Highly purified eicosapentaenoic acid and
`docosahexaenoic acid in humans have similar
`triacylglycerol-lowering effects but divergent effects on
`3
`serum fatty acids 1
`-
`
`Sameline Grimsgaard, Kaare H B¢naa, John-Bjame Hansen, and Arne Nord¢y
`
`ABSTRACT
`To compare the effects of highly purified ethyl
`ester concentrates of eicosapentaenoic acid (EPA) and docosa(cid:173)
`hexaenoic acid (DHA) on serum lipids. apolipoproteins, and serum
`phospholipid fatty acids in humans, we conducted a double-blind,
`placebo-controlled, parallel design intervention study. Healthy
`nonsmoking men (n = 234) aged 36-56 y were randomly assigned
`to dietary supplementation with 3.8 g EPNd, 3.6 g DHNd, or
`4.0 g com oil/d (placebo) for 7 wk. Serum triacylglycerols de(cid:173)
`creased 26% (P < 0.000 I) in the DHA group and 21 % (P =
`0.0001) in the EPA group compared with the corn oil group.
`Although not significant, net decreases in serum triacylglycerols
`were consistently greater in the DHA group across all quartiles of
`baseline triacylglycerol concentrations. Serum high-density-li(cid:173)
`poprotein cholesterol increased 0.06 mmol/L (P = 0.0002) in the
`DHA group. In the EPA group, serum total cholesterol decreased
`0.15 mmol/L (P = 0.02) and apo!ipoprotein A-I decreased 0.04
`g/L (P = 0.0003). In the DHA group, serum phospholipid DHA
`increased by 69% and EPA increased by 29%, indicating retro(cid:173)
`conversion of DHA to EPA. In the EPA group, serum phospho(cid:173)
`lipid EPA increased by 297% whereas DHA decreased by 15%,
`suggesting that EPA is not elongated to DHA in humans. The
`serum phospholipid ratio of n- 3 to n-6 fatty acids increased in
`both groups, whereas the relative changes in n-6 fatty acids
`suggested possible alterations in liver desaturation activity in the
`DHA group. We conclude that both DHA and EPA decrease serum
`triacylglycerols, but have differential effects on lipoprotein and
`Am J Clin Nutr
`fatty
`acid metabolism
`in humans.
`1997 ;66:649-59.
`
`KEY WORDS
`Fatty acids, n-3 fatty acids, eicosapent(cid:173)
`aenoic acid, docosahexaenoic acid, triacylglycerols, phospho(cid:173)
`lipids, randomized controlled trials
`
`INTRODUCTION
`
`oils varying in dosage form, total dose of fatty acids, and
`relative content of DHA and EPA. Examination of these data
`shows that the most consistent effect of n- 3 fatty acids on
`cardiovascular disease risk factors is a reduction in serum
`triacylglycerol concentration, whereas reported effects on
`other variables are less consistent (3-5). It is possible that
`the inconsistencies derive from chance findings in small(cid:173)
`scale studies or differences in study design. However, they
`may also be attributed to varying metabolic effects of DHA
`and EPA.
`Animal studies showed that EPA and DHA accumulate in
`different compartments in the body and thus may be subject
`to differences in both metabolism and effects (6--8). DHA
`selectively attenuated expression of proatherogenic and
`proinflammatory proteins in human endothelial cells, sug(cid:173)
`gesting a beneficial effect of DHA on atherosclerosis (9),
`whereas EPA may be a more potent platelet inhibitor than
`DHA (10, 11). In vitro studies indicate that EPA and DHA
`have different effects on triacylglycerol synthesis ( 12), and
`it was suggested that EPA is primarily responsible for the
`hypotriacylglycerolemic effect of n -3 fatty acids both in
`rats ( 13) and humans (14 ). The extent to which these reports
`can be generalized is constrained by limitations in study
`design, however. Knowledge of the specific effects of EPA
`and DHA is needed to target n- 3 supplements for specific
`effects. Long-term studies with adequate sample size com(cid:173)
`paring the biological effects of pure DHA and EPA in
`human volunteers have not been reported (10, 14-16). We
`therefore conducted a double-blind, randomized, placebo(cid:173)
`controlled, parallel design intervention study to evaluate
`effects of dietary supplementation with highly purified EPA
`or DHA on serum lipids, apolipoproteins, and serum phos(cid:173)
`pholipid fatty acid composition.
`
`Accumulating evidence indicates that fish oil, rich in
`eicosapentaenoic acid (EPA; 20:5n- 3) and docosahexae(cid:173)
`noic acid (DHA; 22:6n-3) of the n-3 family, can modify a
`variety of cellular processes associated with lipid metabo(cid:173)
`lism, atherosclerosis, hypertension, thrombosis, and inflam(cid:173)
`mation (I). The amount and the ratio of DHA to EPA in
`different marine sources vary considerably (I, 2). Earlier
`studies of n -3 fatty acid supplementation in humans used
`
`1 From the Institute of Community Medicine and !he Institute of Clinical
`Medicine, University of Troms0, Troms0. Norway.
