Human and Clinical Nutrition
Supplementation with an Algae Source of
Docosahexaenoic Acid Increases (n-3) Fatty Acid Status
and Alters Selected Risk Factors for Heart Disease in
Vegetarian Subjects1'2
JULIE A. COHQÃœER AND BRUCE J. HOLÃœB3
Department of Human Biology and Nutritional Sciences, University of Guelph,
Guelphi, Ontario, Canada NI G 2W1
ABSTRACT The purpose of this double-blind study
was to investigate the influence of dietary supplemen
tation with an algae source of docosahexaenoic acid
[DMA;22:6(n-3)], devoid of any eicosapentaenoic acid
[EPA; 20:5(n-3)], on serum/platelet DHA status, the
estimated retroconversion of DHA to EPA, and risk
factors for heart disease in vegetarian subjects.
Healthy vegetarians (12 male, 12 female) consumed
nine capsules daily of either DHA (1.62 g/d) or corn
oil for 6 wk. Consumption of DHA capsules increased
DHA levels in serum phospholipid by 246% (from 2.4
to 8.3 g/100 g fatty acids) and in platelet phospholipid
by 225% (from 1.2 to 3.9 g/100 g fatty acids). EPA
levels increased in serum phospholipid by 117% (from
0.57 to 1.3 g/100 g fatty acids) and in platelet phos
pholipid by 176% (0.21 to 0.58 g/100 g fatty acids)
via metabolic retroconversion; the estimated extent of
DHA retroconversion to EPA was 11.3 and 12.0%,
based on the serum and platelet analyses, respec
tively. Arachidonic acid [AA; 20:4(n-6)J levels in serum
and platelet phospholipids decreased moderately dur
ing the trial period (DHA group) as did both docosa-
pentaenoic acids |22:5(n-6) and 22:5(n-3)|. Although
no significant changes were found in the total and LDL-
cholesterol levels with DHA supplementation, the total
cholesterohHDL-cholesterol ratio showed a moderate
decrease over time as did the LDL-cholesterol:HDL-
cholesterol ratio and serum triglycérideconcentra
tions. DHA supplementation did not alter the various
thrombogenic factors measured. In conclusion, DHA
supplementation markedly enhanced the DHA status
(of serum and platelets), provided for the formation
of substantial EPA, and lowered the total and LDL-
cholesterohHDL-cholesterol ratios. J. Nutr. 126:
3032-3039,1996.
INDEXING KEY WORDS:
•humans •vegetarian •DHA supplementation
•cholesterol •thrombogenic factors
0022-3166/96 $3.00 ©1996 American Institute of Nutrition.
Manuscript received 5 June 1996. Initial review completed 5 July 1996. Revision accepted 22 August 1996.
3032
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In contrast to the omnivorous diet, the vegan diet is
devoid of the long-chain (n-3) fatty acids, eicosapentae
noic acid [EPA,4 20:5(n-3)] and docosahexaenoic acid
[DHA, 22:6(n-3)], commonly found in fish and fish oils.
Although vegans consume no EPA or DHA (Pan et al.
1993, Sanders et al. 1978), vegetarians consuming eggs
(Simopoulos and Salem 1992) consume small amounts
of both. Thus, in the vegetarian diet, a-linolenic acid
[aLNA, 18:3(n-3)] is the major dietary (n-3) fatty acid.
The efficiency of conversion of this fatty acid to DHA,
in healthy adults, via desaturation and elongation, ap
pears to be ~5% (Emken et al. 1994). Various studies
have confirmed that vegans/vegetarians have lower se
rum and/or platelet phospholipid levels of DHA than
do omnivores (Reddy et al. 1994).
DHA is found in high levels in the brain and retina,
where it functions in mental performance and visual
acuity, respectively (reviewed in Neuringer et al. 1988).
Several investigators have shown that dietary fish/fish
oils containing EPA plus DHA may help reduce the
risk for developing cardiovascular diseases (CVD) (for
review see Herold and Kinsella 1986). Fish oils con-
1Supported by the Heart and Stroke Foundation of Ontario. J.A.C,
was a recipient of a Heart and Stroke Foundation of Ontario research
fellowship.
2The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 USC section 1734
