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The Journal of the American Nutraceutical Association www.ana-jana.org
Vol. 2, No. 3, January 2000 Reprint
A Peer-Reviewed Journal on Nutraceuticals and Nutrition
Mark Houston, MD
Editor-in-Chief
ISSN-1521-4524
REPRINT OF ORIGINAL RESEARCH ARTICLES
Consumption of Aphanizomenon flos-aquae
Has Rapid Effects on the Circulation of
and Function of Immune Cells in Humans
Gitte S. Jensen, PhD, et al.
_______________
Favorable Effects of Blue-Green Algae
Aphanizomenon flos-aquae on Rat Plasma Lipids
Rafail Kushak, PhD, et al.
ORIGINAL RESEARCH
Consumption of Aphanizomenon flos-aquae
Has Rapid Effects on the Circulation and
Function of Immune Cells in Humans
A novel approach to nutritional mobilization of the immune system
Gitte S. Jensen, PhD,*1Donald I. Ginsberg,1Patricia Huerta,1Monica Citton,1Christian Drapeau, MS2
1Department of Surgery, McGill University, Montreal, Quebec
2Research and Development, Cell Tech, Klamath Falls, Oregon
ABSTRACT
Objective: To examine the short-term effects of con-
sumption of a moderate amount (1.5 grams) of the blue-
green algae Aphanizomenon flos-aquae (AFA), on the
immune system.
Methods: Using a crossover, placebo-controlled, ran-
domized, double-blind design, 21 volunteers were studied,
including 5 long-term AFA consumers.
Results: Consumption of a moderate amount (1.5
grams) of the blue-green algae Aphanizomenon flos-aquae
results in rapid changes in immune cell trafficking. Two
hours after AFA consumption, a generalized mobilization of
lymphocytes and monocytes, but not polymorph nucleated
cells, was observed. This included increases in CD3+,
CD4+, and CD8+ T cell subsets and CD19+ B cells. In
addition, the relative proportions and absolute numbers of
natural killer (NK) cells were reduced after AFA consump-
tion. No changes were observed in the relative proportions
of naïve versus memory T cells, neither in the CD4 nor the
CD8 fractions. A mild but significant reduction in phago-
cytic activity was observed for polymorph nucleated cells.
When freshly purified lymphocytes were exposed to AFA
extract in vitro, direct activation was not induced, as evalu-
ated by tyrosine phosphorylation and proliferative activity.
Discussion: The changes in immune cell trafficking
displayed a high degree of cell specificity. Long-term
consumers responded stronger with respect to altered
immune cell trafficking. In vitro, AFA did not induce a
direct activation of lymphocytes. These data support a sig-
naling pathway from gut to CNS to lymphoid tissue. The
signals from CNS may be crucial for the rapid changes in
the general distribution and specific recruitment we
observed. Moderate anti-inflammatory modulation may
account for the modification of phagocytic activity.
Conclusion: Consumption of AFA leads to rapid
changes in immune cell trafficking, but not direct activation
of lymphocytes. Thus, AFA increases the immune surveil-
lance without directly stimulating the immune system.
KEYWORDS: Lymphocyte trafficking, natural killer
cells, phagocytes.
INTRODUCTION
Blue-green algae are among the most primitive living
organisms on Earth. Though they are technically classified
as bacteria, they share properties with bacteria and with
plants. They contain many biologically active substances
that have beneficial effects on human health. Thus, a large
research interest in the use of blue-green algae for food sup-
plementation has emerged. Several blue-green algae,
including Aphanizomenon flos-aquae (AFA) have pro-
nounced antibacterial properties1and have protective
effects in the classical AMES test.2The blue-green algae
Spirulina has documented antiviral3,4 and anticancer5,6
properties. In addition, Spirulina subspecies have effects
50 JANA Vol. 2, No. 3 January 2000
* Correspondence:
Dr. Gitte S. Jensen
Surgical Research Labs H6.33
Royal Victoria Hospital
687 Pine Avenue West
Montreal Quebec H3A 1A1 Canada
Phone: (514) 842-1231, ext 4497
Fax: (514) 843-1411
Email: gjense@po-box.mcgill.ca
Reprinted with permission from the Journal of the
American Nutraceutical Association.
