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NEW ENZYMATIC
OMEGA-3 FATTY ACIDS
SCIENTIFICAL UPDATE
The main objective of this presentation is to show our experience in the use of omega-3 fatty acids
In this respect it suits to place initially the concept of Omega-3 as a nutritional and
therapeutic value.
It is an admitted fact by the scientific community that the ingestion of polyunsaturated fatty acids (PUFAs) Omega-3 represents a benefit to health in general. A great
number of studies have shown the importance of Omega-3 fatty acids in the diet, even
though these micronutrients are not considered essential as they are synthesized in
low amounts by the body.
Different basic, clinical and epidemiologists studies have reported important benefits
by the ingestion of Omega-3 fatty acids in cardiology, neurology, oncology, inflammatory bowel disease, ophthalmology… Plus, it is critical to bran development
and function.
NUTRITIONAL AND MEDICAL BENEFITS OF OMEGA-3
MEDICAL SPECIALITY
POTENTIAL REDUCTION IN RISK OR IMPACT
Cardiology
All cause mortality, sudden cardiac death, ischemie stroke, triglyceride reduction (especially DHA), possible HDL increase but generally does
not impact overall lipid levels in low-to-moderate amounts (large intakes from supplements can significantly reduce triglycerides), blood
pressure and heart rate reduction.
Dermatology
Psoriasis, skin cancer.
Gastroenterology
Colorectal cancer, inflammatory bowel disease (Crohn disease and ulcerative colitis).
Immunology/infectious
disease/nephrology
Asthma, hepatitis, IgA nephropathy.
Neurology
Alzheimer’s disease (especially DHA), dementia, autism, attention deficit-hyperactivity disorder.
Gynecology-Pediatrics
Maternal and child health (especially DHA), cognitive (improved intelligence quotient) and visual impact in newborns and adolescents, improves gestation length, size, and potentially a variety of other outcomes, such as postpartum depression.
Oncology
Breast cancer, cachexia (reduces weight loss), colorectal cancer, endometrial cancer, leukaemia, melanoma, ovarian cancer, pancreatic cancer,
prostate cancer, renal cell cancer, DHA-paclitaxel combination drug in phase I/II trials, and may improve the impact of other standard chemotherapy drugs (5-fluorouracil, cyclophosphamide) from laboratory studies.
Ophthalmology
Macular degeneration, retinitis pigmentosa.
Orthopaedics
Osteoporosis/fracture, inflammatory/joint conditions (arthritis).
Psychiatry
Depression, bipolar disorder, schizophrenia, attention deficit-hyperactivity disorder.
Rheumatology
Rheumatoid arthritis, osteoarthritis, osteoporosis.
Surgery
Improve postoperative healing and other outcomes but may also cause unwant blood thinning if supplements taken preoperatively.
Urology
Prostate cancer, prostatitis, benign prostatic hyperplasia.
DISADVANTAGES
However
A recent meta-analysis done by Hooper et.al conclude that it is been impos-
The studies previously showed different deficiencies in its exposition related
sible to document, in an objective and statistically significant way, a beneficial
with the no-separation of the interventions done with the different Omega-3
effect in the consumption of PUFAs Omega-3 in the mortality related with car-
acids or mixtures of the same ones in shape of fish oil or nourishing enriched
diovascular or cancer events1.
supplements.
Also it isn’t been documented, in an objective and statistically significant way,
It also has been observed a lack of homogeneity as for the duration and doses
the Omega-3 effect in the reduction of the risk in developing cancer2.
in each treatment.
It neither has been value the importance of the chemical structure in which
has been administrated the Omega-3 acid: trigliceric (TG), ethyl ester (EE) or
free fatty acid (FA).
1
Hooper L. et al. Risks and benefits of omega 3 fats for mortality, cardiovascular disease and cancer: systematic
review. BMJ 2006; 332:752-760; Balk E.M. et al. Effects of omega-3 fatty acids on serum markers of cardiovascular disease risk: A systematic review. Atherosclerosis 2006; 189:19-30.