`2 Supported by grants from the Norwegian Research Council. Pronova
`Biocare AS, and Odd Berg Medical Research Foundation.
`·' Address reprint requests to S Grimsgaard, Institute of Community
`Medicine, University of Troms!l. N-9037 Troms0, Norway.
`Received July 16. 1996.
`Accepted for publication April 18. 1997.
`
`Am J Clin Nutr 1997:66:649-59. Printed in USA. © 1997 American Society for Clinical Nutrition
`
`649
`
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`

`650
`
`GRIMSGAARD ET AL
`
`SUBJECTS AND METHODS
`
`Subjects and experimental design
`
`In 1986--1987, 21 826 subjects, 81.3% of the men aged
`20--0 I y old and the women aged 20--56 y old Ii ving in the
`municipality of Trams¢, participated in a health survey (visit I)
`( 17). All subjects completed a questionnaire about previous
`disease, use of drugs, and diet and smoking habits, and their
`height, weight, blood pressure, and nonfasting serum lipid
`concentrations were measured. Four hundred seven men be(cid:173)
`tween the ages of 35 and 55 were selected according to the
`following criteria: they reported being healthy nonsmokers, did
`not use nonprescribed or prescribed drugs, and consumed less
`than four fish dishes per week in their usual diet. They also had
`serum cholesterol concentrations < 8.0 mmol/L, diastolic
`blood pressure < 95 mm Hg, and systolic blood pressure <
`160 mm Hg. These men were then asked in 1993 to undergo a
`clinical examination that included a complete medical history,
`physical examination, and laboratory tests.
`Among the 349 men who responded to the invitation, 251
`subjects filled the above-mentioned criteria and were recruited
`into the present study. They had no cardiovascular, liver, or
`renal disease; bleeding disorder; diabetes mellitus; psycho(cid:173)
`pathologic disease; alcoholism; or other disease that can influ(cid:173)
`ence blood pressure, lipid metabolism, or hemostasis. They
`were not consuming special diets and did not expect to change
`their diet or lifestyle during the study period. Their mean
`(± SD) age was 44 ±. 5 y (range: 36--56). The study was
`approved by the regional board of research ethics, and each
`subject gave informed consent.
`The study was performed according to Good Clinical Prac(cid:173)
`tice requirements ( 18). It began with a 4-mo run-in period
`during which subjects were asked to continue their usual diet
`and living habits and during which their blood pressure and
`fasting serum lipid concentrations were measured on two oc(cid:173)
`casions (visit 2 and visit 3). Each subject's average intake of
`nutrients was calculated on a fourth visit. At the beginning of
`the run-in period and throughout the study, participants were
`instructed not
`to
`ingest cod
`liver oil or other fish-oil
`supplements.
`For entry into the intervention phase of the study, a subject's
`mean serum triacylglycerol concentration during the run-in
`period had to be < 5.0 mmol/L and mean serum cholesterol
`concentration< 9.5 mmol/L. Among the 251 subjects, 2 were
`smokers, 2 had serum glucose or triacylglycerol concentrations
`above the inclusion criteria, 2 used cardiovascular drugs, I
`consumed more than three fish dishes per week, and I 0
`dropped out during the run-in period for personal reasons.
`Thus, 234 men entered the double-blind, parallel group inter(cid:173)
`vention trial, which lasted for 7 wk. Computer-generated ran(cid:173)
`dom numbers were used to assign the participants to either
`4.0 g 95% ethyl ester EPA/d, 4.0 g 90% ethyl ester DHA/d, or
`4.0 g com oil/d. The dietary supplements were administered in
`indistinguishable soft gelatin capsules that each contained 1.0 g
`oil and 4-6 IU vitamin E as an antioxidant (Table 1). Each
`individual was asked to ingest two capsules in the morning and
`two capsules at night. The dietary supplements were manufac(cid:173)
`tured by Pronova Biocare AS, Oslo.
`Participants were examined after an overnight fast between
`0800 and 1130 on two separate occasions separated by an
`
`TABLE I
`Composition of dietary supplements'
`
`Constituent
`
`22:6n- 3 Ethyl ester (mg)
`20:5n-3 Ethyl ester (mg)
`18:2n-6 (mg)
`18:1n-9 (mg)
`Vitamin E (IU)
`p-Anisidine value
`Peroxide value
`
`DHA
`
`889
`18
`()
`()
`4-6
`<35
`<0.01
`
`Com oil
`
`0
`0
`559
`259
`3.7
`
`EPA
`
`12
`941
`0
`0
`4--6
`<35
`<0.01
`
`1 Dietary supplements were given in indistinguishable, oblong, soft
`gelatin capsules of 1.4 g average weight. DHA. docosahexaenoic acid
`supplement: EPA. eicosapentaenoic acid supplement.