January 2000
on the immune system, by enhancing the phagocytic activ-
ity in macrophages7,8, inhibiting allergic reactions in
rodents,9-11 and by enhancing antigen-specific antibody pro-
duction and proliferative responses in chickens.8Other
algae contain sulfolipids with potent anti-viral properties.12
Thus, blue-green algae species contain phytochemicals that
are potent modulators of certain immune functions.
The trafficking of immune cells between various loca-
tions is an important aspect of the healthy immune system,
as part of scavenging for invading pathogens, infected or
transformed cells. The various cell types that constitute our
immune system are present throughout almost all tissues in
our body. The absolute and relative amounts of trafficking
immune cells in the blood is rapidly altered in response to
chemical messenger molecules.The monitoring of these
changes are widely used to evaluate the short-term immune
changes to various physical, chemical, and psychological
stressors. The various populations of immune cells in nor-
mal blood is depicted in Figure 1, along with the surface
markers used for their identification.
Trafficking cells re-circulate between various anatomi-
cal locations by entering the blood stream. In order for the
cells to exit the blood and enter a new anatomical location,
they must be able to adhere and migrate. In almost all tissue
(spleen and liver being exceptions), the cells must perform a
specific series of tasks in order to transmigrate: 1) Slow
down the speed by forming loose adhesion on the vessel
wall, and rolling along the endothelial surface, 2) Form a
strong adhesion onto the endothelium, and 3) Migrate
through the endothelial barrier and the underlying basement
membrane.13-15 These events are mediated by a combination
of chemotactic factors and adhesion molecules. The circu-
lating cells are able to “sense” sites of cellular recruitment
via chemokines bound to the endothelium or secreted into
Vol.2,No. 3 JANA 51
Figure 1: Schematic diagram of the relative proportions of white
blood cells and the markers associated with their identification.
the lumen of the blood vessel. Alarge number of chemokines
are known, and they are able to activate cell subsets in a high-
ly selective manner.16 Of interest for our data are the
chemokines involved in recruitment of natural killer (NK)
cells into tissue. Seven out of 8 tested C-C chemokines
induced chemotaxis of NK cells,17 as well as fractalkine.18
Another chemokine of interest is lymphotactin, which elicits
a migratory response in NK and T cells, while having no
effect on monocytes and neutrophils.19 Thus, mechanisms
are in place to mediate highly selective patterns of migration
and recruitment of specific leukocyte subpopulations.
The re-circulation pattern of immune cells varies in a
circadian pattern, which is dependent on neuro-endocrine
signals. In one study, a clear circadian rhythm is seen for T
cell subsets, but not for NK cells,20 whereas another study
reported a clear increase of NK numbers and activity in the
morning.21 It is believed that high levels of cortisol in the
beginning of the day interfere with interleukin-2 production
and enhances the migration of lymphocytes from the blood
into tissues. Other mechanisms of inducing high levels of
cortisol (stress, exercise),22-24 as well as injection of hydro-
cortisone25 have similar inhibitory effects on lymphocyte
migration. Importantly, species variations exist, and stress
experiments in rodents cannot directly be compared to
human studies. The different physiological responses to
various stressors in the human system may be difficult to
understand in the light of how apparently similar stressors
are perceived in laboratory animals.