solely to indicate this fact.
3 To whom correspondence and reprint requests should be ad
dressed.
4Abbreviations used: AA, arachidonic acid; ACD, acid citrate dex
trose; CVD, cardiovascular disease; DHA, docosahexaenoic acid;
EPA, eicosapentaenoic acid; aLNA, a-linolenic acid; PPP, platelet-
poor plasma; PRP, platelet-rich plasma; PUFA, polyunsaturated fatty
acids; TxA2, thromboxane A2; TxB2; thromboxane B2.
 DMA AND HEART DISEASE RISK FACTORS
taining EPA plus DHA lower serum triglycéridelevels
in humans (for review see Harris 1989) and reduce
blood platelet reactivity (for review see Weaver and
Holub 1988). These latter effects have often been attrib
uted indirectly to EPA due to its predominance in most
fish oil supplements/concentrates studied to date. Iso
lated studies with DHA-enriched preparations con
taining significant levels of EPA have shown a reduc
tion in platelet aggregation (von Schacky and Weber
1985) and a lowering of serum triglycérideconcentra
tions in human volunteers (Sanders and Hinds 1992).
DHA has been found to inhibit human platelet reactiv
ity in vitro (Gaudette and Holub 1991).
Although vegetarians tend to be at overall decreased
risk for CVD (Dwyer 1988), due partly to their lower
serum cholesterol levels, their thrombogenic risk fac
tors may be significant (Pan et al. 1993). Considering
also the lower DHA status in vegetarians, it was of
interest to determine the effect of oral supplementation
with a DHA-rich/EPA-free source (from micro-algae,
non-fish derived) of triglycéride oil (DHASCO™) in
this group. In addition to evaluating the potential for
DHASCOâ„¢ to enhance the DHA and EPA (via retro-
conversion) status in vegetarians, the influence of di
etary DHA on conventional (serum total cholesterol,
lipoproteins and triglycérides) and thrombogenic car
diovascular risk factors (platelet aggregation, throm-
boxane production, serum viscosity, factor Vile and fi-
brinogen levels) was determined. Measures of total cho-
lesterol:HDL-cholesterol and LDL-cholesterol:HDL-
cholesterol ratios were of particular interest because
recent reports indicate that they are superior predictors
of coronary heart disease risk compared with total cho
lesterol and LDL-cholesterol levels (Kinosian et al.
1994 and 1995).
SUBJECTS AND METHODS
Subjects and experimental design. The subjects
were 24 healthy vegetarians (12 male, 12 female) se
lected from the Guelph community who reported hav
ing no meat, poultry or fish for a period of at least 6
mo. Approval for this double-blind study was granted
by the Human Ethics Committee of the University of
Guelph and written informed consent was obtained
from each subject. The 24 subjects (aged 29.6 ±1.7 y,
mean ±SE)were randomly assigned (12/group) to the
two supplementation groups, DHA-supplemented and
control (6 males, 6 females each). The DHA-enriched
encapsulated triglycéride oil, DHASCO™ (algae de
rived), and placebo (corn oil) capsules were kindly pro
vided by David Kyle of Martek Biosciences, Columbia,
MD. As confirmed by quantitative gas-liquid chro-
matographic analyses, the DHASCOâ„¢ oil contained
DHA as the major fatty acid (39 g/100 g fatty acids)
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3033
TABLE 1
Subject characteristics and estimated dietary intake during
docosahexaenoic acid or placebo supplementation!
VariableHeight,
mWeight, 0.067.7
±
kgBMI, 3.422.9
±
kg/m?Protein,
2 1.011.8
±
energyFat,% of ±0.625.4
energyCarbohydrate,
% of 1.758.4
±
energyAlcohol, % of 3.14.2
±
% of energyControl1.7 ±1.9DHASCO™1.767.122.910.822.963.60.
1.3
1 Values are reported as means ±SEM,n = 12 (height, weight,
BMI) or n = 10 (protein, fat, carbohydrate, and alcohol as a percentage
of energy intake). No significant differences between the groups were
found for the above variables.
2 BMI, body mass index.