`
`interval of 3-5 d, both at baseline (visits 5 and 6) and after 7 wk
`of supplementation (visits 7 and 8). At each visit blood pres(cid:173)
`sure was measured and blood samples were collected. Partic(cid:173)
`ipants were asked to abstain from alcohol and strenuous exer(cid:173)
`cise for 48 h before the visit. A telephone interview was
`performed in the middle of the intervention period to monitor
`study compliance, side effects, and intercurrent disease. Com(cid:173)
`pliance was assessed by counting leftover capsules and was
`calculated as the percentage of the prescribed capsules taken.
`We also measured serum phospholipid fatty acid concentra(cid:173)
`tions at baseline and at the end of intervention.
`
`Clinical and laboratory measurements
`
`Height was measured during the run-in period and weight
`was measured at baseline and after the intervention period on
`an electronic scale with subjects wearing light clothing and no
`shoes. Before the intervention each subject's habitual nutrient
`intake was assessed during a 1-h interview by a cenified
`clinical nutritionist using the dietary history method. Food
`models and containers were used to estimate quantities. Dietary
`constituents were calculated from standard food tables that also
`cover individual fatty acids by using a specially designed
`computer program ( 19-22). Each subject completed a self(cid:173)
`administered questionnaire at baseline and during the last week
`of the intervention to monitor food habits and physical activity
`during the intervention. Participants were asked how many
`times they ate fish or meat for dinner and how many units of
`alcohol they consumed during the past week (one unit of
`alcohol equals 9 g). Those who reported being physically
`active four or more times weekly for :?: 20 min, leading to
`sweating or shortness of breath, were categorized as active;
`those reporting 1-3 times weekly were categorized as moder(cid:173)
`ately active; and those reporting 0 times weekly were catego(cid:173)
`rized as sedentary.
`Blood samples were drawn from an antecubital vein into an
`evacuated tube system using minimal stasis. Serum was pre(cid:173)
`pared by clotting whole blood in a glass tube (Becton Dickin(cid:173)
`son, Meylan Cedex, France) at room temperature for I h and
`then centrifuging the sample at 2000 X g for 15 min at 22 °C.
`One-milliliter aliquots of serum were transferred into sterile
`2-mL cryovials (Coming, Park Ridge, IL), flushed with nitro(cid:173)
`gen, and stored at - 70 °C. Blood for plasma preparation was
`collected into vacutainers (Becton Dickinson) containing 0.129
`mol sodium citrate/L (blood:anticoagulant = I 0: I). Plasma
`was prepared by centrifugation at 2000 X g for 15 min at
`22 °C, transferred into sterile cryovials in aliquots of I mL,
`
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`

`EFFECTS OF INDIVIDUAL n-3 FATTY ACIDS
`
`651
`
`flushed with nitrogen. and stored at - 70 °C. All blood samples
`were analyzed after completion of the intervention period and
`before the randomization code was broken.
`Serum lipids were analyzed on a Hitachi 737 Automatic
`Analyzer (Boehringer Mannheim, Mannheim, Germany) with
`reagents from the manufacturer. Total cholesterol was mea(cid:173)
`sured with an enzymatic colorimetric method (CHOO-PAP)
`and high-density-lipoprotein (HDL) cholesterol was assayed by
`the same procedure after precipitation of lower-density lipopro(cid:173)
`teins with heparin and manganese chloride. Serum triacylglyc(cid:173)
`erol concentrations were determined with an enzymatic color(cid:173)
`imetric
`test
`(GPO-PAP). Low-density-lipoprotein
`(LDL)
`cholesterol was calculated according to the Friedewald formula
`(23 ). Apolipoprotein A-I and apolipoprotein B-1 were mea(cid:173)
`sured immunochemically by rate nephelometry using the Array
`Protein System from Beckman Instruments Inc (Brea, CA).
