Favorable effects of blue-green algae Aphanizomenon flos-aquae on rat plasma lipids

Abstract

Background: Polyunsaturated fatty acids (PUFAs) are essential for human health. There are indications that the lipid fraction of blue-green algae Aphanizomenon flos-aquae contains about 50% PUFA and may be a good dietary source of PUFA. The purpose of this study was to investi-gate the effect of diets supplemented with algae on blood plasma lipids. Methods: Rats were fed with four different semisyn-thetic diets: 1) standard, with 5% soybean oil; 2) PUFA-free with 5% coconut oil; 3) PUFA-free with 10% algae; 4) PUFA-free with 15% algae. After 32 days the levels of plas-ma fatty acids, triglycerides, and cholesterol were studied. Results: Rats fed the PUFA-free diet demonstrated an absence of linolenic acid (LNA) in plasma; however, sup-plementation with algae resulted in the same level of LNA as controls, increased levels of eicosapentaenoic acid and docosahexaenoic acid, and a decreased level of arachidonic acid. Dietary supplementation with 10% and 15% algae decreased the plasma cholesterol to 54% and 25% of the control level, respectively (p<0.0005). Plasma triglyceride levels decreased significantly (p<0.005) after diet supple-mentation with 15% algae. Conclusion: Algae Aphanizomenon flos-aquae is a good source of PUFA and because of potential hypocholesterolemic properties should be a valuable nutritional resource.

Full text

January 2000 Vol.2, No. 3 JANA 59
ORIGINAL RESEARCH
Favorable Effects of Blue-Green Algae
Aphanizomenon flos-aquae on Rat Plasma Lipids
Rafail I. Kushak, PhD,1* Christian Drapeau, MS, Elizabeth M. Van Cott,1
Harland H. Winter1
1Combined Program in Pediatric Gastroenterology and Nutrition and Division of Laboratory Medicine
Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Background: Polyunsaturated fatty acids (PUFAs) are
essential for human health. There are indications that the
lipid fraction of blue-green algae Aphanizomenon flos-
aquae contains about 50% PUFAand may be a good dietary
source of PUFA. The purpose of this study was to investi-
gate the effect of diets supplemented with algae on blood
plasma lipids.
Methods: Rats were fed with four different semisyn-
thetic diets: 1) standard, with 5% soybean oil; 2) PUFA-free
with 5% coconut oil; 3) PUFA-free with 10% algae; 4)
PUFA-free with 15% algae. After 32 days the levels of plas-
ma fatty acids, triglycerides, and cholesterol were studied.
Results: Rats fed the PUFA-free diet demonstrated an
absence of linolenic acid (LNA) in plasma; however, sup-
plementation with algae resulted in the same level of LNA
as controls, increased levels of eicosapentaenoic acid and
docosahexaenoic acid, and a decreased level of arachidonic
acid. Dietary supplementation with 10% and 15% algae
decreased the plasma cholesterol to 54% and 25% of the
control level, respectively (p<0.0005). Plasma triglyceride
levels decreased significantly (p<0.005) after diet supple-
mentation with 15% algae.
Conclusion: Algae Aphanizomenon flos-aquae is a good
source of PUFAand because of potential hypocholesterolemic
properties should be a valuable nutritional resource.
INTRODUCTION
Previous research identified the important role of
dietary polyunsaturated fatty acids (PUFA) in human
health. A deficiency in n-3 PUFA has been linked to
immunosuppression,1arthritis,2cardiovascular diseases,3-6
mental7,8 and dermatological9problems. Human and ani-
mal models containing n-3 PUFAs have anti-inflammatory
activity2,10,11 that may be mediated by decreasing the
arachidonic acid level and thereby suppressing the produc-
tion of specific cytokines.12 Furthermore, n-3 fatty acids
have been shown to decrease certain cancer risks,13,14 pre-
vent platelet aggregation,6,15 and to lower blood choles-
terol, possibly by stimulating its excretion into bile.3,16
The North American diet is believed to be deficient in
PUFA, especially in n-3 fatty acids.17 Dietary supplementa-
tion with fish oil rich in n-3 eicosapentaenoic (EPA) and
docosahexaenoic acids (DHA) has been recommended as a
potential treatment for hypercholesterolemia.15,18 Much
empirical evidence over the past decade suggests that
Aphanizomenon flos-aquae (Aph. flos-aquae), a blue-green
alga growing naturally in Upper Klamath Lake, Oregon,
may be a good dietary source of PUFA. Nearly 50% of the
lipid content of dried Aph. flos-aquae (5% to 9% of total dry
weight) is composed of PUFA, mostly n-3 α-linolenic acid.