followed by myristic acid (21%), palmitic acid (16%),
oleic acid (11%) and lauric acid (8%); no EPA was de
tected. Linoleic acid (53%), oleic acid (21%) and pal
mitic acid (10%) predominated in the vegetable oil
(corn oil) placebo. Each DHASCOâ„¢ capsule contained
0.5 g oil and was estimated to provide 180 mg DHA.
The experimental group consumed nine DHA-con
taining capsules per day (total 1.62g DHA/d) with their
meals, and the control group consumed nine vegetable
oil placebo capsules per day. Each group consumed the
capsules for a period of 42 d beginning on d 0. After 42 d
of capsule ingestion, both groups completed a washout
period for 21 d during which there was no supplementa
tion. Subjects were weighed on each visit (d 0, 21, 42
and 63) and height was measured at entry; there were
no significant differences in these or other characteris
tics between the groups at entry ¡Table1). The amount
of fat consumed in encapsulated form (4.5 g/d) repre
sented <10% of the dietary fat intake. There were no
differences between daily energy intakes (8372 kj/d) or
intakes of saturated fat, cholesterol, monounsaturated
fat or polyunsaturated fat between the two groups (data
not shown). The weight of the subjects was not affected
throughout the supplementation period in either group.
All dietary records were analyzed by the Can West Diet
Analysis-Plus program which includes comparison
with the Canadian Recommended Nutrient Intakes
(West Publishing, St. Paul, MN). Only 20/24 subjects
completed the dietary questionnaire. All subjects com
pleted the study. Compliance was monitored by de
termining the fatty acid composition of serum and
platelet phospholipid at 0, 6 and 9 wk (3 weeks postsup-
plementation) and from a capsule count at the end of
the study.
Blood collection. At d 0 (presupplementation), 21
and 42 (supplementation), and 63 (washout), blood was
collected by antecubital venipuncture from fasting sub
jects into siliconized tubes containing no anticoagulant
(for serum isolation), or the anticoagulants acid citrate
 3034
dextrose (ACD) [25 g/L Na citrate, 20 g/L dextrose, 14
g/L citric acid (all from Fisher Chemicals, Nepean,
Canada)], orNa citrate (3.2%). Whole blood was treated
as follows: Tube without anticoagulant. Whole blood
was centrifuged at 1250 x g for 15 min to obtain serum.
Serum was used for measurement of plasma total-,
HDL- and LDL-cholesterol levels, triglycéride levels,
serum total phospholipid fatty acid content and serum
viscosity. Serum was stored at -20°C until all samples
were collected and thawed just before analyses of lip-
ids. Serum was stored at room temperature until vis
cosity was analyzed (see below).
Tubes containing Na citrate. Whole blood was cen
trifuged at 200 X g for 17 min to obtain platelet-rich
plasma (PRP) which was removed, and the remaining
blood was centrifuged at 1250 x g for 15 min to obtain
platelet-poor plasma (PPP). The PRP was used in aggre
gation studies and the PPP was used for analyses of
factor VII and fibrinogen levels. The PPP was stored
below -20°C until analysis by Med Chem Laboratories
(Scarborough, Ontario).
Tube containing ACD. Washed platelet suspensions
were prepared according to the method of Turini et al.
1994.
Platelet aggregation. Platelet aggregation was per
formed within 2 h of blood collection. Platelets were
counted in PRP using a Coulter Counter, model ZM
(Coulter Electronics, Burlington, Canada) and adjusted
to a final concentration of 2.0-2.5 x IO11 platelets/L
using autologous PPP. Aliquots (0.5 mL) of adjusted PRP
were preincubated for 1 min in siliconized cuvettes with
stirring at 900 rpm at 37°Cin a dual-channel 800B aggre-
gometer (Payton Instruments, Ion Trace, Scarborough,
Canada) before addition of the aggregating agent. Colla
gen (Hormone-Chemie, München,Germany) was added