`Fatty acids were measured by extracting total lipids from 500
`µL serum according to Folch et al (24), with phosphatidylcho(cid:173)
`line diheptadecanoyl added as an internal standard (P-5014;
`Sigma Chemical Company, St Louis), chloroform:methanol
`(2:1, by vol) as a solvent, and butylated hydroxytoluene (75
`mg/L) as an antioxidant. Total phospholipids were separated by
`solid-phase extraction with NH 2 columns (size 3 cc; Analytiche
`Bond Elut LRC; Varian, Harbour City, CA) (25). followed by
`transmethylation with boron trifluoride, extraction into hexane,
`and evaporation to dryness. The fatty acid methyl esters were
`dissolved in hexane and analyzed by gas-liquid chromatogra(cid:173)
`phy (Shimadzu GC-14 A; Shimadzu Corporation, Kyoto,
`Japan) fitted with a capillary column (CP-Sil 88: length: 50 m,
`internal diameter: 0.25 mm) obtained from Chrompack Inc
`(Raritan, NJ). Retention times and response factors for each
`fatty acid were determined using standards obiained from Nu(cid:173)
`Chek Prep (Elysian, MN). The results were integrated on a
`Shimadzu C-R4A integrator. Fatty acid concentrations are re(cid:173)
`ported as µmo! fatty acid/L serum.
`
`Statistical analysis
`All results are expressed as means ::!:: SDs. On examination
`of the frequency distributions. all variables except serum tri(cid:173)
`acylglycerol and certain lifestyle factors such as level of phys(cid:173)
`ical activity and fish, meat, and alcohol consumption were
`normally distributed at baseline and at the end of intervention.
`Serum lipid concentrations at baseline and at the end of the
`intervention were calculated as the mean of the values obtained
`at visits 5 and 6 and the mean of the values obtained at visits
`7 and 8, respectively. Change was calculated as the value
`obtained after intervention minus the value obtained at base(cid:173)
`line. Percentage change was calculated as the group-wise mean
`percentage change from baseline. Because of missing values,
`change could not be calculated for some individuals. Analysis
`of changes in serum lipids, serum phospholipid 16: 1n-7, and
`sum of serum phospholipid fatty acids are therefore based on
`222, 217, and 209 subjects, respectively. Two influencing
`outlying values were excluded from the analysis of desatura(cid:173)
`tion indexes.
`To evaluate within-group change, we used paired t tests for
`normally distributed variables, the Wilcoxon signed-rank test
`for ordinal and non-normally distributed variables, and the
`chi-square statistic for categorical variables. One-way analysis
`of variance was used to evaluate whether change differed
`between groups; the F test was used for normally distributed
`
`variables and the Kruskal-Wallis test for ordinal and non(cid:173)
`normally distributed variables. Between-group comparisons of
`change were done by contrasting groups in the SAS general
`linear model procedure when the overall F test was significant
`at P < 0.05 (26). We did not adjust for multiple comparisons
`(27). Results were considered significant when the two-sided P
`value was < 0.05. Caution should be applied when interpreting
`P values in the present study because three contrasts were
`tested. When applying the Tukey multiple-comparison proce(cid:173)
`dure (28), the 95% CI included the null value of no effect for
`those contrasts for which the unadjusted P value was > 0.03.
`Correlations were tested by computing Pearson or Spearman
`correlation coefficients.
`
`RESULTS
`
`Three of the 234 subjects who were randomly assigned to a
`study arm dropped out during the intervention period. One
`subject in the DHA group was found to have fat intolerance
`after cholecystectomy, one subject in the EPA group developed
`diarrhea, and one subject in the corn oil group experienced
`vertigo and vomiting that was considered unrelated to the
`dietary supplements. Two individuals in the DHA group, three
`in the EPA group, and two in the corn oil group were excluded
`from the analysis. The reasons for exclusions were possible
`l ), poor compliance with study protocol
`renal disease (n =
`(n = I), initiation of a vasoactive drug (n = I), cancer surgery
`(n = I), and change in amount of physical activity during the
`intervention (n = 3). Thus, 224 subjects are included in the
`present analysis. Mean ages of the subjects were 43 ::!:: 5, 44 ::!::
`5, and 45 ::!:: 6 y and mean body mass indexes (in kg/m 2
`) were
`24.9 ::!:: 2.6, 25.6 ::!:: 2.9, and 24.6 ::!:: 2.7 in the DHA, EPA, and
`corn oil groups, respectively.
`There were no significant changes in hematology, blood
`chemistry (electrolytes, alanine aminotransferase, y-glutamyl
`transferase, alkaline phosphatase, albumin, bilirubin, creati(cid:173)
`nine, and C-reactive protein), serum glucose, or plasma-active
`renin after dietary intervention with DHA. EPA, or com oil
`(data not shown).
`
`Compliance and side effects
`The mean number of days in the study was 49 ::!:: 5, 48 ::!:: 3,
`and 48 ::!:: 4 d in the DHA, EPA, and com oil groups, respec(cid:173)
`tively. Percentage compliance was slightly poorer in the DHA
`group (91 ::!:: 6%) compared with the EPA and com oil groups
`(both 94 ::!:: 6%). There were no within-group correlations
`between compliance and change in serum DHA, EPA, or
`linoleic acid concentrations.
