Biomarkers of DHA status

ARTICLE IN PRESS Prostaglandins, Leukotrienes and Essential Fatty Acids 81 (2009) 111–118

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Biomarkers of DHA status Connye N. Kuratko , Norman Salem Jr. Martek Biosciences, Inc., 6480 Dobbin Road, Columbia, MD 21045, USA

abstract Docosahexaenoic acid (DHA, 22:6n-3) is a long chain omega-3 fatty acid that is the primary n-3 fatty acid found in the central nervous system where it plays both a structural and functional role in cells. Because the tissues of interest are generally inaccessible for fatty acid analysis in humans and because precise DHA intake is difficult to determine, surrogate biomarkers are important for defining DHA status. Analysis of total lipid extracts or phospholipids from plasma or erythrocytes by gas chromatography meet the criteria for a useful biomarker of DHA status. Furthermore, both plasma and erythrocyte DHA levels have been correlated with brain, cardiac, and other tissue levels. Use of these biomarkers of DHA status will enable future clinical trials and observational studies to define more precisely the DHA levels required for either disease prevention or other functional benefits. & 2009 Elsevier Ltd. All rights reserved.

1. Introduction

2. Biological markers

Docosahexaenoic acid (DHA, 22:6n-3) is well documented as the primary omega-3 long chain polyunsaturated fatty acid (n-3 LCPUFA) in the central nervous system. DHA comprises over 97% of n-3 PUFA in brain and is concentrated in aminophospholipids of cell membranes [1–3]. In the retina, over 95% of n-3 PUFA is DHA and this high concentration appears to be essential for optimal neuronal and retinal function [3,4]. The unique motional and biophysical properties of the DHA molecule as a part of the neuronal lipid membrane appears to play a major role in nerve function, a role for which other polyunsaturated fatty acids cannot substitute [5–8]. As a component of heart tissue membranes, DHA may function independently or in combination with eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6) to influence endothelial function and heart rate variability [9]. DHA is a component of all mammalian cell membranes. Clearly, the level of DHA accretion is under control of the specific tissue and cell type, but it also reflects dietary intake [10]. Therefore, an understanding of the relationship of DHA intake to the distribution of DHA in tissues is essential to the understanding of dietary influence on the functional and health outcomes of the brain, eye, and heart. Since access to these tissues is problematic in even the most sophisticated clinical trials, intermediate biomarkers of DHA status are needed.

The National Cancer Institute defines a biomarker as a biological specimen that may be a marker of exposure to a substance, the metabolism of the substance, or the interaction of exposure and metabolism that has a known relationship to the risk of disease [11]. Nutritional biomarkers serve not only as measures of dietary intake, but also as indicators of nutrient metabolism [12]. Characteristics for choosing useful nutritional biomarkers have been described by several authors [12–14]. Acceptable biomarkers for fatty acid status are ones for which: (1) the method of biomarker measurement is standardized, specific and sensitive; (2) the biological material used for biomarker determination is easily obtainable; (3) a correlation between the nutrient biomarker and intake of the nutrient is established; (4) the relationship of the biomarker status and nutrient intake is sensitive and specific; and (5) the biomarker status shows an association with important clinical outcomes. These characteristics are discussed with regard to the use of plasma phospholipid DHA and erythrocyte DHA as biomarkers of DHA status.

 Corresponding author. Tel.: +1 443 542 2552; fax: +1 410 997 7789.

E-mail address: [email protected] (C.N. Kuratko). 0952-3278/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2009.05.007

2.1. The method of measurement is standardized The measurement of the relative proportions of individual fatty acids in plasma or serum and erythrocytes has become a common and well-documented analytical procedure. These procedures generally involve separation of the lipid fraction of interest [15,16], transmethylation reaction to form fatty acid methyl esters, identification of individual fatty acids via peak identification using gas chromatography and a known standard [17], and the quantification of the DHA peak area [18,19]. Peak areas may be converted to weight % with appropriate response factors where

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Table 1 Studies reporting correlation coefficients between specified DHA intake and plasma or erythrocyte DHA. Citation

DHA source

N

DHA intake (g/d)

Plasma/serum DHA levels (mmol/L)

RBC DHA levels

Correlation coefficient

p-Value

Confidence interval

Andersen [39]

FFQ

0.46 (median)

2.91

–

0.52

o0.001

0.39, 0.63

Kobayashi [40]