at two levels with the final concentrations of 2 and 10
mg/L. The platelets were allowed to aggregate for 2 min
following the addition of agonist and then the level of
aggregation was measured (Born 1962).
Cholesterol and triglycéride measurement. Total
cholesterol was measured enzymatically with a diag
nostic test (Sigma Diagnostics Procedure No. 352, St.
Louis, MO). HDL-cholesterol was isolated by using a
dextran sulfate and Mg ion solution to precipitate the
VLDL and LDL from the serum sample. The HDL frac
tion was then assayed by an enzymatic assay (Sigma
Diagnostics Procedure No. 352-3). Triglycéride was
measured enzymatically with a diagnostic test (Sigma
Diagnostics Procedure No. 339). LDL-cholesterol was
calculated using the formula validated by Friedewald
et al. (1972). Analyses of all samples for each volunteer
were performed in a single assay.
Total phospholipid analysis. The fatty acid compo
sitions of total phospholipid from serum and washed
platelet suspensions were determined following lipid
extraction, TLC, transmethylation of fatty acid resi
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CONQUER AND HOLUB
dues and gas liquid chromatography by procedures sim
ilar to those previously described (DonadÃ-oet al. 1994).
Fibrinogen, factor Vile and serum viscosity. Fibrin
ogen, factor Vile and serum relative viscosity were ana
lyzed by Med-Chem Laboratories LTD. Serum relative
viscosity was determined on serum (maintained at
room temperature) within 16 h of blood withdrawal.
The relative viscosity is the flow time of specimen in
seconds/flow time of water in seconds in an Oswald
Viscosimeter tube (Gradwohl 1970). The concentration
of fibrinogen in plasma samples was determined by
a diagnostic test comparing thrombin time values of
diluted plasma samples (diluted with Ortho Q.F.A.
Buffer, Ortho Diagnostic Systems, Johnson and John
son, Raritan, NJ), to which Ortho Q.F.A. Thrombin was
added, to a reference curve (Clauss 1957). The log of
the thrombin time value is inversely proportional to
the log of the fibrinogen concentration. To determine
factor Vile levels, prothrombin time was determined
using Thromborel S (Behring Diagnostics, Westwood,
MA) in conjunction with plasma deficient in factor Vile
(Quick 1935).
Thromboxane production. Aliquots of PRP (0.5
mL) were stimulated with collagen (2 mg/L) for 2 min
and reactions stopped by the addition of 250 ¿¿mol/L
(final concentration) indomethacin (Sigma) and 4.5 g/
L NaCl (final concentration) and were immediately
chilled on ice. Samples were centrifuged at 2000 x g
for 15 min at 0°C.The supernatant was removed and
stored at -20°C until analysis using the Thromboxane
B2 Biotrack enzyme immunoassay system (Amersham
Canada, Oakville, Ontario, Canada).
Statistical analysis. All data are reported as means
±SEM.Data that were not normally distributed were
transformed (log or square) before analysis to reach nor
mality. When observations were missing, least-squared
means were calculated so that means could be com
pared. Split-plot design, including time and treatment
as factors, was used in all analyses before examination
of specific differences by Fisher's protected LSD. Spe
cific differences for preplanned comparisons between
treatment means as well as comparison between di
etary intakes and subject characteristics were exam
ined by f test (Kirk 1968). Statistical analyses were done
using the SAS system (SAS Institute, Cary, NC).
Changes within groups as well as comparisons between
groups were made.
RESULTS
The fatty acid compositions of the serum total phos
pholipid (controls and DHA-supplemented) at entry
(wk 0) and after 3 and 6 wk are given in Table 2. The
levels of the various (n-3) polyunsaturated fatty acids
(PUFA) were not different between the two groups at
 DHA AND HEART DISEASE RISK FACTORS 3035