The recruitment of NK cells is very sensitive to cate-
cholamines, especially epinephrine.26 The catecholamines
have a negative effect on the adhesion of NK cells to the
vessel walls, and causes the NK cells to detach. The
changes in NK cell trafficking is not accompanied by
changes in adhesion molecule expression on the circulating
NK cells. The catecholamine-induced accumulation of NK
cells in the blood was identical in normal and splenec-
tomized donors, indicating that the spleen was not the rele-
vant reservoir of NK cells.27 Several studies have reported
a stress-mediated increase in numbers of B and NK cells in
blood.28,29
Throughout the body, many nerve factors are able to
function as chemokines, and immune cells express recep-
tors for neurotransmitter molecules. Only some cytokines
are regulated by cortisol, and a hierachy of cortisol-sensi-
tivity has been proposed.30 The bi-directional relationship
between neuronal and immunological systems extends to
the lymphoid tissues. In addition to the well characterized
central nervous system regulation of adrenals, nerve termi-
nals invade all lymphoid tissue, and synapse-like forma-
tions can be seen between nerve endings and immune cells
in bone marrow, lymph nodes and spleen.31 Neuronal con-
trol of haematopoiesis has been studied in detail, and a
complex feedback system exists, involving multiple
cytokines and neurotransmitters.32,33 Neuropeptide Y is an
January 2000
example of a neurotransmitter that is directly able to upreg-
ulate adhesion molecules on human endothelial cells.34
The central nervous system regulation of immune sur-
veillance is of functional importance. In mice, when sig-
naling from the sympathetic nervous system to the periph-
ery was interrupted prior to injection of NK-sensitive tumor
cells, the numbers of metastases were significantly
increased.35 As the NK activity was not altered, nor was the
ability to respond to tumor antigens, one possible explana-
tion is that the sympathetic nervous system regulates either
NK trafficking or matrix deposition in tissue, thereby regu-
lating the ability of NK cells to migrate to the vicinity of
tumors. This was partially confirmed by demonstrating that
the sympathetic nervous system modulates lymphocyte
recruitment into lymph nodes.36
Consumption of the blue-green algae AFA has
increased, and despite a large number of anecdotal reports
on health benefits, studies of the exact mechanisms of
AFA's effects on immune function were needed. In a pre-
vious brief report, we presented preliminary data to show
that AFA induced a rapid induction of NK cell recruitment
into tissue in humans.37 Since then, we have analyzed the
migratory patterns of multiple white blood cell types in a
total of 21 study subjects. Upon oral consumption of 1.5
grams AFA, we observed immediate changes in several
specific immune parameters.
MATERIALS AND METHODS:
Subjects: Twenty-one non-hospitalized volunteers were
analyzed in a double-blinded cross-over fashion, upon
informed consent. The volunteers had no known acute or
chronic infections. Five were long-term AFA consumers, 2
were occasional AFA consumers, and the remaining 14 had
never before consumed AFA. Occassional consumers had pre-
viously used AFA daily for at least 6 weeks continuously, but
were not consuming AFA regularly during the weeks leading
up to this study. No volunteer had taken AFA for at least 24
hours prior to being studied. Ten volunteers were male, and
eleven were female. The age range was 20-52 years.
Study design: Each volunteer was studied on two
separate days. Any volunteer was always studied at the
same time on the two study days, to eliminate the circadian
influence on the data. The volunteers were asked to con-
sume the same breakfasts at the same times on the two
study days, and not to consume any other vitamin prepara-
tions or nutraceuticals for at least 12 hours before the study.
The volunteers were required to sit quiet for 45 minutes
prior to study start, so that any prior walking or other exer-
cise did not affect the relative proportions of leukocytes.
The first blood sample was taken, and the substance was
given. Until the sampling of the second blood sample 2
hours later, the volunteer was required to remain quiet and
avoid any extensive walking.
52 JANA Vol. 2, No. 3
Consumables and reagents: Both AFA and placebo
were provided by Cell Tech (Klamath Falls, Oregon). The
dose given to the volunteers was 1.5 grams, which is the
recommended dose for daily supplementation. A list of
monoclonal antibodies used for immunostaining and flow
cytometry is listed in Table 1.