`Side effects were mild and transient and for most individuals
`faded 1-2 wk after the start of the intervention. Fifty-eight
`percent of subjects in the DHA group and 57% in the EPA
`group experienced belching after initiation of the dietary sup(cid:173)
`plements compared with 4% in the corn oil group. A taste of
`fish oil during the intervention was reported by 67% of subjects
`in the DHA group, 65% in the EPA group, and 3% in the corn
`oil group.
`
`Diet, body weight, and physical activity
`The DHA. EPA. and com oil groups were well balanced at
`baseline. Total fat accounted for 30% of energy intake in all
`
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`

`652
`
`TABLE 2
`Composition of background diet'
`
`Daily nutrient intake
`
`DHA
`(n = 72)
`
`GRIMSGAARD ET AL
`
`fish dishes weekly before or during the intervention. There was
`good agreement between measures of alcohol consumption
`obtained by the nutritionist during the run-in period and by the
`self-administered questionnaire at baseline (r = 0.73, P =
`0.0001).
`
`Com oil
`(n = 77)
`
`10 370 :':: 2561
`103 :!: 23
`335 :':: 80
`24.l :!: 7.3
`5.82 :!: 6.28
`81.2 :!: 32.1
`314 :':: 102
`33.6 :!: 12.4
`27.6 :!: 11.3
`13.5 :':: 8.4
`
`0.40 = 0.15
`10.2 = 7.0
`0.18 = 0.20
`
`0.34 :!: 0.32
`2651 :':: 1902
`
`1003 = 955
`1.66 = 0.38
`
`Energy (kl)
`Protein (g)
`Carbohydrate (g)
`Fiber (g)
`Alcohol (g)
`Total fat (g)
`Cholesterol (mg)
`Saturated fat (g)
`Monounsaturated fat (g)
`Polyunsaturated fat (g)
`P:S
`18:2n-6 (g)
`20:5n-3 (g)
`22:6n-3 (g)
`/3-Carotene (µg)
`2634 :':: 1284
`993 :!: 717
`Retinal (µg)
`Thiamine (mg)
`1.67 :!: 0.35
`1.76 :!: 0.43
`2.20 :!: 0.70
`Riboflavin (mg)
`2.29 :!: 0.67
`2.31 :!: 0.72
`Niacin (mg)
`23.6 :':: 4.8
`23.2 :':: 4.8
`23.8 :!: 5.2
`78.6 :!: 35.5
`88.9 :!: 45.0
`Vitamin C (mg)
`90.0 :!: 1.6
`Vitamin D (µg)
`6.08 :':: 9.77
`5.33 :!: 4.16
`5.65 :':: 3.81
`4.87 :!: 1.48
`Vitamin E
`4.96 :!: 1.34
`5.33 :!: 1.53
`1 x :':: SD. DHA. docosahexaenoic acid group; EPA, eicosapentaenoic
`acid group; P:S, ratio of polyunsaturated to saturated fatty acids.
`
`EPA
`(n = 75)
`10223 :':: 2170
`103 :!: 22
`324 :':: 74
`23.2 :!: 6.7
`6.39 :!: 6.56
`81.6 :!: 23.9
`327 :!: 89
`34.3 :!: 10.9
`28.0 :!: 8.7
`12.7 :':: 4.6
`0.39 :!: 0.13
`9.50 :!: 3.70
`0.19 :':: 0.18
`
`0.35 = 0.28
`
`10 877 :+:: 2455
`107 :!: 25
`349 :':: 87
`25.2 :!: 7.7
`7.04 :!: 7.00
`85.9 :!: 27.0
`334 :!: IOI
`35.4 :!: 11.4
`29.6 :!: 9.6
`14.0 :':: 6.1
`
`0.40 = 0.13
`10.7 = 5.1
`
`0.19 :':: 0.21
`0.36 :!: 0.32
`2749 :':: 1905
`
`1031 = 697
`
`Serum lipids and apolipoproteins
`Serum mean (95% CI in parentheses) triacylglycerol con(cid:173)
`centrations decreased 0.22 mmol/L (0.15, 0.29) in the DHA
`group and 0.15 mmol/L (0.06, 0.24) in the EPA group
`(Table 4). In the corn oil group serum triacylglycerols
`increased 0.11 mmol/L (0.03, 0.19). Compared with change
`for the corn oil group, serum triacylglycerols decreased 26%
`in the DHA group and 21 % in the EPA group. The differ(cid:173)
`ence between the DHA and EPA groups was not significant
`(P = 0.14). However, net decreases in serum triacylglycer(cid:173)
`ols were consistently greater in the DHA group than in the
`EPA group across quartiles of baseline triacylglycerol con(cid:173)
`centrations (Table 5). In the EPA and DHA groups there
`were no correlations between changes in individual n - 3
`changes
`in
`serum
`triacylglycerol
`fatty
`acids
`and
`concentrations.