2
MacLean CH et al. Effects of omega-3 fatty acids on cancer risk: a systematic review. JAMA 2006; 295:403-15)
recommendations
Considering this controversy, the opinion of different experts is that the present recommendations
in the Omega-3 fatty acids consumption would be maintain but considering a series of criteria that
would have to fulfil Omega-3 fatty acids for their nutritional and therapeutic application, as they
are:
· PUFAs Omega-3 manufactured according to pharmaceutical standards that include quality
control steps to insure purity and potency.
· Use compounds containing at least 60% concentration in PUFA Omega-3 of all fatty acids.
· The ratio of Omega-3/Omega-6 must be greater than 50:1.
These recommendations agree with the conclusions of a workshop about fatty acids Omega-3 recently
published in the Journal American Clinical Nutrition magazine, that also stands out as an important
point in the need to achieving more clinical tests that would permit to calibrate the real potential of the
fatty acids Omega-3 in a preventic as well therapeutic level.
Which Omega-3 fatty acid?
In agreement with these criteria the first point to solve is the election of the
better adapted PUFA Omega-3 for a determined treatment. The three fatty
25
acids of the series 3 more important, in a physiological level, are:
g/100 g
20
· The alfa-linolenico (ALA)
15
· The eicosapentaenoic acid (EPA)
· The docosahezaenoico (DHA)
10
5
The DHA in the most abundant fatty acid Omega-3 in membranes and is present in all organs. Is particularly the most abundant in the central nervous sys-
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tem, in sperm and in the retina, also the most variable among the organs.
Only minute quantities of ALA and EPA are present in tissues, and the content
of DHA is generally between 5-30 times greater than the content of EPA in the
majority of organs. Also, the DHA is about a 100 times more abundant than
ALA
EPA
DHA
Cross study analysis of fatty acid concentrations in tissues from adults from the United
States, Canada, Australia and Europe.
the EPA in the brain and in the retina.
Which Omega-3 fatty acid?
Although the three fatty acids are metabolically related, several studies have demonstrated that the administration of ALA doesn’t generate the needed physiological
α-Linolenic acid (18:3n-3)
quantities of DHA and EPA. About the administration of DHA or EPA, the last recommendations suggest that the more efficient way for the increment of the concentrations of an Omega-3 acid is the administration of the specific acid already told.
Stearidonic (18:4n-3)
Anyhow, in a more general way, the DHA administration guarantees a basic concentration of EPA, whereas the exclusive administration of this diminishes the
(20:4n-3)
DHA concentration.
In humans, the endogenous capacity to convert ALA to DHA or EPA is very low or
EPA (20:5n-3)
absent, for this reason, the consumption of DHA in the diet may be critical for maintaining the adequate concentration of DHA in the cellular membranes. BrJ Nutr2002;
DPA (22:5n-3)
88:355–363.
Retroconversion of DHA to EPA: in the DHA group, it’s concentration increased by
DHA (22:6n-3)
69% and EPA increased by 29%. In the EPA group, EPA increased by 297% whereas
DHA decreased by 15%. AmJ ClinNutr1997; 66:649-59/ AmJ ClinNutr2000; 71:1085–94/
Am J Clin Nutr 2004;79:765–73.
Which Omega-3 fatty acid?
The DHA is the Omega-3 fatty acid in the central nervous system.
On the other hand, from a nutritional point of view, whose aim is to recover the deposit of Omega3 fatty acids, the DHA administration guarantees an intervention in the central nervous system
since the concentration of EPA in these tissues is purely nominal.
Diets that contain 9,5% DHA, completely suppresses N-Methyl-N-nitrosourea induced retinal degeneration, whereas the diet containing 4,75% DHA (EPA + DHA group) or only EPA did not have protective
effect. Exp Eye Res 2003; 77:167–173.
DHA is the precursor of the docosatrienes and the D series resolvins that each regulates the processes
associated to the inflammation and its final resolution. J Biol Chem 2003; 278:14677–14687.
DHA released from membrane phospholipids is the endogenous precursor to a neuroprotective signalling response to ischemia-reperfusion. J Biol Chem 2003; 278: 43807–43817.