CD# Clone Specificity Source
CD3 SK7 TCR complex Becton-Dickinson
CD4 SK3 helper/inducer T cells Becton-Dickinson
CD8 SK1 cytotoxic T cells Becton-Dickinson
CD11a 25.3 Alpha-L chain (Beta-2 integrin) Immunotech
CD11b D12 Alpha-M chain (Beta-2 integrin) Becton-Dickinson
CD14 MoP9 PI-anchored receptor, binds LPS Becton-Dickinson
CD18 7E4 Beta-2 subunit (Integrin) Immunotech
CD19 89B (B4)B cell surface molecule Coulter
CD29 3S3 Beta-1 subunit (Integrin) Serotec
CD44 F1044-2 H-CAM pgp-1 Serotec
CD49d L25 Alpha-(VLA)-4 chain (integrin) Becton-Dickinson
CD62L TQ1 L-selectin Coulter
Purification of mononuclear cells: Fourteen ml of
heparinized or EDTA blood was drawn from a peripheral
vein. The blood was layered onto a Ficoll gradient and cen-
trifuged to purify the peripheral blood mononuclear cells.
Cells were washed, and used for direct immunofluores-
cence labeling. Samples were fixed in 1% formalin and
stored cold and dark prior to flow cytometric analysis.
Flow cytometry: Data were acquired and stored on
list mode for subsequent data analysis. The CellQuest soft-
ware (Becton Dickinson) was used for acquisition and
analysis. During analysis, electronic gating was used to
eliminate red cells and clumps from the analysis.
Data analysis: The relative proportions of mono-
cytes, B and T lymphocytes and T cell subsets were calcu-
lated based on positivity for the MoAbs listed in Table 1.
The relative proportion of of NK cells was calculated by
excluding monocytes and large granular cells from the
analysis, then excluding the CD3+ cells, and evaluating the
proportion of CD56+ cells in the sample. The number of
CD3-CD56+ small lymphocytes was then related to the
total number of PBMC. Changes were calculated by com-
paring the AFA- and placebo-induced values for each vol-
unteer. Figure 1 gives a representation of the various cell
types tested, their relationship and the marker used for
quantification. By combining the relative proportions with
actual cell counts, the absolute numbers of peripheral blood
mononuclear cells and PMNs were calculated in 12 volun-
teers. Also, the changes in absolute numbers of the follow-
ing subpopulations were calculated: monocytes, CD3+ T
Table 1: List of monoclonal antibodies used in this study.
January 2000 Vol.2, No. 3 JANA 53
cells, CD19+ B cells, CD4+CD45R0-/+ and
CD8+CD45R0-/+ subsets.
Purification of neutrophils: Seven ml of heparinized
whole blood was mixed with 1.5 ml of 6% dextran70 in
0.9% saline at room temperature. Sedimentation was
allowed for 1 hour. The leukocyte rich supernatant was har-
vested and the cells pelleted by centrifugation. The pellet
was resuspended in 2 ml phosphate buffered saline, which
was then layered on top of 3 ml of Ficoll-Hypaque. Gradient
centrifugation was performed, and the pellet was resuspend-
ed in 0.5 ml of phosphate buffered saline. The remaining red
blood cells were lysed by hypotonic shock for 25 seconds,
after which isotonicity was restored. Cells were washed,
resuspended in RPMI, and kept on ice until use.
Assay for PMN phagocytic activity: The ability of
PMN cells to kill Staphylococcus Aureus bacteria was per-
formed as follows: S. Aureus (frozen aliquots) were
defrosted and washed. The bacteria were then opsonized
with pooled human serum for 30 minutes in a 37oC shaking
water bath. PMN cells and bacteria were added to a series
of tubes, and incubated in a 37oC shaking water bath. At
the following time points: 5, 15, 30, and 45 minutes a tube
was removed, immediately placed on ice, and 0.5 ml
icecold serum added in order to stop further phagocytic
activity. The tubes were centrifuged in the cold for 5 min-
utes at 3000 rpm, and the supernatant was decanted. The
pellet was stained with Acridine Orange (14.4mg/L) for 1
minute. One ml of icecold buffer was added, and cells were
washed 3 times. Cells were resuspended in cold buffer and
kept on ice until microscopic examination. A wet mount
slide was prepared from each tube for examination in a UV
microscope at 100 times magnification. The proportion of
phagocytic PMN were evaluated by counting 100 PMN,
and counting how many of these cells contained at least 3
bacteria (whether bacteria were live or dead). During the
examination, the total number of live versus dead bacteria
was counted in 50 PMN.
