933-2874/$ - see front matter © 2013 National Lipid Association. All rights reserved. http://dx.doi.org/10.1016/jjad.2013.05.001

KEYWORDS:

n-3 Fatty acids;

Erythrocytes;

Plasma;

Biomarker;

Biovariability

The Omega-3 Index is a validated biomarker of tissue membrane omega-3 (n-3) polyunsaturated fatty acid (PUFA) status.1 The test analyzes the level of eicosapentae-noic acid (EPA) plus docosahexaenoic acid (DHA) in red blood cell (RBC) membranes and expresses the result as a percentage of total fatty acids (FAs). The choice of RBCs was based on several factors, including previous literature that linked RBC EPA + DHA to risk of sudden cardiac death,2 the ready availability of this biological membrane in blood samples, and the high correlation between RBC and myocardial EPA + DHA levels.3,4 In addition, RBCs are a more stable, within-person biomarker of n-3 status than plasma, the latter having 4 times greater biological variability.5 Just as the RBC-based hemoglobin A1c assay is a more stable marker of glycemic status than fasting/fed serum glucose, the Omega-3 Index appears to be a more stable marker of n-3 status than plasma-based assays. Although stability has been documented over a 6-week period,5 the acute effects of a dose of EPA + DHA, whether from supplements or fish, on n-3 biostatus has received only limited attention. A single dose of these FAs clearly raises plasma EPA and DHA levels, peaking at approximately 5 hours after administration,6 but the effect of such a dose on RBC EPA and DHA content are unknown. The hypothesis tested in this study was that RBC EPA + DHA levels would be less affected by an acute load of n-3 FA than would plasma EPA + DHA levels.

There were both scientific and practical reasons for undertaking this study. Scientifically, it is not known how rapidly n-3 FA, on entering the plasma compartment in chylomicrons, become incorporated into cell membranes. Once in the nonesterified FA fraction, exchange is known to occur between DHA and RBC membranes, beginning approximately 8 hours after ingestion, with the predominant delivery form being albumin-bound DHA-lysophos-phatidyl choline,7 but the detailed time course in vivo is not clear. From a practical point of view, now that medical testing for n-3 FA status has become more common, clinicians have a choice of whether to use RBC- or plasma-based assays, and, for the latter, FA content can be expressed as a percentage of total FA or as a concentration. Different assays have different performance characteristics, fasting requirements, and sensitivities to perturbation with exogenous n-3 FA. Understanding these issues can help clinicians choose the appropriate testing method.

Methods Subjects

Inclusion criteria were healthy male and female subjects of any race, older than 18 years, with body mass index (BMI; calculated as weight divided by height; kg/m2) between 18 and 35. Subjects were recruited by a company-wide e-mail solicitation, and initially screened

by telephone interview. Subjects who had taken fish oil supplements in the past 30 days, or who reported eating >1 entree of ''oily fish'' (ie, salmon, herring, sardines, al-bacore tuna, mackerel) per week over the prior month were excluded. In addition, subjects with any known condition that would impair fat absorption (eg, cystic fibrosis, abetalipoproteinemia, chronic pancreatitis, pancreatic li-pase deficiency syndrome) were also excluded. The first 20 subjects who qualified were e-mailed a copy of the informed consent document (which had been approved by the Copernicus Group Institutional Review Board). They subsequently attended an initial visit at which time the document was reviewed and informed consent was obtained by signature.

Procedures

The initial laboratory visit took place in the morning after at least an 8-hour fast at which time a baseline blood sample was drawn. Immediately after the blood draw, the subjects were given a standardized breakfast plus 4 enteric-coated fish oil capsules, each containing 900 mg of EPA (647 mg) 1 DHA (253 mg; Simply Natural Triple Strength; Sam's Club). This dose provided a total of 3.6 g of EPA 1 DHA as ethyl esters, which is equivalent to the n-3 FA dose one would get in approximately 6 ounces of farmed salmon. It is also approximately the dose for n-3 acid ethyl esters approved by the Food and Drug Administration; thus, there were no safety concerns. Subjects returned to the laboratory 2, 4, 6, 8, and 24 hours after the test meal for subsequent blood draws.