Dietary Record (4, 7-day weighed records)

87

0.9

7.3 (mean) 4.1–11.3 (range)

–

0.43

o0.001

–

Kuriki [41]

Dietary Record (7 day, weighed record)

94

0.62670.299 (men) 0.247 (women)

4.2701.11 (men)

0.574 (men)

o0.05

–

4.970.13 (women)

0.303 (women)

o0.05

FFQ

65

Lucas [42]

125

–

–

0.36

o0.05

–

–

–

0.40

o0.05

–

0.0, 0.52, 1.04, or 1.56 g/d,

–

–

0.71 at 6 wk 0.72 at 12 wk

o0.001 o0.001

–

0.14 (men) 0.16 (women)

–

–

0.42

o0.01

– –

Milte [43]

Fish oil

Ma [44]

FFQ

3570

Norrish [45]

FFQ

480

0.11

–

–

0.32

–

Poppitt [46]

High SFA diet x 21 d Crossover High USFA x 21 d

20a

0.570.1

–

2.7770.51

0.369 (Day 21)

o0.05

20a

0.670.1

–

2.8270.53

0.394 (Day 21)

o0.05

53

0.134

2.02 (0.71 SD)a

4.06 (1.12 SD)a

0.48 (Plasma) 0.39 (RBC)

o0.001 (Plasma) o0.01 (RBC)

306

a

0.48 (Plasma) 0.56 (RBC)

o0.01 o0.01

Sullivan [47]

Sun [48]

a

FFQ

FFQ

67

0.11570.97 (calc from total fish) 0.12870.99 (total marine)

0.29

1.56

a

a

3.71

% Total fatty acids.

required, or to concentrations when referenced to an internal standard. Inaccuracies can result from improperly handled samples; poor recovery of the lipid fraction; or poor analytical technique in using the gas chromatograph. In general, however, the methodology is widely used, well understood, and is currently undergoing adaptations that will allow high-throughput protocols and fast gas chromatography as well as streamlined procedures for sample collection and storage [20,21].

2.2. The method of measurement is specific and sensitive The method of gas chromatography is both specific and sensitive for the determination of individual fatty acids, including DHA. Modern capillary columns are highly efficient and provide for complete separation of DHA from other fatty acids [20,21] for measurement of values as low as 0.01 wt%. Gas chromatography is also specific, capable of distinguishing DHA as a separate and unique peak when compared to known standards.

2.3. Biological material is easily obtainable DHA status of the central nervous system and other internal organs is of primary interest, but is not accessible under most study conditions. The biomarkers of DHA status most frequently used and most accessible are blood components which include total lipid and phospholipid-rich extracts of plasma and erythrocytes. Less frequently used tissues and blood components for

biomarker determination include adipose tissue, buccal cells, sperm, platelets and leukocytes [22–28]. DHA levels are lower in plasma and erythrocytes than in neural tissues. Plasma levels of DHA are approximately 2–6% compared to 14% or more in parts of the brain [1]. In plasma, DHA is divided unequally between a large lipoprotein pool and a smaller nonesterified fatty acid pool, representing 4–6% of the total [29]. Lipoprotein-carried DHA is found esterified to phosphatidylcholine, phosphatidylethanolamine, cholesterol, and in triglycerides [29]. These pools of DHA are purported to supply DHA for brain, retina, liver, and other tissues. Research is ongoing to determine the effectiveness of each form as a supply for tissue accretion [30–32]. Study designs have reported the time course for DHA incorporation and washout in plasma and/or erythrocytes. In 1997, Katan et al. fed 58 healthy men varied doses of DHA in fish oil capsules for 12 months followed by a 6 months washout period. The study reported DHA incorporation, along with other fatty acids, into cholesterol esters, erythrocyte membranes, and adipose tissue [10]. DHA incorporation into cholesterol esters reflected DHA intake over the previous 1–2 weeks period; erythrocyte DHA reflected intake over the past 1–2 months; and adipose tissue over a period of years. The 6-month washout period planned in the study was not adequate for a return of DHA to baseline in the two high-dose groups [10]. Other investigators have also found that a prolonged wash out period is required following DHA supplementation [33–35]. Skeaff et al. [36] demonstrated that the time course for DHA accretion was similar in both plasma and erythrocytes. Red blood

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cells have a lifespan of approximately 90 d and reflect the dietary intake for many nutrients over that time period. This may not be the case for lipids, however. The half-life of many fatty acids in the membranes of erythrocytes is relatively short, 7–9 h [37,38]. The absolute quantity of fatty acids differs in erythrocyte membrane and plasma, but an exchange of fatty acids continuously occurs during circulation. Erythrocyte fatty acids may respond to diet in a very similar time course as plasma [36,38].