TABLE2
Fatty acid composition of total phospholipid in human serum before and after supplementation with docosahexaenoic acid or placebo1

Control DHASCOâ„¢

Week

Fatty acids

g/100 g fatty acids

0.4313.1
±
16:018:018:118:2(n-6)18:3|n-3)20:3(n-6)20:4|n-6) 0.5a,b± 0.4a,b13.2
± 0.6a,b12.8
± 0.5a,b12.4± 0.4b±0.3»±
0.4a,b14.2
± 0.3a,b± 0.3b12.6
± 0.4a,b13.2
± 0.4a,b12.3 ±
0.6b22.8± 0.6a,b±0.5b±0.04±0.lc0.3c,d0.05a0.03b0.03bO.OSb±
0.6a,b23.90.262.89.00.640.270.200.972.19.50.070.230.5b0.03O.lc0.4c,dO.Oga0.03b0.03b0.06b0.2a0.6b
± 0.6b22.0± 0.7a,b21.3± 0.4a±
0.8a,b0.27
± ±0.7a0.25 0.7a,b±0.03±0.2a±
0.7a,b0.212.27.31.00.030.030.400.03O.lb0.3bO.lbO.OlaO.OlaO.O
±
0.052.7
± 0.033.2±
O.lc8.9
± 0.2<a9.7
±
(AA)220:5|n-3) 0.2C0.630.270.210.912.29.20.070.08a0.03b0.03b0.08b0.2a0.6bO.Ola0.24
± 0.4d0.57 0.2a±0.2b±
(EPA)22:4[n-6)22:5(n-6)22:5(n-3)22:6(n-3) 0.07a0.29
0.04C0.29 O.Ola±
0.04C0.92 O.Ola±
0.06b2.4 O.OSa±
(DHA)|n-6)/(n-3)EPA/AADHA/AA22:5(n-6)/22:6(n-3)26.40.2a±0.5b± 0.2a8.9± 0.3b3.3± 0.4b±
0.6b0.06
± 0.2a0.14± 0.2a±
O.Ola± O.Ola0.25
± O.Olb1.1 ± 0.02C±
0.02a0.10
± O.Ola± 0.03a0.12
± 0.06b0.003
± 0.06C±
O.Olb26.9 O.Olb26.7
±O.Olb27.212.713.023.90.242.79.30.540.250.180.902.19.90.060.230.08±
±O.Olc27.6 ±O.OOia27.812.311.721.70.212.16.51.30.030.030
O.OOia

1 Values are reported as means ±SEM,n = 12. Differing superscripts in a row indicate significant differences, P < 0.05.
~ AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaeneoic acid.

entry and did not change in the control group; however,
the DHA rose dramatically (from 2.4 to 8.3 g/100 g
fatty acids or 246% overall by wk 6) in the DHASCOâ„¢-
supplemented group as did EPA (from 0.57 to 1.3 g/
100 g fatty acids). The 22:5(n-3) showed a significant
decrease upon DHA supplementation (from 0.92 to
0.43 g/100 g fatty acids) as did all of the 20/22 carbon
PUFA of the (n-6) series [arachidonic acid (AA), 20:3,
22:4, 22:5] but not 18:2(n-6) (linoleic acid). In the case
of platelet phospholipids (Table 3), the marked rise in
DHA with supplementation up to 6 wk (from 1.2 to
3.9 g/100 g fatty acids) and in EPA (from 0.21 to 0.58
g/100 g fatty acids) was also accompanied by significant
decreases in 22:5(n-3) as well as AA, 22:4(n-6) and
22:5(n-6) but not 18:2(n-6) or 20:3(n-6). The fatty acid
alterations in serum/platelet phospholipid reversed to
wards entry (wk 0) values at 3 wk following cessation
of the DHA supplementation (data not shown).
The effects of DHA supplementation on the serum
lipids and lipoproteins are given in Table 4. Although
no significant alteration was found in the total- and
LDL-cholesterol levels with DHA supplementation, a be 0, 33 (based on 4.5 eggs/wk) and 78 mg, respectively
significant decrease in the total cholesterol:HDL-cho-
lesterol ratio (by 16%) as well as the LDL-choles-
terol:HDL-cholesterol ratio (by 22%) was exhibited in
6 wk. Furthermore, the triglycérideconcentrations de
creased significantly (P < 0.05) within 3 and 6 wk (by
22 and 16%, respectively) in the DHA-supplemented
group.
Collagen-induced platelet aggregation as well as col
lagen-induced thromboxane A2 (TxA2) [measured as
thromboxane B2 (TxB2)] formation, serum viscosity, fi-
brinogen and factor Vile levels were not significantly
altered by DHA supplementation (Table 5).
DISCUSSION
Despite consuming as much or more plant-derived
(n-3) (as aLNA) relative to omnivores (for example,
-3.6 vs. 2.7 g/d) (Pan et al. 1993, Râperet al. 1992),
vegetarians have a significantly lower DHA status
based on measured DHA levels in serum, platelets and
breast milk (Reddy et al. 1994, Sanders et al. 1978). The
limited conversion of LNA to DHA in humans in vivo
(Emken et al. 1994) and the absence of DHA in plant-
based foods likely accounts for these differences. The
approximate daily intake of DHA in the diet of vegans,
ovo-lacto vegetarians, and omnivores is estimated to
(Ferrier et al. 1995, Pan et al. 1993, Râperet al. 1992);
the vast majority of the latter intake is in the form of
fish (Raper et al. 1992).
Our double-blind trial in vegetarian subjects demon
strated that a novel algae-derived source of DHA, free
of EPA, which provided 1.62 g of DHA/d could more
than triple the DHA content of the serum and platelet

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 3036 CONQUER AND HOLUB

TABLE 3
Fatty acid composition of total phospholipid in human platelets before and after supplementation with docosahexaenoic acid or placebo!