The synthesis of 10,17S-docosatriene, neuroprotectin D1, protect retinal cells from oxidative-stress-induced apoptosis, and we predict that it will similarly protect neurons. Proc Natl Acad Sci USA 2004; 101:
8491–8496.
bioavailability
Another interesting aspect that can condition the effectiveness of a certain
treatment is the bioavailability of Omega-3 fatty acids. Numerous animal studies exist that have demonstrated that the acylglycerol are the more effective
CONCENTRATION OF
N-3 PUFA (%)
% BIOAVAILABILITY*
25-30%
74-100%
Ethyl esters
Up to 85%
21-57%
Free fatty acids
Up to 65%
51%
Min 70-75%
98%
TYPE OF OIL
Fish Oil
Re-esterified
triglycerides
transporters for the fatty acids. Even more, the acylglycerols are considered
to be the chemical form more favourable from a nutritional view, greater than
the ethyl esters of unsaturated fatty acids which present a spoilt intestinal absorption of the n-3 fatty acids observed as it’s been demonstrated in laboratory animals. It has also been shown that ethyl esters of unsaturated fatty
acids hydrolyze at a slower rate than their corresponding acylclycerols.
The study on humans done by a Sigma-Tau Pharmaceutical company demonstrated that the triglycerides obtained by reeserification show a bio-
In vivo study * on 24 healthy subjects comparing a single oral administration of n-3 PUFA in different chemical structures, demonstrated higher plasma levels of total n-3 PUFA in the group of
subjects treated with extra-refined oil.
availability similar to the natural fish oil and greater than free fatty acids;
*Extract of SIGMA-TAU publication.
whose only advantage is the purity that can achieve but against it generates
that even generate digestive problems; and also greater than the ethyl esters,
ethanol that can be toxic in high doses.
10
Bioactivity - safety
CHEMICAL DHA TG
ALGATRIUM PLUS
The industrial synthesis of triglycerides can be done chemically or in an
enzymatic way. The differences between chemical and enzymatic process will
• Extreme pH
• High temperatures (distillation)
• Chemical reactives
• Physiological pH
• Low temperatures
• No chemical reactives
establish the final qualities for each product. The use of chemical traditional
methods (chromatographic separation, molecular distillation, etc.) to produce
concentration of PUFAs Omega-3 implicate the use of extreme PH and high
temperatures, that can destroy partially the structure of the double bonds
• Low triglycerides level
• Secondary reaction molecules
• Chromatography purification
• High triglycerides level
• No secondary reaction molecules
• No need further purification
natural all-cis of PUFAs Omega-3 by oxidation, isomerization or migration.
On the other hand, the mild conditions (temperatures less than 50º C, pH
6-8, and less chemicals) used in enzymatic hydrolysis provide a promising
• Double bonds migration
• Double bonds oxidation
• Isomerizations Cis/Trans
• Preservation of double bonds
• No oxidation of double bonds
• No isomerizations Cis/Trans
alternative that could also save energy and increase product selectivity.
The preservation of the double bonds, will confer to their molecule its specific
bioactivity.
ALGATRIUM PLUS
Specification
Acid Value
Antioxidant content
Unit
Limits
Mg KOH/g
3.0 max.
g/100g
1.0 - 1.1
Many studies show that the bioactivity of Omega-3 oils, and mainly DHA, is
directly related with it’s content with regard to the total fatty acids, which can
compete with the Omega-3 in a concrete metabolism. To obtain the maximum
Density at 20º C
Kg/l
0.930 - 0.940
Fatty acid DHA (C22:6n3)
%FA
70 min.
Fatty acid DPA(C22:5n3)
%FA
8 max.
Fatty acid EPA (C20:5n3)
%FA
10 max.
Fatty acid Omega-3
%FA
85 min.
Meq O2/Kg
5.0 max.
g/100g
0.10 max.
Peroxide value
Residue of ignition
Specifications of Omega-3 oil used in these studies.
12
level of bioactivity, Brudy has manage its efforts in two basic goals:
· To have the maximum concentration of DHA in the same
triglyceride.
· To reduce the EPA content as much as posible.
Even more, this oil obeys with all the legal requirements such as peroxides,
acid, antioxidant content, heavy metal levels…
ALGATRIUM PLUS
90
80
Of all oils that are in the market, Algatrium Plus (DHA 70e) is the enriched oil
70
with a global omega-3 content and in higher DHA.