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system based on Norwegian food tables. The subjects also provided 14-day weighed diet records for comparison. The Spearman correlation coefficient between the estimated DHA intake from the questionnaire and serum DHA was 0.52 (95% CI 0.39–0.63, po0.001) at a mean daily DHA intake of 460 mg/d (0.43% of total dietary fat). Kobayashi et al. [40] used multiple 7-day weighed dietary records from 87 male subjects in Japan to estimate DHA intake from Japanese food composition tables. The mean intake was 900 mg DHA/d (1.7% total fatty acids). The mean serum phospholipid DHA composition was 7.3% of total fatty acids. Pearson’s correlation of DHA intake expressed as % total dietary fatty acids in serum was 0.61 (po0.001). Both Kobayashi and Andersen found a stronger correlation between serum levels and dietary DHA expressed as percent of the total fat in the diet than as DHA expressed as a level of intake. The subjects in Japan not only had a greater overall DHA intake than the Norwegian subjects described by Andersen, but also a 3-fold higher DHA intake expressed as percent of the total fat in the diet. These factors may play a significant role in the ultimate correlation with blood parameters. Kuriki et al. [41] estimated DHA intake from 7-day weighed diet records from 94 Japanese dietitians, 15 males and 79 females. The mean DHA intake was 626 mg/d in men and 571 mg/d in women, representing 1.28% and 1.26% of total dietary fatty acids, respectively. The intake was significantly correlated with plasma DHA levels, r ¼ 0.574 (men) and r ¼ 0.303 (women) when expressed as an absolute dietary intake and r ¼ 0.681 (men) and r ¼ 0.379 (women) when DHA is expressed as % of total dietary fatty acids. Lucas et al. [42] estimated DHA intake from a food frequency questionnaire using the Canadian Nutrient Data File in 65 middleaged women. The DHA intake from total marine sources in these subjects was positively correlated with erythrocyte DHA, r ¼ 0.40 (po0.05). Milte et al. [43] supplemented 67 adult men and women with 0, 520, 1040, or 1560 mg DHA/d. Correlation of DHA intake and erythrocyte DHA composition was highly significant (r ¼ 0.72, po0.001). Ma et al. [44] estimated DHA intake from a 66-item food frequency questionnaire administered to 3570 free-living middleaged adults. Dietary DHA, expressed as percent of total fatty acids, was correlated with plasma phospholipid DHA (r ¼ 0.42, po0.01).

2.4. Correlation with dietary intake is established Most studies designed to describe a clinical outcome related to DHA intake show its relationship to blood biomarkers. For this review, a systematic search was conducted to obtain studies specifically designed to report a correlation or dose effect of DHA intake with blood biomarker status. This search included human studies in the PubMed database through January 12, 2009 and included the following terms: docosahexaenoic acid; biological markers; docosahexaenoic acid/administration and dosage/analysis/ blood; erythrocytes/chemistry; erythrocyte membranes/chemistry; fatty acids, unsaturated/analysis, blood; fatty acids, omega-3/ analysis, blood. The search yielded 97 citations which reported results in plasma and 99 citations which reported results in erythrocytes. A first level screening for relevancy included only those articles which reported data for: healthy human subjects (exclusion of studies in disease populations); a defined level of DHA intake (excluding reports of fish intake only); a defined level of plasma or erythrocyte composition (reported numerically or graphically); and statistical analyses for dose effect or correlation. Only triglyceride sources of DHA supplementation were considered. The results of the overall search appear in Tables 1–3. Table 1 shows the results of studies which reported correlations between DHA intake and erythrocyte or plasma DHA concentration. Eight of the nine studies report the correlations to be statistically significant and showing a positive relationship with correlation coefficients ranging from 0.32 to 0.72. One study did not report the statistical significance of the correlation. Andersen et al. [39] measured DHA concentration in total serum lipids of 125 Norwegian men. DHA intake was estimated from a 180 item food frequency questionnaire using a software

Table 2 Studies reporting effect of DHA dose on erythrocyte DHA (mmol/L). Reference

DHA (g/d)