`Serum total cholesterol decreased 0.15 mmol/L (P < 0.05) in
`the EPA group and apolipoprotein A-I decreased 0.04 g/L (P <
`0.001, Table 4 ). These changes differed significantly from both
`the DHA and the corn oil groups. In the DHA group, HDL
`cholesterol increased 0.06 mmol/L (P < 0.001 ), differing sig(cid:173)
`nificantly from both the EPA and com oil groups. Hence, in
`both the EPA and DHA groups there was an increase in the
`ratio of HDL cholesterol to apolipoprotein A-I and a decrease
`in the ratio of total cholesterol to HDL cholesterol.
`
`Serum phospholipid fatty acid concentrations
`In the total study group (n = 224), the correlations between
`dietary intake and serum phospholipid concentrations of DHA
`and EPA at baseline were r = 0.39 and r = 0.35, respectively
`(both P = 0.0001). The mean of individual ratios of dietary
`DHA to EPA at baseline was 2.5 ::!:: 1.2, whereas the serum
`phospholipid ratio of DHA to EPA was 3.8 ::!:: 1.6 (P = 0.0001,
`for the difference between the ratios), indicating accumulation
`of DHA relative to EPA in serum phospholipids.
`
`groups. Dietary intake of DHA and EPA at baseline accounted
`for 0.7% of total fat intake. Differences in nutrient intake
`between the DHA, EPA, and com oil groups were minor and
`not significant (Table 2). No significantly different within- or
`between-group changes were found with respect to body
`weight, physical activity, or food habits during the intervention
`(Table 3). Body weight increased by 0.6 kg in the com oil
`group and by 0.7 kg in the DHA and EPA groups. There was
`a nonsigniflcant increase in the percentage of participants who
`reported being sedentary after compared with before the inter(cid:173)
`vention. Alcohol, meat, and fish consumption (dinner meals)
`increased slightly but not significantly during the intervention.
`None of the participants reported consuming more than three
`
`TABLE 3
`Body weight and lifestyle factors at baseline and change after 7 wk of supplementation with docosahexaenoic acid (DHA), eicosapentaenoic acid
`(EPA), or com oil'
`
`DHA (n = 72)
`
`EPA (n = 75)
`
`Body weight (kg)
`Fish consumption (dishes/wk)
`Meat consumption (dishes/wk)
`Teetotalers(%)
`Alcohol consumption (g/wk)1
`Physical activity
`Sedentary (%)
`Moderate(%)
`Active(%)
`
`Baseline
`
`Change
`
`80.0 :!: IO.a1
`2.10 :!: I.OJ
`2.46 :!: 1.31
`4
`45.3 :!: 44.3
`
`22
`69
`9
`
`0.7 :!: l.2
`0.06 :!: 1.13
`0.24 :!: 1.53
`0
`0.3 :!: 5.1
`
`3
`-6
`3
`
`1 There were no significant difference< among groups.
`2 x :!: SD.
`-'Teetotalers were excluded from analysis of alcohol consumption.
`
`Baseline
`
`82.6 = 10.0
`
`2.16 :!: 1.05
`2.56 :!: 1.39
`I
`59.6 :!: 63.9
`
`26
`59
`15
`
`Change
`
`0.7 :!: 1.4
`0.16 :!: 1.06
`0.16 :!: 1.23
`0
`-0.8 :!: 6.5
`
`-I
`0
`
`Com oil (n = 77)
`Baseline
`Change
`
`79.5 :!: 9.4
`2.03 :!: 1. IO
`2.93 :!: 1.28
`4
`55.5 :!: 50.8
`
`0.6::: I.I
`0.21 :!: 1.29
`0.15 ~ 2.01
`0
`1.5 :!: 7.0
`
`25
`54
`21
`
`6
`
`-7
`
`ICOSAPENT DFNDTS00006139
`
`Hikma Pharmaceuticals
`
`IPR2022-00215
`
`Ex. 1007, p. 4 of 11
`
`

`

`[!] The American Journal of' Clinical Nutrition
`
`TABLE 4
`Serum lipids and apolipoproteins at baseline and change after 7 wk of supplementation with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or com oil