The standardized meals provided for breakfast, lunch, and dinner (and evening snack) contained no fish, and subjects were instructed to take no other fish oil pills during the study. The food was provided on the basis roughly of BMI, with subjects with a BMI < 27 receiving a total of 1800 kcal for the day and subjects with a higher BMI, 2200 kcal. Lunch and dinner were consumed after the blood draws at 4 and 8 hours, respectively. The final sample was drawn after a 10-hour overnight fast.

Laboratory methods

Blood was drawn into 5-mL ethylenediaminetetra-acetic acid tubes and centrifuged immediately to separate RBCs from plasma. Aliquots of packed RBCs and plasma were transferred to cryovials and stored at — 80°C until they were shipped on dry ice to OmegaQuant Analytics (Sioux Falls, SD) for analysis. Total cholesterol and triglycerides assays were performed with standard automated enzymatic methods on a Roche/Hitachi P-Modular system with Roche reagents (Roche Diagnostics, Indianapolis, IN). Low-density lipoprotein cholesterol and high-density lipoprotein cholesterol were measured with direct enzymatic assays from Randox (County Antrim, United Kingdom) on a Roche/Hitachi P-modular system. Plasma glucose was measured with enzymatic methods on a

Beckman Coulter AU5842, hemoglobin A1c was measured by ion-exchange high-performance liquid chromatography on a Bio-Rad Variant Turbo II, and complete blood cell count was measured on Beckman Coulter Uni-Cel DxH800.

FA analysis

Samples were thawed, and an aliquot of plasma was combined (1:40 parts) with the derivatizing reagent [boron trifluoride in methanol (14%), toluene, and methanol (35:30:35 parts)], shaken, and heated at 100°C for 45 minutes. After cooling, 40 parts of both hexane and distilled water were added. After briefly vortexing, the samples were spun to separate layers, and an aliquot of the hexane layer that contained the FA methyl esters was extracted. Diheptadecanoyl-phosphatidyl choline was included in the plasma analysis as an internal standard for calculating concentrations. RBCs were analyzed similarly with the exception that only boron trifluoride in methanol (14%) was used for derivatization, and the samples were heated for only 10 minutes. Gas chromatography was performed with 100-m SP-2560 column in a Shimadzu 2010 instrument equipped with a flame ionization detector as described previously.8 FAs were quantified by 3-point calibration curves and reported as percentage of composition (ie, each FA as a percentage of the total identified FAs) for both plasma and RBCs, and, in addition, as a concentration (mg/mL) in plasma. Coefficients of variation (CVs) for control samples were 2.5%, 2.0%, and 5.0% for these analytes, respectively.

Statistical methods

General linear mixed models were used with estimation to analyze the mean response profiles in FA changes over time for plasma and RBCs. The responses over time were all normalized to their baseline value, and because the distribution was right-skewed, a natural log transformation was used to improve normality and homoscedas-ticity of the residual errors. The repeated measures covariance structure that minimized Akaike Information Criterion9 was selected as follows: Toeplitz for plasma concentration, first-order autoregressive for plasma percentage, and compound symmetry for RBC percentage. The Toeplitz and autoregressive structures are banded, with the later imposing a power lag function. The compound symmetry structure provides a single correlation between any 2 time points. The model parameter estimates were exponentiated and reported as percentage of changes from baseline. To determine which time points were different from baseline for each sample type, Dunnett adjustment was used to control for multiple comparisons. Dunnett adjusted P values < .05 were considered statistical significance. Analyses were performed with SAS software version 9.3. (SAS Institute).

Results

A total of 20 volunteers participated in this study. All laboratory values were within normal ranges (Table 1). The mean total energy consumed for the study day was 1936 + 80.4 kcal, and the mean total fat intake was 66 ± 4.3 g (or 31% of kilocalories). A total of 19% of the day's kcal and fat (ie, 362 + 77 kcal and 13 + 4 g, respectively, excluding the kilocalories and fat from the supplements) were consumed at breakfast when the fish oil capsules were taken.