Barcelo-Coblijn [49]

0.126 0.252

Brown [34,35]

Baseline

Time 1

Time 2

Time 3

Time 4

Time 5

Time 6

p-Value

3.870.6 4.070.5

3.970.6 3.870.6

3.870.5 4.270.4

o0.05 o0.05

3.270.7 3.070.5

3.670.7 3.570.7

3.770.6 3.870.5

3.870.6 4.170.4

0.41 0.99 0.58

5.2971.29 5.6971.46 4.7770.93

5.8371.25 6.5071.34 6.1970.66

6.2471.13 6.8771.31 –

6.6071.27 7.3371.13 –

– – –

– – –

– – –

o0.001 o0.001 o0.001

Cao [50]

0.864

Graphical data

–

–

–

–

–

–

o0.001

Geppert [52]

0.94 0.94 0.94

4.470.2 6.570.3 1.3870.07

7.970.2 12.170.3 3.7870.13

Total lipids PE PC

– – –

– – –

– – –

– – –

o0.001 o0.001 o0.001

Katan [10]

0.16 0.33 0.49

4.670.6 4.770.6 4.470.4

4.670.5 4.870.5 4.870.5

– – –

– – –

– – –

– – –

– – –

– – –

Milte [43]

0.52 1.04 1.56

Graphical data Graphical data Graphical data

– – – – – – – – DHA rose by 78% with the 6 g/d of DHA-rich fish oil

– –

Vidgren [53]

0.15 Fish 1.68 DHASCO 0.95 FO

– – –

– – –

NS o0.05 NS

Graphical data Graphical data Graphical data 3.070.5 3.270.9 3.270.6

Graphical data Graphical data Graphical data 4.570.1 6.171.2 4.770.8

– – –

– – –

– – –

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Table 3 Studies reporting effect of DHA dose on plasma or serum DHA. Reference

DHA (g/d)

Baseline

Time 1

Time 2

p Value

Cao [50]

0.864

Graphical data

Conquer [51]

0.75 1.5 0.75 1.5

2.470.2 2.770.5 0.3870.5 0.2970.08

5.470.2 6.370.7 0.7470.15 1.770.8

Geppert [52]

0.94

2.870.1

7.470.2

o0.001

Katan [10]

0.16 0.33 0.49

0.670.2 0.670.2 0.570.2

0.970.2 1.170.3 1.270.2

– – –

Vidgren [53]

0.15 Fish 1.68 algal oil 0.95 fish oil 0.15 Fish 1.68 algal oil 0.95 fish oil

0.670.2 0.570.2 0.670.1 3.870.8 3.570.9 3.470.6

0.970.3 1.070.3 1.170.2 5.271.2 6.571.3 5.570.8

o0.0014 o0.0014 o0.0014 o0.014 o0.0014 o0.0014

o0.0001 6.470.3 7.970.4 1.370.2 2.270.4

o0.05 o0.05 o0.05 o0.05

 vs baseline. 4

vs control.

Norrish et al. [45] recorded DHA intake and erythrocyte phosphatidylcholine levels in controls as well as cases in a study designed to determine the relationship between prostate cancer risk and DHA. In the controls (n ¼ 480), a correlation of r ¼ 0.32 was obtained between erythrocyte phosphatidylcholine DHA and daily intake of DHA as estimated from the amount of total fish consumed. Poppitt et al. [46] reported erythrocyte fatty acids in 20 healthy men on a strictly controlled diet containing 40% energy from either a high saturated fat or high unsaturated fat diet. The high saturated fat diet contained 500 mg/d DHA and the high unsaturated fat diet contained 600 mg/d DHA. Pearson correlations between diet and erythrocyte DHA composition after 21 days on either the high saturated fat diet or high unsaturated fat diet were 0.369 and 0.394, respectively (po0.05). Sullivan et al. [47] reported both red blood cell and plasma DHA as part of a study to validate a food frequency questionnaire. There were significant Spearman’s correlation coefficients between the DHA intake calculated from the food frequency questionnaire and erythrocyte and plasma DHA, r ¼ 0.39 and 0.48, respectively (po0.01 and o0.001). Mean DHA intake calculated from the food frequency questionnaire in the 53 subjects was 109 mg/d. Sun et al. [48] estimated the DHA intake of 306 US women from a food frequency questionnaire. DHA in erythrocytes and plasma were measured and correlated with intake. Erythrocyte DHA concentrations were better correlated with intake (r ¼ 0.56) than were plasma DHA concentrations (r ¼ 0.48). The mean DHA intake of these subjects was 0.29% of total fat. As a group, these studies show significant and positive correlations between DHA intake and blood parameters. Furthermore, DHA appears to show the strongest correlation when considerations are made regarding the background diet and correlations are made with DHA as a percent of total dietary fatty acids. Tables 2 and 3 show results from studies which reported a dose–response relationship between DHA intake and plasma/ serum or erythrocyte DHA. In a study designed to define any potential bioequivalence of flaxseed oil and fish oil, BarceloCoblijn et al. [49] supplemented firefighters in North America with 126 or 252 mg DHA/d from fish oil (n ¼ 11 and 10, respectively) for 12 weeks. The fatty acid composition of erythrocyte membrane was reported on weeks 0, 2, 4, 6, 8, 10,