In our experiments using rats as the animal model,
Aph. flos-aquae not only served a source of dietary PUFA
but also significantly lowered blood cholesterol and triglyc-
eride levels.
METHODS
Animals: Thirty-two adult male Sprague-Dawley rats
were randomly distributed into 4 groups. Animals were
placed into individual wire cages, and maintained at 22° C
with a 12-hour light-dark cycle. Food and water were sup-
plied ad libitum. For 32 days the animals were fed with the
following semipurified test diets based on the American
* Correspondence:
Rafail Kushak, PhD., Dr. Sci.
Pediatric Gastroenterology & Nutrition
Massachusetts General Hospital
55 Fruit Street, VBK 107
Boston, MA 02114-2698
Phone: (617) 726-7451
Fax: (617) 724-2710
E-mail: Kushak.Rafail@mgh.harvard.edu
Reprinted with permission from the Journal of the
American Nutraceutical Association.

January 200060 JANA Vol. 2,No. 3
Institute of Nutrition (AIN-76) standard:
1. Standard diet containing 5% soybean oil (SBO);
2. PUFA-deficient diet containing 5% coconut oil (PUFA-D);
3. PUFA-deficient diet containing 10% algae (Alg10);
4. PUFA-deficient diet containing 15% algae (Alg15).
The algal material used in this study was supplied by Cell
Tech (Klamath Falls, OR) and contained 6.3% lipids. Feed
was provided by Purina Test Diets (Richmond, IN).
After the feeding trial, the animals were fasted
overnight and euthanised by carbon dioxide inhalation.
Plasma was collected by heart puncture in a tube containing
100 µl 0.5 M EDTA (pH 8.0), centrifuged at 3,000 g for 15
minutes, and stored at -80°C.
Lipid Analysis: Blood fatty acid analysis was per-
formed using a direct transesterification method19 as modi-
fied by Mosers.20 In brief, 250 µl of plasma was vortexed
with 1 ml methanol:methylene chloride (3:1). 50 nmol of
17:0 free fatty acid (internal standard) in 50 µl of hexane
was added to this mixture. Under continuous vortexing 200
µl of acetyl chloride was added and the mixture was incu-
bated in the oven at 75oC for one hour. After cooling for 15
min at room temperature 4 ml of 7% potassium carbonate
was added, vortexed, and then 2 ml of hexane was added.
The mixture was vortexed for 60 sec and then centrifuged
at 1750 g for 10 min at 4oC. The hexane layer was removed,
2 ml of acetonitrile was added and the mixture was cen-
trifuged at 1120 g for 5 min at 4oC. The hexane layer was
removed, dried under nitrogen to a final volume of approx-
imately 100 µl, and 1 µl of the sample was used for analy-
sis. Fatty acid identification was performed on a Hewlett-
Packard 5890 series II model gas chromatograph-mass
spectrometer GC-MC with a Hewlett-Packard 5971 mass
spectrometer (Hewlett-Packard, Wilmington DE). Soybean
and coconut oils were methylated by acid methanolysis
before fatty acid analysis. The algae material was soaked in
methanol, extracted and then methylated by acid methanol-
ysis prior to fatty acid analysis.
Plasma triglycerides and cholesterol were measured on
the automated clinical chemistry analyzer Roche BHO/H917
using corresponding Boehringer Mannheim kits.
Statistics: Statistical difference between groups was
determined using unpaired Student’s t-test. Difference in
fatty acid profiles was evaluated using repeated measures
analysis and contrast tests21. For all analysis, differences of
p<0.05 were considered statistically significant.
RESULTS
Dietary Fatty Acids: Fatty acid composition of Aph.