60
50
40
DPA
30
EPA
20
Algatrium Plus
10
OMEGA-3
0
P
M
HSO
Comparative figure of different omega-3 oils in the market.
DHA 50 CHEM ALGATRIUM PLUS
ALGATRIUM PLUS
OMEGA-3 % OMEGA-3
Synthesis
INSA
MG
DG
CHOL
PLM
TG
EE
GTOTAL
A. PLUS
70
ENZYMATIC
0,5
n.d.
7,1
n.d.
n.d.
86,5
5,9
93,6
FISH
20
Tuna
4,7
n.d.
2,2
4,2
n.d.
88,9
n.d.
91,1
DHA
50
Chemical
2,7
4,3
10,3
3,5
22,8
40,1
14,0
53,2
DHA
50
Chem-Bpm
2,1
n.d.
18,8
n.d.
n.d.
66,4
5,2
85,2
Especificaciones del aceite omega-3 utilizado en estos estudios.
14
Oil bioavailability is directly proportional to the type
glycerides are triglycerides and without mono-
of molecules which compose it, specially to its lipid
glycerides). The chemical DHA has a low level of
compose. Natural oils contains more of 90% in glyc-
TG (<50%), greater ethyl ester levels, and high
eride forms, what it gives a maximum bioavailability.
level of polymers (15-20%), which are not absorbed
Algatrium Plus it’s very similar to a natural oil (
and must be eliminated by column chromatography
composition up to 90% of total glycerides, 85% of
( Chem-Bpm).
LEGISLATION
100
90
80
Only natural fish or algae oils that have not been re-esterified fulfil food
70
legislation, and therefore are food. The chemical DHA doesn’t fulfil Europe-
60
an Pharmacopoeia requirements, above all related to the level of triglycerides,
50
total glycerides and its content in polymers compounds. It is necessary a pu-
40
rification by column chromatography for it to fulfil European Pharmacopoeia
requirements. Algatrium Plus has the same composition than natural oils
30
and therefore it’s considered food.
20
10
0
INS.
MG
Structural composition.
DG
PLM
FA
TG
EE
GTotal
Algatrium Plus
Pharmacopoeia
DHA 50% Chemical
DHA 50% Chemical-PURIFIED
Antitumoral activity
COMPOSITION
STRUCTURE
IC50 (μM)
healthy cells
cancer cells
safety
ratio
%DHA
FORESKIN RENAL
HeLa
FA
70
123,1 ±9,0
n.d.
72,2 ±5,8
1,7
EE
70
566,3 ±53,9
72,6 ±8,3
71,0 ±9,7
7,98 -1,02
TG (Algatrium Plus)
70
716,7 ±41,9
392,7 ±37,5
58,6 ±8,8
12,23 -6,70
TG (Chemical)
50
1956,0 ±27,8
413,6 ±25,6
564,0 ±69,6
3,47 -0,73
TG (Fish)
20
2267,6 ±27,9
3116,0 ±158,2
1379,4 ±286,4
1,64 -2,26
Effects on concentration and chemical structure. Cellular toxicity.
16
In order to value Algatrium Plus’ activity, out first objective was to
cells with ethyl esther (EE), free fatty acid (FA) or triglyceride (TG).
determine its cytotoxic antitumoral capacity. In this we performed a
But in healthy renal cells, the EE form has the same toxicity as can-
comparative study using tumour cells (HeLa) and healthy cells (Fore-
cer cells and in skin cells, the FFA form shows the more cytotoxicity,
skin and renals) as control. We have done dose-response studies to
being the TG, the form with a best safety ratio. Using a TG form, a
determine the IC50 value as cytotoxic criteria (it’s the concentration
decrease of DHA content to 50 or 20% (the last was the concentra-
that kills up to 50% of the cells). We’ve analyzed two variables, the
tion in the fish oil), diminishes the cytotoxic effect in tumour cells.
effect of lipid structure and of DHA content. At a DHA content of
These results show that the best way is the TG form with a high DHA
70% of all fatty acids, the same cytotoxicity was observed in tumour
content (Algatrium Plus).