and 12 of the experiment. At both levels of fish oil supplementation, erythrocyte DHA was significantly different from baseline by week 4 at a level which persisted throughout the supplementation. However, there were no statistical differences in erythrocyte DHA according to the level of supplementation. Brown et al. [35] supplemented healthy men with 410 or 990 mg DHA/d from fish or a combination of fish and fish oil for 6 weeks (n ¼ 4 per group). Both doses resulted in a significant increase in erythrocyte DHA composition. The crossover design of the study demonstrated that greater than 18 weeks was necessary for erythrocyte DHA levels to return to baseline following supplementation. In 2006, Cao et al. [50] supplemented healthy adults with 864 mg/d DHA from fish oil and measured plasma phospholipid and erythrocyte DHA. Plasma phospholipid DHA increased 1.9% per gram of dietary DHA by 8 weeks, the majority of which occurred by 4 weeks. Individuals with high baseline DHA incorporated additional DHA at a slower rate than those with low initial levels. DHA concentrations tended to plateau at 6%. The loss of DHA following the discontinuation of supplementation occurred in 2 phases; a steeper slope occurred in the first 2 weeks followed by a more gradual decline over the subsequent 6 weeks. In plasma phospholipids, DHA showed a greater increase and a more rapid washout period than in erythrocytes. Erythrocyte membranes, therefore, appeared to be a better index for monitoring long-term intake of omega-3 fatty acids, whereas plasma phospholipids were more sensitive to short-term change. Conquer et al. [51] supplemented subjects with 0.75 or 1.5 g DHA/d for 6 weeks (n ¼ 6 and 7, respectively). The DHA serum phospholipid levels were determined at baseline and at week 3 and week 6 of the supplementation. Both groups had a higher concentration of DHA at 3 weeks; and at week 6 the high DHA group had higher serum phospholipid DHA levels than the lower DHA group. The additional 0.75 g in the high DHA group resulted in an additional 23% rise in DHA over the lower DHA group. Results indicated that serum phospholipid levels began to plateau at the higher dose. Geppert et al. [52] supplemented 55 healthy vegetarians with 940 mg DHA/d for 8 weeks. This level significantly increased DHA in total lipid, phosphatidylethanolamine, and phosphatidylcholine fractions of erythrocytes. Katan et al. [10] used fish oil to supplement subjects with 171, 342, or 513 mg DHA/d for 12 months. The incorporation of DHA into erythrocytes, compared to EPA, was somewhat erratic but from extrapolated data in the report, DHA increased an additional 1% in total fatty acids for each gram consumed. Vidgren et al. [53] supplemented 59 healthy males with 0, 90, 950, or 1680 mg DHA/d from a variety of sources. After 14 weeks, each of the three groups fed some form of DHA showed increased DHA in plasma phospholipids. DHA incorporation into erythrocytes, however, was variable.

2.5. Specificity of the relationship between DHA intake and plasma or erythrocyte status In defining the specificity of a biomarker for DHA intake, the degree to which the precursors ALA and EPA support DHA content must be considered. Several published reviews concerning the metabolism of ALA to EPA, DPA n-3, and ultimately DHA are consistent in their findings [54]. ALA shows only minimal conversion to DHA in humans. While this conversion prevents total depletion of DHA from blood lipids, supplementation with additional ALA fails to increase DHA levels. Similar results are seen with increased intake of EPA or other precursor n-3 fatty

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acids. Only DHA in the diet results in a significant increase in blood and tissue levels of DHA [54].