flos-aquae, soybean oil and coconut oil used in this study is
represented in Table 1. The composition of soybean and
coconut oil in the present study is close to that found in the
TABLE 1
Fatty acid composition (% of total fatty acids)
of soybean oil, coconut oil, and algae
Fatty Acid Source of Fatty Acids
Soybean oil Coconut oil Algae
Caprylic (8:0) - 9.70 -
Capryc (10:0) - 7.50 -
Lauric (12:0) - 42.10 -
Myristic (14:0) - 22.40 9.10
Palmitic (16:0) 14.69 18.20 36.60
Palmitoleic (16:1) - - 11.90
Margaric (17:0) - - 0.89
Stearic (18:0) 5.40 - 2.70
Oleic (18:1) 26.80 - 6.70
Linoleic (18:2n-6) 44.40 - 7.40
Linolenic (18:3n-3) 8.00 - 22.30
Arachidic (20:0) 0.35 0.14 -
Arachidonic (20:4n-6) - - 0.65
Eicosapentaenoic (20:5n-3) - - 0.08
Behenic (22:0) 0.33 - -
Total polyunsaturated 52.40 - 30.43
Total saturated 20.77 100.04 49.29
Table 2
Lipid composition (%) of experimental diets
Indices Diets
SBO PUFA-D Alg10 Alg15
Oil Source
Soybean oil 5.00 0.00 0.00 0.00
Coconut oil 0.00 5.00 4.50 4.250
Algae 0.00 0.00 10.00 15.00
Total fat 5.00 5.00 5.13 5.20
Fatty Acid Content
Linoleic acid 2.22 0.00 0.05 0.07
(18:2n-6)
Linolenic acid 0.40 0.00 0.14 0.21
(18:3n-3)
Total polyunsaturated 2.62 0.00 0.19 0.28
(PUFA)
Lauric acid (12:0) 0.00 2.11 1.89 1.79
Myristic acid (14:0) 0.00 1.12 1.07 1.04
Palmitic acid (16:0) 0.73 0.91 1.05 1.12
Stearic acid (18:0) 0.27 0.00 0.02 0.03
Oleic acid (18:1) 1.34 0.00 0.04 0.06
Total saturated (SFA) 1.00 4.14 4.03 3.95
PUFA/SFA 2.62 0.00 0.05 0.07
n-6/n-3 5.55 - 0.36 0.36

January 200064 JANA Vol. 2,No. 3
were similar in rats fed SBO and algae supplemented diets,
there were significantly higher blood levels of EPA in the
rats fed the Aph. flos-aquae diet. It has been previously sug-
gested that increased dietary SFA increased the rate of con-
version of LNA to EPA, whereas increased dietary n-6
PUFA decreased this conversion by 40-50%.23 This dual
effect could explain the fact that rats fed algae supplement-
ed diets, which contained significantly more SFA, had high-
er blood levels of EPA than rats fed the SBO diet, which
contained significantly more LA.
When the two main plasma n-6 PUFA (LA and AA)
were analysed as profile, there was a very good positive
correlation between LA dietary intake and the total level of
n-6 PUFA. However, the n-6 PUFA profiles in rat plasma
were different between the various groups. Supplementing
diets with algae led to a dose-dependent decrease in plasma
AA and concomitant accumalation of of LA. This could be
due to Aph.flos-aquae’s content of phycocyanin.
Phycocyanin, the blue pigment in blue-green algae, was
recently shown to have significant anti-inflammatory prop-
erties24,25 which seemed to be mediated by an inhibition AA
metabolism.26 The presence of phycocyanin in the algae
supplemented diets may have inhibited AA synthesis and
consequently promoted the accumulation of LA.
This study suggests that Aph. flos-aquae has significant
hypocholesterolemic properties when compared to soybean
oil. Many studies have demonstrated the hypocholes-
terolemic properties of n-3 PUFAs16,27,28 and the negative
correlation between PUFA/SFA ratio and blood cholesterol
levels.29,30 In this study, cholesterol levels were positively
correlated with the PUFA/SFA ratio. The main SFA present
in the diet of the algae-treated groups were lauric, myristic
and palmitic acids, which were all demonstrated to promote
hypercholesterolemia to some degree.31-33 This suggests that
the hypocholesterolemic effect of Aph. flos-aquae is likely to
be mediated by factors other than its fatty acid content.
Specifically Aph. flos-aquae contains a significant amount
of chlorophyll (1-2% dry weight) which was shown to stim-
ulate liver function, and increase bile secretion34. A synthet-
ic derivative of chlorophyll was shown to reduce blood cho-
lesterol.35 Therefore, it is possible that Aph. flos-aquae
chlorophyll is responsible for the increased liver function
and secretion of cholesterol into bile. Spirulina, another
blue-green algae, was also shown to affect cholesterol
metabolism by increasing HDL levels. 36 According to other
sources37, hypocholesterolemic effect of blue-green algae
(Nostoc commune) is related to their fibers.
In conclusion, this study demonstrated that Aph. flos-
aquae is a good source of PUFA with strong hypocholes-
terolemic properties. Aph. flos-aquae's ability to increase
serum level of LNA, EPA, DHA, and lower level of AA in
rats makes it a good candidate for future nutritional
research in humans.
ACKNOWLEDGMENT
We are indebted to Dr. David J. Schaeffer, for assis-
tance in statistical analysis and to Dr. M. Laposata for the
critical review of the manuscript. We are also grateful for
the grant from the Clinical Nutrition Reseach Center
at the Massachusetts General Hospital (P30 DK40561).