Statistical analysis: Standard statistical analysis was
performed using NNCS software. Paired t-test was used to
determine statistical significance. Values that were outside
two interquartile ranges from the 25th and 75th percentiles
were considered extreme outliers and were removed from
the analysis. The removal of outliers did not change the
actual conclusion.
RESULTS. Immediate mobilization of mononuclear
cells into the blood: The absolute cell counts before and
after consumption of either AFA or placebo were monitored
in 12 volunteers. The consumption of AFA resulted in
increased blood cell counts when compared to placebo.
The polymorph nucleated cell (PMN) population did not
change, whereas the lymphocyte (Ly) and monocyte
(CD14) subsets increased (Figure 2A). Within the lympho-
cyte subpopulation, the increase was observed in all of the
following T cell subsets: CD3+, CD4+, CD8+, as well as in
Figure 2A: AFA-induced changes in blood leukocyte populations.
The histogram shows the % change of polymorph nucleated cells
(PMN), monocytes (CD14), and lymphocytes (Ly). Black columns
represent the mean values of placebo, and the white columns repre-
sent the mean values of AFA. The bars indicate the standard error of
the mean.
the CD19+ B cell population (Figure 2B).
The relative proportions between naïve (CD45A+) and
memory (CD45R0+) T cells was monitored in all 21 sub-
jects, for both the CD4+ helper and CD8+ cytotoxic T cell
subsets. Despite a tendency for a shift towards less naive
and more memory T cells in the blood, no significant
changes were seen in naive versus memory T cell subsets.
Specific recruitment of CD3- CD56+ small lympho-
cytes from the blood: In all 21 study subjects, the propor-
tional changes of NK cells was examined. Two hours after
AFA consumption, the relative proportion of CD3- CD56+
natural killer cells was decreased, when compared to place-
bo (p<0.03). The effect was specific for small NK cells
Figure 2B: AFA-induced changes in lymphocyte sub-popula-
tions. The histogram shows the % change of total T cells (CD3), T
cell subsets (CD4, CD8), and B cell (CD19) lymphocyte popula-
tions. Black columns represent the mean values of placebo, and the
white columns represent the mean values of AFA. The bars indicate
the standard error of the mean.
54 JANA Vol. 2, No. 3 January 2000
Figure 4: Changes in natural killer cells (NK cells) in % of the
starting value. Black columns represent the mean values of place-
bo, and the white columns represent the mean values of AFA. The
bars indicate the standard error of the mean.
(low forward/side scatter properties), as the subset of cells
defined as large granular lymphocytes (CD14-negative,
large granular cells) was not affected (data not shown).
Long-term consumers produced a more pronounced
response than naive volunteers. When the volunteers were
grouped into long-term AFA consumers and naive volun-
teers, naive volunteers displayed a minor reduction in NK
cells after AFA consumption, whereas long-term consumers
displayed a pronounced reduction (p <0.005).
Adhesion molecule expression on circulating leuko-
cytes: We examined the expression of a series of adhesion
molecules on the surface of monocytes, B and T cells before
and after AFA exposure in vivo and in vitro. The following
adhesion molecules and subunits were examined: CD62L,
CD11a, CD11b, CD18, CD29, CD44, and CD49d. The flu-
orescence intensity was monitored by % positive, as well as
mean and median fluorescence values. Short-term incuba-
tion (90 minutes) in vitro with AFA extract resulted in a
moderate loss of CD62L on B as well as T cells, and a weak
upregulation of CD11b, but no other changes in the expres-
sion of the following adhesion molecules: CD11a, CD18,
CD29, CD44, and CD49d. Analysis of adhesion molecule
expression on lymphocytes from volunteers 2 hours post
AFA consumption showed moderate changes in CD62L
expression, but no other changes (data not shown).