Control DHASCO

Week

Fatty acid

g/100 g fatty acids

0.4«17.4
±
16:018:018:118:2|n-6]20:3|n-6)20:4jn-6| 0.4a,b16.8
± 0.3b16.3
±
0.4a17.5± ±0.3a16.9 0.6a17.9
±
0.5a,b6.1
± 0.3a,b6.7
± 0.5b6.7
±
±0.3a1.3 ±0.2b1.3 0.3a,b1.5
±
0.6a,b24.2
± ±0.1a,b25.5 ±0.lb21.7
(AA)220:5(n-3) 0.6b0.24
± 0.4b0.25
± 0.730.58
±
(EPA)22:4(n-6)22:5(n-6|22:5|n-3)22:6(n-3)
0.04a2.1
± 0.0432.1
± O.OSb1.2
±
O.lb0.19
± O.lb0.17
± O.lb0.28
± :0.01 ±O.la
0.04b1.71.111.60.010.04O.lbO.la0.5CO.OaO.Oa0.17
± ±0.05b1.71.111.90.010.04O.lbO.la0.5C0.03O.Oa0.14
0.03C1.71.111.20.010.05O.lbO.la0.5CO.OaO.Oa0.25
± 0.0030.66
± O.Ola0.71
±
0.0533.8
± 0.0733.9
±
(DHA|(n-6)/(n-3)EPA/AADHA/AA22:5(n-6)/22:6(n-3)16.8 ±0.lb6.7 0.2b6.1
±
0.3a0.02
± 0.330.03
±
O.Ob0.16
± O.Oc0.18
±
O.Ob0.00
± O.Oc0.00
±
0.03b,c17.2 0.04b17.117.416.75.71.124.40.290.4a,b0.4a0.4a0.5aO.la0.8b0.0432.2
0.03b18.016.717.56.81.423.80.481.30.3b0.230.5a,b0.2bO.l3,b0.4b0.
±O.OOa17.8
0.03C17.116.917.06.01.323.60.212.40.181.71.210.70.010.050.3aO.Sa0.43,b0.2aO.la,b0.7b0
±O.OOa

1 Values are reported as means ±SEM,n = 12. Differing superscripts in a row indicate significant differences, P < 0.05.
2 AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.

phospholipids within 3 wk and maintain these levels
over the 6 wk supplementation period (Tables 2 and
3). Previous studies have shown that fish oils, which
contain EPA plus DHA, raise both EPA and DHA levels
in the platelet and serum phospholipids of omnivorous
subjects (Herold and Kinsella 1986, Weaver and Holub
1988). The (n-3) fatty acid level in serum phospholipid
is regarded as a useful biochemical index of the dietary
intake and overall nutritional status for EPA/DHA (Bo-
naa et al. 1992). The level of DHA attained (8.3 g/100
g total fatty acids in serum phospholipid) is above the
levels reported in free-living omnivorous subjects (Bo-
naa et al. 1992). The net rise in EPA in serum phospho
lipid (0.7 g/100 g fatty acids) was ~one eighth that of
the corresponding DHA rise (Table 2). The EPA rise
would reflect fatty acid retroconversion from DHA
(Voss et al. 1992) with an estimated efficiency of 11.3
and 12.0% based on the mol/100 mol values for serum
and platelets, respectively (net rise in EPA/net rise in
EPA + DHA as %) as derived from the data in Tables
2 and 3. Interestingly, the decrease in the (n-6) polyun-
saturates [20:4(n-6), 22:4(n-6) and 22:5(n-6)] in platelet
and serum phospholipids (Tables 2 and 3), as observed
in our study as well as with fish oils having EPA plus