2.6. Sensitivity of the relationship between DHA intake and plasma or erythrocyte status Vidgren calculated a dose–response curve based on mg/ml plasma phospholipid DHA of three groups given various amounts of DHA from different sources. Conversion of ALA to a small amount of DHA was apparent, as DHA was measurable even with zero dietary intakes. The curve was steepest with low intakes of DHA and followed a logarithmic form; the higher the intake of DHA the lower the proportional increase in concentration of DHA in plasma phospholipid [53]. This type of dose–response is in agreement with data presented by Arterburn et al. in 2006 [55]. Arterburn reported plasma phospholipid fatty acid concentrations from 16 different supplementation groups ranging from 0.2 to 6 g DHA/d for 1–6 months duration. Results demonstrated that plasma phospholipid DHA concentrations increased in a dose-dependent manner up to doses of approximately 2 g/d. Above that dose, plasma DHA concentrations approached saturation but continued to increase incrementally. The author created a cross-study meta-regression, dose–response analysis of the effect of DHA supplementation on plasma phospholipid DHA concentrations in those 16 supplementation studies. A logarithmic curve fit was generated from the changes from baseline. The equation for the curve was y ¼ 1.742 ln(x)+4.023, r2 ¼ 0.91 [55]. These studies confirm that both erythrocyte and plasma phospholipid DHA rise in relation to dietary intake. Significant differences are seen compared to baseline with as little as 126 mg supplemental DHA/d after 4 weeks. Few of the studies, however, reported the precise differences between doses. The true relationship between DHA intake and incorporation into plasma phospholipids appears to reflect a logarithmic curve. The curve never achieves a true ‘zero’ due to a minimal amount of DHA conversion from ALA and from low level intake of DHA from various food sources. The proportional increase in plasma DHA as the result of DHA intake is greatest at lower intakes and appears to plateau at an intake of approximately 2 g DHA/d. Several environmental factors influence the sensitivity of blood biomarkers for DHA intake. First, a precise intake of DHA is often difficult to define. Intake from fish, the primary source of dietary DHA, is often difficult to accurately describe. Cold-water fatty fish such as tuna and salmon provide as much as 200–400 mg per ounce of cooked fish compared to a lean white fish which may only provide 50 mg or less of DHA per ounce. A fish sandwich from a fast food restaurant may provide negligible amounts of DHA [56]. Second, national nutrient databases are incomplete and inaccurate with regard to long chain polyunsaturated fatty acid composition, making it difficult to accurately assess DHA intake using these resources. For example, red meats including organ meats, do not report DHA values in many large databases. In addition, DHA is added to foods, animal feed, and supplements, making a precise estimate of dietary intake difficult [56]. The food matrix in which DHA is consumed does not appear to affect DHA status in blood parameters. Arterburn et al. [18] recently reported bioequivalence of DHA from food and supplement. In the study, DHA obtained from an algal oil supplement was compared to the same amount of DHA provided in cooked salmon. Both plasma and erythrocyte DHA levels were measured using gas chromatography. The food and supplement matrix provided DHA equally when measured in erythrocyte or plasma phospholipids.

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Other environmental/dietary factors lead to a decrease in DHA intake or absorption, an increase in its turnover and loss within tissues, or an increase in DHA excretion. Such factors include alcohol consumption [57,58], consumption of trans fats [59], and smoking [60]. Recent data suggests that individual genetic variations influence DHA status as well as intake. Evidence shows an association between variants of the fatty acid desaturase 1 enzyme (FADS1) and desaturase 2 (FADS2) on deposition of long chain polyunsaturated fatty acids into plasma phospholipids, erythrocyte membranes, and breast milk lipids [61]. Polymorphisms of the FADS gene cluster with minor allele homozygote and heterozygous pairs are associated with varying degrees of long chain n-3 and n-6 fatty acid endogenous production. To date, there is no evidence to support an increase in ALA to DHA conversion resulting in increased DHA status in subjects with modified FADS gene cluster. Similarly, there is no evidence to support significant decreases in blood DHA levels as the result of a partial inhibition of ALA conversion to longer chain n-3 fatty acids. Studies show that intake of preformed DHA has the largest influence on DHA status since conversion of ALA to DHA is minimal in humans [54].