REFERENCES
1. De Wille JW, Fraker PJ, Romsos DR. Effect of essen-
tial fatty acid deficiency and various levels of dietary
polyunsaturated fatty acids on humoral immunity in
mice. J Nutr 1979;109:1018-1027.
2. Kremer JM, Lawrence DC, Jubiz W. Different doses of
fish-oil fatty acid injection in active rheumatoid arthri-
tis: a prospective study on clinical and immunological
parameters. In: Galli C, Simopoulosap C. (Eds)
Dietary LW3 and LW6 Fatty Acids: Biological Effects
and Nutritional Effects and Nutritional Essentiality
1989; Plenum Publishing, New York pp. 343-350.
3. Simopoulos AP Omega 3 fatty acids in health and dis-
ease and in growth and development. Am J Clin Nutr
1991:54;438-463.
4. Kromhout D. Fish (oil) consumption and coronary heart
disease. In: Galli C, Simopoulosap C. (Eds) Dietary
LW3 and LW6 Fatty Acids: Biological Effects and
Nutritional Effects and Nutritional Essentiality 1989;
Plenum Publishing, New York pp. 273-282.
5. Moller JM, Nielsen GL, Ekelund S, Scmidt EB,
Dyerberg J. Homocysteine in Greenland Inuits.
Thromb Res 1997;86:333-335.
6. Nordoy A, Simonsen T. Dietary n-3 fatty acids, experi-
mental thrombosis and coronary heart disease in man.
In: Lands, Biloxi WEM. (Eds) Proceedings of the
AOCS Short Course on Polyunsaturated Fatty Acids
and Eicosanoids 1987: Mississippi, American Oil
Chemists Society, pp. 25-34.
7. Hibblen JR, Salem NJ. Dietary polyunsaturated fatty
acids and depression: when cholesterol does not satis-
fy. Am J Clin Nutr 1995;62:1-9.
8. Stevens, LJ, Zentall SS, Deck JL, Abate ML, Watkins
BA, Lipp SR, Burgess JR. Essential fatty acid metab-
olism in boys with attention-deficit hyperactivity disor-
der. Am J Clin Nutr 1995;62:761-768.
9. Wright, S. and Burton, J.L. Oral evening primrose seed
oil improves atopic eczema. Lancet 1982:1120-1122.
10. Kremer JM, Jubiz W, Michalek A, Rynes RI,
Bartholomew LE, Bigaoutte J, Timchalk M, Beeler D,
Lininger L. Fish-oil fatty acid supplementation in
active rheumatoid arthritis. A double-blind controlled,
crossover study. Ann Intern Med 1987;106:493-503.
11. Raederstorff D, Pantze M, Bachmann H, Moser U Anti-
inflammatory properties of docosahexaenoic and eicosapen-
taenoic acids in phorbol-ester-induced mouse ear inflamma-
tion. Int Arch Allergy Immunol 1996;111:284-290.

January 2000 Vol.2, No.3 JANA 65
12. Enders S, Ghorbani R, Kelley VE, Georgilis K,
Lonnemann G, van der Meer JWM, Cannon GJ, Rogers
TS, Klempner MS, Weber PC, Schaefer EJ, Wolff SM,
Dinarello CA. The effect of dietary supplementation
with n-3 polyunsaturated fatty acids on the synthesis of
interleukine-1 and tumor necrosis factor by mononu-
clear cells. N Engl J Med 1989;320:265-271.
13. Narisawa T, Fukaura Y, Yazawa K, Ishikawa C, Isoda Y,
Nishizawa Y. Colon cancer prevention with a small
amount of dietary perilla oil high in alpha-linolenic
acid in an animal model. Cancer 1994;73:2069-2075.
14. Stoll BA. Essential fatty acids, insulin resistance, and
breast cancer risk. Nutr Cancer 1998;31:72-77.
15. Smith DL, Wills AL, Nguyen N, Conner D, Zahedi S,
Fulks J.Eskimo plasma constituents, dihomo-γ-
linolenic acid, eicosapentaenoic acid and docosa-
hexaenoic acid inhibit the release of atherogenic mito-
gens. Lipids 1989;24:70-75.
16. Balasubramaniam S, Simons LA, Chang S, Hickie JB.
Reduction in plasma cholesterol and increase in biliary
cholesterol by a diet rich in n-3 fatty acids in the rat. J
Lipid Res 1985;26:684-689.
17. Stoll, BA Western nutrition and the insulin resistance
syndrome: A link to breast cancer, Eur J Clin Nutr
1999;53:83-87.