Baseline levels of the 5 major classes of FA (saturated, monounsaturated, n-6 and n-3 PUFAs, and trans) are presented in Table 2 along with the EPA + DHA values. The within-subject and between-subject variability (both expressed as the CV) differed substantially for each FA class by sample type. FA levels in RBCs had approximately 2-fold lower between-person variability than did plasma FA (percentage) for saturated, monounsaturated, and n-6 amounts; however, they were similar for n-3 PUFAs and trans FAs. Greater variability was observed in concentrations than in relative amounts. For example, the CV for saturated FAs expressed as concentrations was approximately 14-fold higher than the CV for this class in RBC membranes. For monounsaturated FAs, the CVs of these 2 expressions of FA status differed by 5-fold, and for the n-6 PUFA class, by 6-fold. For the n-3 PUFA the between-person variability across sample types was similar. Compared with the RBC-based metric, the within-person variability for the plasma percentage-based metric was 2- to 5-fold higher for all FA classes except trans FA, which was 10-fold higher. For the plasma concentration-based metric, it was 5- to 8-fold higher for all FA classes except

Table 1 Subject Characteristics (Men, n = 2;Women, n = 18)

Mean SD Healthy Range

Age (y) 32 8.5 —

Race, n

African American 9 —

White 9 —

Asian 2 —

Total cholesterol (mg/dL) 185 47.5 <200

LDL cholesterol (mg/dL) 113 46.9 <130

HDL cholesterol (mg/dL) 58 13.9 $40

Triglycerides (mg/dL) 105 72.6 <150

Non-HDL cholesterol (mg/dL) 128 46.8 <130

Hemoglobin A1c (%) 5.3 0.4 #5.6

Glucose (mg/dL) 81 7.5 <100

White blood cells (X103/mL)* 6.8 2.7 4.0 to 10.5

Red blood cells (X106/mL)* 4.5 0.4 4.1 to 5.6

Hematocrit (%)* 41 4.3 36 to 50

Hemoglobin (g/dL)* 13 1.4 12.5 to 17.0

Platelets (X103/mL)* 251 46.2 140 to 415

HDL, high-density lipoprotein; LDL, low- density lipoprotein.

Table 2 Abundance and Variability of Major FA Classes by Sample Type (n = 20)

Saturated Monounsaturated Omega-6 PUFA Omega-3 PUFA trans FA EPA 1 DHA

Red blood cell

Mean (% of total FA) 40.4 13.4 36.9 7.5 1.4 4.8

SD (% of total FA) 0.9 1.2 1.5 1.6 0.3 1.5

Within subject CV (%)* 1.5 1.4 2.0 2.7 1.8 3.1

Between subject CV (%) 2.3 9.0 4.0 21.4 21.2 31.8

Plasma

Mean (% of total FA) 28.4 20.4 45.8 3.5 1.1 2.3

SD (% of total FA) 1.7 3.3 4.1 0.9 0.3 0.8

Within subject CV (%)* 3.3 5.9 3.4 11.6 18.1 15.7

Between subject CV (%) 5.9 16.3 9.1 25.0 29.9 34.7

Plasma

Mean (mg/mL) 834.2 613.6 1325.5 98.9 33.3 64.1

SD (mg/mL) 260.4 277.9 334.5 27.1 13.0 20.8

Within subject CV (%)* 12.1 14.9 9.2 16.7 21.9 19.3

Between subject CV (%) 31.2 45.3 25.2 27.4 39.1 32.4

CV, coefficient of variation;DHA, docosahexaenoic acid;EPA, eicosapentaenoic acid;FA, fatty acid;PUFA, polyunsaturated fatty acid;SD, standard

deviation.

*Within-subject CV was calculated over the 6 time points; all other measures are at baseline.

the trans and monounsaturated FAs, which were > 10-fold higher.

For the FA metric of primary interest in this study, EPA + DHA, the individual subject data (and group means with 95% confidence interval [CI]) for all 3 sample types across the 24-hour period are shown in Figure 1. When expressed as percentage of changes from baseline, both of the plasma-based metrics showed significant increases over the study period, but the RBC-based metric did not (Fig. 2). At 6 hours after load, the plasma concentration of EPA 1 DHA had increased by 47% (95% CI, 24% to 73%), the plasma EPA 1 DHA as a percentage of total FA by 19% (95% CI, 4.7% to 36%), and the RBC EPA 1 DHA percentage composition by —0.6% (95% CI, —2.6% to 1.5%). At 24 hours all sample types had significant increases in EPA 1 DHA of 28%, 30%, and 2.8%, respectively.