2.7. Blood biomarkers of DHA status may correlate with brain DHA composition Although brain growth and remodeling may be relatively inactive during adulthood, the human brain is able to tenaciously retain DHA within its membrane structures throughout life. A continuous turnover of DHA occurs in the adult [31]. There are functional outcomes associated with low levels of DHA in the adult brain, whether induced experimentally in animals or as the result of disease in humans. Low blood levels of DHA are associated with an increased risk for disease, including those of neurological [62–64] and psychiatric etiology [65,66]. In a series of studies, Pawlosky et al. fed a diet containing 0.5% ALA and no DHA to juvenile Rhesus monkeys. In addition, half of the monkeys were placed on a protocol which included alcohol consumption. The authors reported data which show brain cortex, liver, and erythrocyte DHA levels decreased with age in those primates that received a diet devoid of DHA. Chronic alcohol consumption led to further DHA decreases. Although a statistical correlation was not reported, it is evident that a positive relationship exists between erythrocyte DHA levels and tissue levels, including the brain cortex and retina [68,69]. These studies demonstrated that although adult brain DHA content is often said to be tenaciously retained, it is still possible to alter adult primate brain DHA by diet. Sarkadi-Nagy reported the results of a study in baboon neonates from fish oil-supplemented mothers which included both term and pre-term neonates [67]. The neonates were assigned to varying feeding groups including breast milk, formula lacking LCPUFA, and formula containing LCPUFA. At 14 days, C13labeled ALA, or LNA were administered and brain, heart, and liver tissues were analyzed for DHA content. Both erythrocyte and plasma levels were correlated with resulting DHA content in brain, retina, and liver. For brain accumulation in baboon neonates, the brain tissue correlation using erythrocyte biomarkers was r2 ¼ 0.86, whereas the correlation with plasma levels was only r2 ¼ 0.58. This reasonable difference suggests that the erythrocyte levels may provide a significantly more efficient biomarker for the actual accumulation of DHA in the neonate brain. Some [67] neurological/cognitive changes associated with aging may be the result of a net loss of DHA during adulthood. In 1991, Soderberg et al. reported the results of autopsies of people

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who died as the result of Alzheimer’s disease (AD). Phosphatidylethanolamine-DHA composition of the frontal gray matter, white matter and hippocampal tissue in these individuals was approximately half of that from subjects without AD [70]. Epidemiological studies report that an increase in fish consumption decreases the risk for development of AD [71,72]. Investigators have successfully used blood biomarkers of DHA status in their studies of AD. In 2006, Schaefer et al. reported that the highest plasma phosphatidylcholine-DHA composition correlated with a 47% reduction in risk of all-cause dementia among 899 subjects from the Framingham cohort [64]. And in 2004, Whalley et al. studied 364 healthy elderly without signs of dementia taken from a cohort of 2000 who had been studied earlier for childhood IQ. The investigator reported that plasma phospholipid DHA was a significant predictor of IQ at age 64 [73]. 2.8. Blood biomarkers of DHA status correlate with cardiac DHA composition Some of the earliest studies relating consumption of fish (long chain omega-3s) and mortality from heart disease were conducted in the Inuit population of Greenland. These studies related higher levels of EPA and DHA in plasma and platelets with reductions in death from heart disease. Today there is a large volume of literature which addresses several mechanisms whereby the n-3 LCPUFA exert their beneficial effects on the cardiovascular system. Particularly regarding the effect that n-3 LCPUFA has on reductions in risk for arrhythmia and sudden cardiac death, the effect may be on the cardiac tissue itself. As with brain tissue, the DHA content of various blood components show close correlation with cardiac DHA. A study by Harris et al. showed that the DHA composition of erythrocytes was highly correlated with the cardiac DHA composition (r ¼ 0.84, po0.01) in subjects who were low and high consumers of n-3 LCPUFA [74]. Additionally, a study by Metcalf et al. was designed to determine the uptake of DHA and EPA in the atrium, erythrocytes, and plasma phospholipids over time. In that study, erythrocyte DHA matched the pattern of change seen in the ultimate accumulation of atrial phospholipids, however the rate of accumulation was much higher initially in the atrial tissue [75]. 2.9. Other n-3 LCPUFA biomarkers In a recent publication, Courville et al. [76] demonstrated strong correlations between maternal plasma, maternal erythrocyte, and cord blood DHA levels (r values ranged from 0.633 to 0.376, depending upon the parameters compared). The results indicate that either plasma phospholipids or erythrocyte phospholipids are important assessment biomarkers of DHA status in maternal and infant nutrition. A mathematical model developed and reported by Lands et al. can be applied to dietary n-3 and n-6 intake for prediction of the fatty acid composition of plasma phospholipids [77]. By using an equation which accounts for the competition of n-3 and n-6 fatty acids, this method predicts plasma fatty acid levels including total n-3 highly unsaturated fatty acids from a known fatty acid intake; or, in reverse, can predict dietary intake from know plasma levels. The model was developed and coefficients assigned so as to fit empirical data generated by nutritional studies of animals and humans [78]. The facility for making such calculations for correlation between diet and fatty acid composition are currently available to the public at http://efaeducation.nih.gov. Epidemiological studies show a reduced risk of sudden cardiac death with increasing intake and blood levels of DHA and EPA [79]. Investigators have developed methods for defining a