18. Clandinin MT, Foxwell A, Goh WK, Layne K, Jumpsen
JA. Omega-3 fatty acid intake results in a relationship
between the fatty acid composition of LDL cholesterol
ester and LDL cholesterol content in humans. Biochim
Biophys acta 1997;1346:247.
19. Lepage G., Roy CC. Direct transesterification of all
classes of lipids in a one step reaction. J Lipid Res
1986; 27:7647-7651.
20. Moser HW, Moser AB. Measurement of very long fatty
acids in plasma. Techniques in Diagnostic Human
Biochemical Genetics: A Laboratory Manual. pp. 177-
191. Willey-Liss, Inc. 1991.
21. SYSTAT, v.8, SPSS, Chicago, IL 1998.
22. United States Department of Agriculture. Agriculture
Handbook. Series No 8-4. Composition of Foods: Fats
and Oils. 1979.
23. Gerster H. Can adults adequately convert alpha-
linolenic acid (18:3n-3) to eicosapentaenoic acid
(20:5n-3) and docosahexaenoic acid (22:6n-3) Int J
Vitam Nutr Res 1998;68:159-173.
24. Romay C, Armesto J, Remirez D, Gonzalez R, Ledon
N, Garcia I Antioxidant and anti-inflammatory proper-
ties of C-phycocyanin from blue-green algae. Inflam
Res 1998;47:36-41.
25. Gonzalez R, Rodriguez S, Romay C, Gonzalez A,
Armesto J, Remirez D, Merino N. Anti-inflammatory
activity of phycocyanin extract in acetic acid-induced
colitis in rats. Pharmacol Res 1999;39:1055-1059.
26. Romay C, Ledon N, Gonzalez R. Further studies on anti-
inflammatory activity of phycocyanin in some animal
models of inflammation. Inflam Res 1998;47:334-338.
27. Ramesha CS, Paul R, Ganguly J. Effect of dietary
unsaturated oils on the biosynthesis of cholesterol, and
on biliary and fecal excretion of cholesterol and bile
acids in rats. J Nutr 1980; 110:2149-2158.
28 . Sugano M, Ide T, Ishida T, Yoshida K. Hypocholesterolemic
effect of gamma-linolenic acid as evening primrose oil
in rats. Ann Nutr Metab 1986;30:289-299.
29. Lee JH, Sugano M, Ide T. Effects of various combina-
tions of omega 3 and omega 6 polyunsaturated fats
with saturated fat on serum lipid levels and eicosanoid
production in rats. J Nutr Sci Vitaminol 1988;34:117-
129.
30. Hirai K, Nakano T, Katayama Y, Amagase S. Serum
cholesterol levels and the ratio of polyunsaturated to
saturated fatty acid as an indicator of lipid metabolism
in rat liver. J Nutr Sci Vitaminol 1985;31:279-289.
31. Nicolosi RJ, Wilson TA, Rogers EJ, Kritchevsky D
Effects of specific fatty acids (8:0, 14:0, cis-18:1, trans-
18:1) on plasma lipoproteins, early atherogenic poten-
tial, and LDL oxidative properties in the hamster. J
Lipid Res 1998;39:1972-1980.
32. Watts GF, Jackson P, Burke V, Lewis B. Dietary fatty
acids and progression of coronary artery disease in
men. Am J Clin Nutr 1996;64:202-209.
33. Salter AM, Mangiapane EH, Bennett AJ, Bruce JS,
Billett MA, Anderton KL, Marenah CB, Lawson N,
White DA. The effect of different dietary fatty acids on
lipoprotein metabolism: concentration-dependent
effects of diets enriched in oleic, myristic, palmitic and
stearic acids. Br J Nutr 1998;79:195-202.
34. Dashwood R, Guo D Protective properties of chloro-
phylls against the covalent binding of heterocyclic
amines to DNA in vitro and in vivo. Princess
Takamatsu Symp 1995;23:181-189.
35. Vlad M, Bordas E, Caseanu E, Uza G, Creteanu E,
Polinicenco C. Effect of cuprofilin on experimental
atherosclerosis. Biol Trace Elem Res 1995;48:99-109.
36. de Caire, GZ, de Cano MS, de Mule CZ, Steyerthal N,
Piantanida M. Effect of Spirulina platensis on glucose,
uric acid and cholesterol levels in the blood of rodents.
Intern J Exper Botany 1995;57:93-96.
37. Hori K, Ishibashi G, Okita T Hypocholesterolemic effect of
blue-green algae, ishikurage (Nostoc commune) in rats fed
atherogenic diet. Plant Food Human Nutr 1994;48:63-70.