Discussion

The purpose of this study was to compare the response of plasma-based with RBC-based markers of n-3 FA status to a pharmacologic dose of n-3 FAs. We found that the 24-hour excursion for plasma (expressed as both percentage and concentration) was significantly greater than that for RBCs. In plasma the peak n-3 FA concentration was observed at 6 hours, whereas the peak as a percentage of total FA was seen at 24 hours. RBC EPA 1 DHA levels were increased from baseline only at 24 hours, but by an order of magnitude less than plasma (approximately 3% vs 30% elevated).

In a previous study we examined the within-person biological variability in EPA 1 DHA by sample type over 6 weeks in volunteers with a stable n-3 FA intake.5 EPA 1 DHA levels as a percentage of total FA were 10 times

less variable when measured in RBCs (CVwithin = 1.3%) than in plasma (CVwithin = 13.6%) (Fig. 3). These values, measured over weeks, were similar to those observed here (2.7% and 15.7%, respectively) which were measured over hours after a large n-3 FA load. Despite the differences in within-person variability, the mean levels for both (fasting) plasma and RBCs were stable over that 6-week study. Therefore, in research studies done in large populations, either metric can provide an estimate of n-3 FA status, but in the clinical setting, where visit-to-visit comparisons are most important, the RBC-based metric is preferred. Another problem with plasma-based metrics is their sensitivity to fasting status. In the earlier study,5 the effects of a high-fat meal (with no fish oil) on EPA 1 DHA levels in RBCs and plasma (again, both expressed as a percentage of total FA) were assessed. Four hours after the meal, no change was observed in the RBC metric, but an 11% decrease (P = .05) was seen in the plasma metric. The latter was caused by the increase in FAs from the meal in the plasma which reduced the n-3 fraction as a percentage of the total. In the present study, the percentage of EPA 1 DHA in plasma increased by approximately 20% after the meal. This is because a large amount of n-3 FA was given with the test meal. Although a large dose of EPA 1 DHA as given here can begin to affect the RBC level after 24 hours, it generally takes 12 to 16 weeks for the EPA 1 DHA level in RBCs to reach a new steady state after a sustained increase in intake10; plasma (phospholipid) levels stabilize in approximate 6 weeks.11 The findings of the present study compliment these longer term data and indicate that the RBC-based metric is not only more stable over time when n-3 FA intake is unchanging but is also essentially unaffected by an acute dose of n-3 FA.

The difference in the FA composition of plasma and RBCs is well known.12-14 Relative to the former, the latter

Figure 1 Effects of a single dose of EPA 1 DHA (3.4 g) taken with breakfast on absolute EPA 1 DHA levels in plasma (expressed as a percentage of total fatty acids, top; and expressed as concentrations, middle) and in RBCs (expressed as a percentage of total fatty acids; bottom) over 24 hours. Individual patient responses (n = 20) are shown (gray lines) and group means with 95% confidence intervals (black lines). DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; RBC, red blood cell.

Figure 2 Effects of a single dose of EPA 1 DHA (3.4 g) taken with breakfast on the percentage of change from baseline (hour 0) in EPA 1 DHA levels in plasma (expressed as a percentage of total fatty acids, top; and expressed as concentrations, middle) and in RBCs (expressed as a percentage of total fatty acids; bottom) over 24 hours (n = 20). Mean estimate and 95% Dunnett-adjusted confidence intervals are shown. *Difference from baseline, P < .05. DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; RBC, red blood cell.

are enriched in PUFAs (both n-6 and n-3) because as membranes, they are comprised almost entirely of phospho-lipids, the lipid class that carries the greatest proportion of long-chain PUFAs.12 With respect to their responses to changes in n-3 FA intakes, all blood-based pools (and cardiac tissue3,4) become enriched with higher intakes to roughly equal extents.15

Although recent meta-analyses of randomized trials have questioned the value of n-3 FA supplementation for reducing the risk of coronary heart disease,16,17 the epide-miologic evidence for a benefit from higher intakes of n-3 FA (which are better correlated with RBCs than