TABLE 4
Effect of supplementation with docosahexaenoic acid or placebo on human serum lipid and lipoprotein levels1

Control DHASCOâ„¢

Week

Variable

Total
mmol/LHDL-cholesterol,
cholesterol, 0.15a1.35
± 0.15a1.40±0.1lb,c2.9
± 0.20a1.20
± 0.26a1.36
± 0.24a1.40
±
mmol/LTotal 0.09b3.0
± O.lOc3.0
± O.lOa3.2
± 0.12b2.8
± 0.13b,c2.7
±
HDL-cholesterol,
cholesterol:
moLtnolLDL-cholesterol, 0.2b2.10
± ±0.2a,b2.02 ±0.2b2.29 ±0.3b1.99 0.2a,b1.97
± 0.2a1.86
±
mmol/LLDL-cholesterol: 0.1831.7
± 0.18a1.6
± 8b1.7±0.1 0.1831.8
± 0.2131.6
± 0.17»1.4
±
HDL-cholesterol,
mol:molTriglycéride, 0.2b,c0.81
± 0.2b0.77
± 0.2b,c0.92
± 0.2C0.96
± 0.2b0.75
± ±0.2»0.80
mmol/L3.83 ±0.08a,b,c3.78 ±0.10a,b4.16±0.19b1.45
±0.14b,c3.63 ±0.1ic3.67 ±0.09a3.63 ±O.lla,b

Values are reported as means ±SEM,n = 12. Differing subscripts in a row indicate significant differences, P < 0.05.

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 DHA AND HEART DISEASE RISK FACTORS 3037

TABLE5
Effect of supplementation with docosahexaenoic acid or placebo on selected thrombogenic risk factors in vegetarians!

Control DHASCOâ„¢

Week

Variable

Platelet
ofmaximumaggregation,2 %
aggregationwith
collagenTxBj
10 mg/ L ±196.3 2.4± 1.9± 3.2±28.2± ±176.1 ±159.2
x10«
production, ng/2.5
plateletsRelative ±1.5 21.8± 24.3± ±1.4 ±1.5
serumViscosity ±2.3 0.1± 0.0± 0.0± ±1.9 ±1.9
water,where
relative to
1.0Fibrinogen,
water =
g/LFactor ±119.9 0.1± 0.1± 0.1± ±103.1 ±122.0
%prothrombin
Vile,
time83.5 7.389.5189.11.52.3125.3±
±4.626.90.00.110.587.3186.01.42.299.3± 10.486.1180.61.51.8117.8±
11.086.9 ±2.917.90.10.15.588.7
±1.724.30.00.18.1

1 Values are reported as means ±SEM,n = 12. No significant differences due to supplementation were found.
2 Values for platelet aggregation are for 2 mg/L collagen as a percentage of maximum aggregation with 10 mg/L collagen.
3 TxB2, thromboxane 82.