Figure 5: Western blotting of tyrosine phosphorylation of pro-
teins extracted from unstimulated lymphocytes (lane 1) versus
lymphocytes incubated with Pokeweed Mitogen (PWM, positive
control, lane 2) or AFA (lane 3: extract 1:5, lane 4: extract 1:25).
Incubation of freshly purified human lymphocytes with AFA
extract did not induce tyrosine phosphorylation. The data are rep-
resentative of 4 similar experiments.
Figure 3: The relative changes in subpopulations of T cells is
shown (mean and SEM for 21 volunteers). The helper (CD4+) T
cell and cytotoxic (CD8+) T cell populations only showed a slight
shift towards less naive and more activated/memory T cells in the
circulation, and no statistical significance was reached.
January 2000 Vol.2, No.3 JANA 55
AFA extract does not activate lymphocytes directly:
We tested whether AFA extract could directly activate lym-
phocytes in vitro. When purified mononuclear cells were
incubated with AFA extract, no activation was seen, as
examined by tyrosine phosphorylation after 1-20 minutes
of AFA exposure (Figure 5) and proliferative responses
after 5 days of AFA exposure in vitro (Figure 6).
Modulation of the phagocytic activity of polymorph
nucleated (PMN) cells: The phagocytic activity of PMN
cells was evaluated, using PMNs from blood samples
drawn before and 2 hours after AFA consumption. The
phagocytic activity was monitored at different times of
incubation. When the study subjects had ingested placebo,
no differences on phagocytic activity was seen. In contrast,
after consumption of AFA, a mild decrease in phagocytic
activity was measured (Figure 7). This effect only reached
levels of significance at longer incubation times (see legend
to Figure 7).
DISCUSSION
Based on the many case reports on beneficial neurologi-
cal and immunological effects of consumption of the blue-
green algae Aphanizomenon flos-aquae, we studied the
immune activation within 2 hours after ingestion of 1.5 grams
AFA. This dose is recommended for food supplementation.
We examined several aspects of immune cell migration and
function. The data presented in this paper indicate a mild, but
consistent effect on the human immune system.
The absolute numbers of circulating leukocyte subsets
was increased. This effect was limited to lymphocytes and
monocytes, whereas polymorph nucleated cells were not
affected. This indicates a selective mobilization of lym-
phocytes and monocytes from primary or secondary lym-
Figure 6: Flow cytometric evaluation of lymphocyte proliferation
after 5 days of culture with no stimulation (top), with AFA water
extract (middle), and Pokeweed Mitogen (PWM, bottom). The X
axis displays fluorescence intensity, where loss of fluorescence
corresponds to proliferative activity. The proliferative indexes for
each culture condition is displayed in upper right corner of each
histogram. The experiment was conducted three times, where all
cultures were performed in triplicate.
Figure 7: Phagocytic activity of polymorph nucleated cells
(PMN) from volunteers before and after placebo or AFA inges-
tion. The phagocytic activity was unaffected by placebo, but was
moderately reduced by AFA, thus resulting in a lower maximum
phagocytic capacity, and a lower phagocytic rate.
January 200056 JANA Vol. 2, No. 3
phoid tissues, into the blood circulation. Thus, more mono-
cytes, B and T cells were released into the blood. In the ini-
tial preliminary study (involving 1 occasional and 4 regular
AFA consumers), AFA consumption induced a substantial
transient recruitment of NK cells in all five volunteers, peak-
ing at 2 hours and rapidly declining.37 In the current analy-
sis of 21 people, there was a specific recruitment from the
blood of small NK cells. It could be argued that AFA only
leads to margination (i.e. lymphocytes sticking to the vessel
walls without transmigration). However, margination is not
a permanent phenomenon, and the on/off rate would allow us
to sample some cells that have marginated and later released
from the blood vessel wall. Such cells would likely demon-
strate altered adhesion profiles, which we did not find. In
addition, as the recruitment of cells from circulation into
lymphoid tissue is highly cell type specific, mediated in part
by cell-type specific chemokines, transmigration would pro-
vide a more plausible explanation.