18 19 20

plasma ) are robust. , Indeed, several studies have specifically linked RBC n-3 FA levels with cardiovascular disease (CVD) outcomes. Most notably, in 1995 Siscovick et al2 were the first to document a strong correlation with RBC EPA 1 DHA and primary cardiac arrest. In 2002, Albert et al21 showed that whole blood EPA 1 DHA (which is highly correlated with RBC levels1) was an independent predictor of risk of sudden cardiac death. Building on this

foundation, the Omega-3 Index was proposed in 2004 as a marker of risk of death from CVD.1,22 This particular assay method has now been widely used in a variety of studies that examined not only CVD but also depression and cognitive function.23-39 Others that used different analytical methods have reported RBC n-3 FA associations with other conditions. For example, in a large cohort in China, RBC DHA was strongly and inversely associated with risk of multiple forms of chronic disease.40 RBC FA patterns have been related to atrial fibrillation,41 longevity,42 literacy/attention deficit hyperactivity disorder,43 breast cancer in Japan,44 prostate-specific antigen levels in Jamaica,45

cognitive function,46,47 hostility,48 eating disorders,49 and

dyslexia.50 Relatively few studies have examined the relations between plasma FA percentage of composition and CVD outcomes, and most studies have come from Ja-pan.51-58 Only one study has compared the percentage of composition of n-3 FA in plasma and RBCs as predictors of coronary heart disease, in this case for nonfatal myocar-dial infarction.58 The former was generally better than the

Figure 3 Levels of EPA 1 DHA in whole plasma (upper) and RBCs (lower; both in percentage of composition) measured in the fasting state once a week for 6 weeks in 21 healthy volunteers. DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; RBC, red blood cell. Data from Harris and Thomas.5

Finally, there is a place for both plasma and RBC n-3 FA analyses (and for whole blood, if it is the only sample type available21,28). As noted earlier, both could potentially be used in population research studies (if fasting status is known) because mean levels are reasonably stable, and these 2 markers are well correlated (particularly as percentages, r = 0.90; not as well for RBC percentage vs plasma concentration, r = 0.73; unpublished observations). Plasma also responds more quickly to a change in n-3 FA intake than does the RBC pool.11 The RBC method, however, has at least 4 characteristics that favor its use in the clinical setting: low within-subject variability, resistance to artificial elevation after an acute n-3 FA dose, much longer half-life,10 and in-sensitivity to fasting status. In addition, the amount of published research that links RBC n-3 FA levels with clinically relevant outcomes is greater than that for plasma. Hence, RBC-based therapeutic targets have a clinical relevance not enjoyed by plasma-based targets.

Conclusion

In conclusion, the RBC fraction appears to be the

preferred sample type for the assessment of n-3 FA biostatus.

latter, but their RBC assay produced EPA and DHA levels that were roughly equal, which is highly suspect as DHA is typically 6-8 fold higher than EPA in RBCs (24-26). This suggests co-elution of a contaminant with EPA in the analysis, making the RBC data questionable in this study. A recent study from the Cardiovascular Health Study that focused on plasma phospholipid n-3 FA percentage (another analyte that correlates well with the RBC metric15) reported a significant inverse relationship between n-3 FA levels and total mortality.59

As alluded to earlier, many of the RBC-based studies23-39 have used the identical laboratory method,8 whereas avariety of methods were used in the plasma-based studies. Without formal interlaboratory comparison, there is no guarantee that values with one method will agree with values of another method; even RBC-based methods vary in output.60 It has been shown that methodologic differences in just the pre-chromatography phase of FA processing can lead to different

values.61

The results of this study have practical implications. For example, most patients are told to increase their intake of oily fish or to start taking fish oil supplements after a blood test reveals suboptimal n-3 FA levels. Although some will comply, others will not but may choose to take a single large dose of fish oil within 24 hours before their appointment in an effort to improve their blood levels and thereby appear to have been compliant. When their blood is drawn at the appointment, their plasma EPA + DHA level will indeed be elevated and could easily mislead the physician, but the RBC level should be unchanged, reflecting the true chronic condition.

Acknowledgments

The authors thank Monique Alston, Kim Slominsky, and Ali Baedeke for their valuable assistance in the planning and execution of this study. This study was supported by Health Diagnostic Laboratory, Inc.

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