`
`DHA (n = 72)
`
`Baseline
`
`Change
`
`EPA (n = 75)
`Baseline
`Change
`
`Com oil (n = 77)
`Baseline
`Change
`
`Frest P1
`
`DHA vs EPA DHA vs com oil
`
`EPA vs com oil
`
`Contrasts between groups: P
`
`TriacylglycemL~ (mmol/L)
`Total cholesterol (mmoVL)
`LDL cholesterol (mmol/L)
`HDL cholesterol ( mmol/L)
`Apolipoprotein A-1 (g/L)
`Apolipoprotein B (g/L)
`HDL:apolipoprotein A-I
`Total:HDL cholesterol
`
`1.24 ± 0.58'
`6.00 ± 0.95
`4.06 ± 0.86
`1.36 ± 0.30
`1.38 ± 0.21
`1.00 ± 0-21
`0.97 ± 0.14
`4.62 ± 1.19
`
`-0.22 ± 0.31·'
`om ± 0.49
`O.o? ± 0.46
`0.06 ± 0.13'
`O.o2 :!: 0.13
`-0.ol :!: 0.11
`0.£)4 ± 0.07.t
`-0.19 ± 0.52'
`
`1.23 ± 0.57
`5.98 ± 0.94
`4.06 ± 0.83
`1.33 ± 0.31
`1.38 ± 0.20
`1.01 ± 0.23
`0.96 ± 0.13
`4.70 ± 1.24
`
`-0.15 ± 0.40'
`-0.15 ± 0.55'
`-0.08 ± 0.48
`0.01 ± 0.12
`-0.04 :!: 0.101
`-0.QJ ± 0.11'
`0.04 ± 0.081
`-0.13 ± 0.47'
`
`1.22 ± 0.55
`6.02 ± l.08
`4.04 ± 0.98
`1.41±0.28
`1.46 ± 0.23
`1.02 ± 0.28
`0.97 ± 0.12
`4.43 ± 1.19
`
`0.11 ± 0.34'
`0.10 ± 0.55
`0.06 ± 0.48
`-0.Dl ± 0.11
`0.00 :!: 0.12
`0.02 :!: 0.11
`-0.01 :!: 0.06
`0.11 ± 0.62
`
`0.0001
`O.DI
`0.10
`0.001
`0.003
`0.05
`0.0001
`0.002
`
`0.14
`0.04
`
`0.009
`0.0008
`
`0.8
`0.4
`
`0.0001
`0.4
`
`0.0005
`0.3
`
`0.0003
`0.0006
`
`0.0001
`0.004
`
`0.4
`0.G2
`
`0.0001
`0.007
`
`1 ANOV A for between-group comparisons of change.
`2 x ±SD.
`J-.5 One·sample t test of difference between baseline and 7 wk: 'P < 0.001. 'P < O.oJ. 'P < 0.0S.
`
`TABLE 5
`Change in serum rriacylglycerol concenlration in docosahexaenoic acid (DHA). eicosapenlacnoic acid (EPA), and com oil groups ac1;ording to quartiles of baseline triacylglycerol concentration
`
`Baseline triacylglycerol
`concentration
`
`I st quartile:
`0.69 (0.34--0_82) mmollL'
`2nd quartile:
`0.96 (0.83-1.09) mmol/L
`3rd quartile:
`l.24 (1.1~1.44) mmol/L
`4th quanile:
`2.01 (1.45-3.61) mmol/L
`
`om± 0.50
`1 Effect anriburable to DHA and EPA: ie. change in DHA group minus change in com oil group and change in EPA group minus change in com oil group.
`1 i; range in parentheses.
`3 .i ::!:: SD.
`
`-0.56 ± 0.35
`
`-0.52 ± 0.46
`
`-0.59 (-29)
`
`DHA (n = 72)
`
`0.00 ::!: 0.13 1
`
`-0.14±0.18
`
`-0.16 ± 0.23
`
`Change in serum triacylglycerol concentration
`EPA (n = 75)
`mmoVL
`
`Com oil (n = 77)
`
`DHA
`
`EPA
`
`Estimated n-3
`
`acid effect'
`
`mmol/L (%}
`
`0.()3 ± 0.21
`
`-0.04 ± 0_26
`
`-0.03 ± 0.34
`
`0.10 ± 0.21
`
`0.15 ± 0.28
`
`0.14 :!: 0.32
`
`-0. JO (--14)
`
`-0.29 (-30)
`
`-0.30 (-24)
`
`-0.o7 (- 10)
`
`-0.19 (-20)
`
`-0.17 (-14)
`
`-0.55 (-27)
`
`ICOSAPENT _DFN DTS00006140
`
`Hikma Pharmaceuticals
`
`IPR2022-00215
`
`Ex. 1007, p. 5 of 11
`
`

`

`654
`
`GRIMSGAARD ET AL
`
`The w1al amount of serum phospholipid fatty acids did not
`change between groups during EPA, DHA, or com oil supple(cid:173)
`mentation (Table 6). Likewise, there were no changes in serum
`phospholipid saturated fatty acids. As for the monounsaturated
`fatty acids, palmitoleic acid ( 16: In - 7) decreased significantly
`by 20% in the DHA group, compared with no change in the
`EPA or com oil groups. Oleic acid ( 18: ln-9) concentrations
`decreased by 11% and 12% in the DHA and EPA groups,
`respectively.