Increase in adhesion molecule expression was previ-
ously observed in a small number of long term con-
sumers.37 The present study reports data from a more thor-
ough evaluation. When examining the profile of adhesion
molecules on the surface of circulating lymphocyte subsets,
we found occasional shifts in adhesion molecule expres-
sion, confirming earlier observations, but in this larger
study we found no consistent differences induced by AFA in
vivo. This evaluation is hampered by the fact that we are not
able to directly sample the cells that have left the circulation
as a result of AFA. Thus, AFA did not uniformly affect the
adhesion profile of all circulating lymphocytes.
The low dose of AFA ingested and the rapidity of the
observed effects do not support a direct effect, where bioac-
tive molecules in AFA would be absorbed into the blood,
and transported to the bone marrow and spleen, and there
result in cellular changes leading to release of cells into the
blood. A more plausible model for explanation is that
neuro- or immune- active substances in AFA leads to trig-
gering of a gut-to-brain activation. It has been reported that
IL-1 beta is able to mediate a gut-to-brain communication
via the abdominal vagus nerve.38,39 Thus, in terms of rapid
modulation of leukocyte re-circulation, a gut-to-brain signal
would result in brain-to-lymphoid tissue signals, including
the rapid release of chemokines. Many neuropeptides are
either chemotactic or immuno-modulatory. As nerve ter-
minals wrap around the high endothelial regions of lym-
phocyte recruitment in the peripheral tissue, a central acti-
vation could rapidly amplify and alter cellular recruitment
in a highly selective manner. In the bone marrow, nerve ter-
minals come in close contact with developing and maturing
cells, and could regulate the volume of cells released into
the blood circulation.
The rapid changes in leukocyte re-circulation were
stronger in long-term AFA consumers. Since the study
design was double-blinded and randomized, the volunteers
Figure 8: Hypothetical model for AFA-induced immuno-modu-
lation. 1: Ingestion of AFA, and release of bioactive phyto-
chemicals in the stomach and/or upper intestine. 2: Release of
cytokine(s) in the gut trigger vagus nerve signals from gut to
CNS. 3: Central nervous system signals to the peripheral lym-
phoid tissues, resulting in altered immune cell trafficking.
were not themselves aware of when they were receiving
AFA versus placebo. Given the suggested CNS-mediated
modulation of the immune system, a conditioning may
have been established in which the CNS may recognize the
stimulation by AFA and in previously exposed consumers
add a conditioned component to the immune activation of
cell trafficking.
During our studies, we have been on guard for obser-
vations that could point in the direction of over-activation
of the immune system. More is not always better. An over-
activation of the immune system could be associated with
circulating immune complexes and increase in inflammato-
ry processes that could be detrimental to health. We found
no indications of a direct activation of any component of
the immune system or a general activation of the immune
system as a whole. The increased trafficking of immune
cells should translate into a better immune surveillance, i.e.
a better and more efficient patrolling of microbial invaders,
as well as virus-infected or transformed cells. We see this
January 2000 Vol.2, No. 3 JANA 57
as very positive for a potential use of AFA in various clini-
cal situations or as a nutritional support for the prevention
of viral infections. This data also points to further research
in a potential role for AFA in cancer prevention.
ACKNOWLEDGEMENTS
This study was funded by Cell Tech, Klamath Falls,
Oregon, and performed in the laboratory of Dr. Gitte S.
Jensen. We are grateful to Ann Griffith for her enthusiastic
help with data entry and analysis, to Christine Ichim for
technical assistance, and to Dr. David Schaeffer, University
of Illinois, for stastistical analysis.
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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,2Elizabeth 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
2Cell Tech, Klamath Falls, Oregon
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 provided by Cell Tech and the grant from the
Clinical Nutrition Reseach Center at the Massachusetts
General Hospital (P30 DK40561).
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