relationship of long chain fatty acids that is predictive of cardiovascular disease risk. One such example is the ‘‘omega-3 index’’ as defined by Harris et al. [74,80]. This index expresses EPA plus DHA in erythrocyte membranes as a percentage of total erythrocyte fatty acids. The erythrocyte membrane is selected due to the ease of access and its reflection of tissue membrane structure. The omega-3 index, as well as EPA plus DHA in plasma and in cheek cells, was tested as a predictor for EPA plus DHA in cardiac tissue. Results showed that the correlation for erythrocytes was r ¼ 0.47, p ¼ 0.03, for cheek cells was r ¼ 0.49 and p ¼ 0.023, and for plasma levels was r ¼ 0.22, p ¼ NS [81]. A second method for defining n-3 LCPUFA status described by Stark et al [82] is a modification of the approach of Lands et al. [77,78] which includes a score for n-3 HUFA (HUFA ¼ highly unsaturated fatty acids; Z20 carbons and Z3 double bonds) [38,83,84]. The score is calculated as a percentage of n-3 HUFA in the total HUFA pool of a particular blood component or tissue. This calculation includes the concentration of n-6 HUFA as the equation’s denominator in order to reflect the competition that long chain n-6 fatty acids exhibit for the sn-2 position of the phospholipid. Results showed that the omega-3 status was more consistent between total lipid extracts and phospholipids when assessed by the n-3 HUFA index compared to the other methods. And, in animal models, the association was stronger between blood status and liver, heart and brain status when assessed by the n-3 HUFA index compared to the Omega 3 Index [38]. A recent publication by O’Brien et al. [85] reported a method of measuring a stable isotope found predominantly in large predatory fish as a predictor of EPA and DHA status. The d15, nitrogen marker was found to be highly correlated with erythrocyte EPA and DHA (r ¼ 0.83 and 0.75 respectively). With an increasing use of algal oil as a source of DHA, the value of this approach is yet to be determined. Both the Omega 3 Index and the n-3 HUFA index show procedural benefits of using total lipid extracts to eliminate the need for laboratory separation of the various phospholipids. This approach facilitates analysis using high throughput technology. Both of these methods are flawed, however, for use as exclusive biomarkers for DHA intake since they represent EPA as well as DHA in their calculation. They reflect DHA status, per se, only in that DHA provides the majority of dietary n-3 LCPUFA in most North American diets. This may not be the case, however, on an individual basis or in other countries. Both indices are based on DHA content of blood fractions measured by gas chromatography and both may prove valuable for use as clinical biomarkers of disease risk.

3. Conclusions An increase in dietary DHA correlates with both phospholipid and total lipid extracts of erythrocytes and plasma DHA content. In addition, erythrocyte and plasma DHA content are highly correlated; and in turn, these blood measures of DHA are predictive of internal organ DHA status. For the nervous system, the degree of correlation is dependent upon age with much better correlation occurring during early neural development. As demonstrated in baboons, during this period of early development, the erythrocyte may be a better biomarker of neonatal brain and retinal DHA status than is plasma DHA levels. In adults, DHA is generally well retained by the brain and retina, even in the face of dietary challenge. However, the adult nervous system is not completely impervious to dietary alteration of DHA status and, thus both plasma and erythrocyte DHA are useful biomarkers during adulthood as well. Conventional and fast gas chromatography provides sensitive and specific methods for measurement

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of DHA in blood samples. These techniques are amenable to high throughput analyses that may be used for large clinical trials and for observational studies. Future studies will further define the precise erythrocyte as well as the precise plasma DHA levels required for disease prevention or functional benefits of DHA intake.

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