`The IOtal serum phospholipid n-6 fatty acid concentra(cid:173)
`tion (sum of 18:2n-6. 20:3n-6, and 20:4n-6) decreased
`more in the EPA (-23%) than in the DHA (-11 %) group.
`In the DHA group, however, the ratio between the individual
`n-6 fatty acids changed more than in the EPA group. The
`ratio of 20:4n-6 + 20:3n-6 to 18:2n-6 can be used as an
`index of '16 desaturation activity because changes in '15
`desaturation will not influence the ratio. The Ll6 desatura(cid:173)
`tion index decreased significantly in the DHA group com(cid:173)
`pared with no change in the EPA or corn oil group (Table
`7). Similarly. the ratio of 20:4n-6 to 20:3n-6 + 18:2n-6
`can be used as an index of A5 desaturation activity. This
`ratio decreased significantly in the DHA group whereas it
`increased significantly in the EPA group. As a result, the
`ratio of arachidonic (20:4n-6) to linoleic (18:2n-6) acid
`decreased in the DHA group and increased in the EPA
`group.
`During the intervention, the serum phospholipid concentra(cid:173)
`tion of n-3 fatty acids increased by 47% in the DHA group
`and by 68% in the EPA group. The concentration of a-linolenic
`acid (18:3n-3) decreased in both the DHA and EPA groups
`compared with the com oil group (Table 6). In the DHA group,
`mean serum phospholipid DHA and EPA concentrations in(cid:173)
`creased significantly by 69% (individual range: - 33% to
`669%) and 29% (individual range: -63% to 557%), respec(cid:173)
`tively, whereas docosapemaenoic acid (DPA; 22:5n-3) de(cid:173)
`creased by 33% (individual range: -72% to 221%). In this
`group, the correlation between the change in serum DHA and
`EPA was r = 0.30 (P = 0.01, Figure 1). In the EPA group,
`serum phospholipid EPA increased by 297% (individual range:
`-2% to 1196%) and docosapentaenoic acid by 130% (individ(cid:173)
`ual range: -9% to 393%). Surprisingly, the serum phospho(cid:173)
`lipid concentration of DHA decreased by 15% (individual
`range: -65% to 85%; P < 0.001) after EPA supplementation.
`The correlation between the change in serum DHA and EPA
`was r = 0.39 (P = 0.0005) during supplementation with EPA
`(Figure 2).
`
`DISCUSSION
`
`Numerous studies have examined the effects of marine
`n-3 fany acids on lipid metabolism, but the separate effects
`of the two major n- 3 fatty acids have remained largely
`unknown. The present report extends previous data by show(cid:173)
`ing that both DHA and EPA lower serum triacylglycerol
`concentrations. DHA may be responsible for the increase in
`HDL cholesterol observed with some n - 3 fatty acid sup(cid:173)
`plements whereas EPA may produce a small decrease in
`serum total cholesterol. Our data further show that DHA and
`EPA produce different effects on the fatty acid composition
`of serum phospholipids. We studied a fairly large sample
`
`recruited from the general population and compliance with
`the study protocol was good. The generalizability of the
`study therefore appears sound.
`It is well established that n- 3 fatty acids lower serum
`triacylglycerols, but this is the first study in humans showing
`that this effect is attributable to both EPA and DHA. Surpris(cid:173)
`ingly. DHA consistently had a more pronounced triacylglycer(cid:173)
`ol-lowering effect than EPA across all baseline concentrations
`of triacylglycerol (Table 5). These observations provide strong
`evidence that DHA has a triacylglycerol-lowering effect of its
`own and is not acting solely after retroconversion IO EPA,
`because if that were the case, DHA would not be more potent
`than EPA. This finding contrasts with previous studies in rats,
`in which dietary supplementation of highly purified EPA low(cid:173)
`ered serum triacylglycerols whereas DHA had a modest effect
`if any ( 13, 29-31 ). The opposing findings may be dose related:
`the DHA and EPA supplements in the rat studies calculated as
`mg· d·- i ·kg body wt·· I are 10- to 30-fold larger than in the
`present study in humans ( 13, 29). The opposing findings may
`also depend on species differences because rats and humans
`differ with respect to lipid metabolism (32). Our finding is
`further opposed by results from a single-blind crossover study
`concluding that EPA is responsible for the triacylglycerol(cid:173)
`lowering effect in humans ( 14). However, the study was small
`with only nine individuals in the DHA group. and the wash-out
`period was 2 wk, which is wo short in dietary intervention
`trials with n-3 fatly acids (33).
`Previous studies suggested that serum HDL cholesterol is
`better maintained with oil rich in DHA than oil rich in EPA
`(2