DHA, was further accompanied by a marked decrease
in docosapentaenoic acid [22:5(n-3)j. The latter fatty
acid generally exhibits considerable enrichment in se
rum and platelet phospholipids when fish oils [provid
ing EPA, DHA and some 22:5(n-3)] are fed (Herold and
Kinsella 1986, Weaver and Holub 1989). Thus, dietary
DHA itself appears to replace both 22:5(n-6) and 22:5(n-
3) in circulating and cellular phospholipids in human
subjects, likely by competition from DHA-containing
precursors, via combined de novo phospholipid biosyn-
thetic pathways and deacylation/reacylation reactions.
Clinical trials with fish oils containing EPA and
DHA (Harris 1989) have generally shown a significant
decrease in serum triglycérideconcentrations and often
a rise in LDL-cholesterol. These trials (Harris 1989)
have employed fish oil concentrates which usually con
tain much more EPA than DHA. Because the EPA-free
DHASCOâ„¢ used in this study as well as the EPA-
containing concentrate of DHA used previously (von
Schacky and Weber 1985) both lowered serum triglyc
érideconcentrations, dietary DHA may produce a tri-
glyceride-lowering effect independent of EPA. The rela
tively minor retroconversion of DHA to EPA is likely
insufficient to account for the observed alterations in
serum lipids and lipoproteins. In our study, the use of
EPA-free DHASCOâ„¢ indicated that dietary DHA itself
can provide a moderate reduction in the total-cho-
lesterohHDL-cholesterol ratio and the LDL-choles-
terohHDL-cholesterol ratio as well as a slight reduction
in serum triglycérideconcentrations of vegetarian sub
jects (Table 4). These changes are consistent with a
favorable influence of dietary DHA on these recognized
lipid/lipoprotein risk factors for cardiovascular disease
(Drexel et al. 1994). Recent publications (Kinosian et al.
1994 and 1995) indicate that the total cholesterohHDL-
cholesterol and LDL-cholesterol:HDL-cholesterol ra
tios are better predictors of heart disease than total
cholesterol or LDL-cholesterol levels alone. Supple
mentation with fish oils containing EPA plus DHA for
several weeks has generally not significantly reduced
LDL-cholesterol:HDL-cholesterol ratios (Harris 1989).
It remains to be studied whether the moderate lipid/
lipoprotein shifts as seen in our vegetarian subjects on
relatively low fat diets (23% of energy) are also seen in
other population and dietary groups.
Although many previous studies, often using intakes
of EPA plus DHA ranging from 3 to 6 g/Ã ,have shown
a significant reduction in collagen-induced platelet ag
gregation and thromboxane formation, this was not ob
served (Table 5) in our subjects consuming 1.62 g
DHASCOâ„¢/d for 6 wk. One previous study, using
much higher levels of DHA (6 g/d) over 6 d, reported
a reduction in collagen-induced aggregation (von
Schacky and Weber 1985). Further, the absence of any
significant effect of DHA supplementation on other
thrombogenic risk factors (viscosity, fibrinogen, factor
Vile) in our trial can be compared with various studies
with fish oils (EPA/DHA) in which either no change
or some beneficial effects have been reported (Harris
1989, Herold and Kinsella 1986, Saynor and Gillott
1992, Weaver and Holub 1988). Future studies which
employ varying dietary levels of DHA, including vary
ing EPA/DHA ratios, will be of interest.
The marked rise in the DHA level of serum and
platelet phospholipids of our subjects is of interest in
view of recent epidemiological studies which have
shown an inverse relationship between DHA levels in
the population (both diet and blood) and the risk of

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 3038 CONQUER
CVD (Leng et al. 1994, Simon et al. 1995). Thus, part
of the cardioprotective effect of fish/fish oils containing
(n-3) PUFA appears due to DHA in addition to EPA.
The cardioprotective effects of (n-3) fatty acids are con
sidered to be mediated by a number of physiological
and biochemical mechanisms; included among these
are studies which indicate that the enrichment of heart
tissue in DHA provides an antiarrhythmic effect (Pepe
and McLennan 1996), which may account for the re
duction in cardiac arrest and sudden cardiac death in
those having a higher DHA status. In addition to the
above, dietary DHA intakes and increased status in
the body have been implicated in favorable effects on
attention-deficit disorders (Stevens et al. 1995), depres
sion and anxiety disorders (Hibbeln and Salem 1995),
as well as protection against breast cancer in postmeno-
pausal women (Zhu et al. 1995).
In conclusion, the consumption of 1.62 g of an ani
mal-free source of DHA per day by vegetarians readily
enhances their DHA status, provides for EPA formation
based on serum and platelet phospholipid analysis, and
exerts moderately favorable (lowering) effects on the
total cholesterol:HDL-cholesterol ratio, the LDL-cho-
lesterol:HDL-cholesterol ratio, as well as serum triglyc
érideconcentrations. The present studies, conducted
with young non-hyperlipidemic vegetarian adults of
both genders, should now be extended to other groups
(e.g., omnivorous normolipidemics and hyperlipid-
emics).
ACKNOWLEDGMENTS
We would like to thank Margaret Berry, Indù Mani
and Melinda Gooderham for their help in all aspects of
this investigation. We would also like to thank David
Kyle and Eeva-Kaarina Koskelo of Martek Biosciences
Corporation (Columbia, MD) for supplying both the
control and DHA capsules for this investigation. Ap
preciation is also extended to Martek for covering the
costs of the fatty acid analyses as provided by Lipid
Analytical Laboratories, University of Guelph Re
search Park (Guelph, Ontario, Canada).
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