Antitumoral adyuvant activity
cell line
HeLa-Epitelial (Dox)
HT-29-Colon (5-FU)
addition
system
DHA content
ALGATRIUM
PLUS μM
IC50 (μM)
Control
0
295,3 ±31,6 (n=8)
Incorporated
1
5
10
30,1 ±5,4 (n=8)
28,7 ±6,7 (n=8)
37,0 ±4,4 (n=6)
Simultaneous
5
184,3 ±16,2 (n=4)
Control
0
23.30 ±3.68 (n=12)
Incorporated
1
5
10
2.80 ±1.10 (n=7)
2.38 ±1.01 (n=4)
3.80 ±1.42 (n=6)
Simultaneous
5
14.80 ±8.01 (n=6)
Effects of DHA about the antitumoral activity of Dozorubicin or 5-fluoroacyl. Cellular toxicity.
Control
DHA
Algatrium Plus
DHA TG 50
Chemic
DHA TG 20
Fish
IC50 (nM)
DHA (μM)
tumoral cell
healthy cell
0
295,3 ±31,6
1158,6 ±369,9
5
28,7 ±6,7
1046,6 ±230,4
10
37,0 ±4,4
1080,5 ±174,2
5
130,9 ±15,6
1163,3 ±156,9
10
106,3 ±20,7
980,1 ±270,9
5
257,4 ±29,6
1157,5 ±124,9
10
236,5 ±15,7
1530,3 ±481,3
Effects of DHA concentration on doxorubicin toxicity.
Another interesting activity of PUFAs Omega-3 in cancer, is its use as adyu-
This potential effect depends on the DHA content, because using chemical oil
vants in conventional quimotherapy. In this, we assess the effects of DHA
(50% in DHA) or fish oil (20% in DHA), it looses the potential effect progres-
at sub-toxic concentrations on Doxorubicin and 5-fluoroacyl cytoxicities. Si-
sively. It is also significant that this effect doesn’t increase the cytotoxicity in
multaneous exposition to DHA and drug decreases the IC50 value in 2-fold.
healthy cells.
On the other hand, if Algatrium Plus was previously incorporated in the cell
membrane, this potential effect increases up to 10 times.
Bioactivity-safety
Pro- or anti-oxidant activity?
In pure chemical systems, the molecules most susceptible to free radicals attack
are polyunsaturated fatty acids (PUFAs) as docosahexaenoic (DHA, 22:6 n-3) acid
and, in general, oxidizability increases as the number of double bonds increases.
In vivo, the same relative oxidative susceptibility is thought to occur.
Then, why the DHA, a molecular target of the free radicals, if the major fatty acid
in tissues exposed to a potential oxidative stress like retinal tissue or in tissues
with a deficient antioxidant cellular system like the central nervous system?
18
Bioactivity-safety
In this context, we studied the influence of the presence of enzymatic DHA in the cellular membrane, about the intracellular generation of free radicals using as an experimental system two human cell lines, fibroblast skin cells ( Foreskin, ATC CRL 2076) and
retinal pigment epithelial cells (ARPE-19, ATCC CRL 2302).
The quantification of generated free radicals has been done by fluorescent methodologies using molecular probes as H2DCF-DA, DHR and DHE.
As exogenous oxidative stress inducers we used AAPH and the xantine/xantine oxidase system, as a endogenous rotenone and antimycine A.
Antioxidant cellular response
skin cells
Fluorescence (UA)
H2DCF-DA
Fluorescence (UA)
retinal cells
-49%
-42%
As it’s shown in the figure, the presence of Algatrium Plus in the cells
membrane diminishes the intracellular generation of free radicals
after putting the cells through an oxidative stress. This behaviour
is independent of the cell in study (skin or retina) and the used marker
0
50
100 150
Time (min.)
200
250
0
50
100
150
Time (min.)
(H2DCF-DA or DHR), this shows us the activation of a general mecha-
200
nism. The level of protection observed it’s also similar to the same oxida-
Fluorescence (UA)
DHR
Fluorescence (UA)
tive stimulus.
-56%
-50%
Control
Algatrium Plus
0
20
50
100 150
Time (min.)
200
250
0
50
100 150
Time (min.)
200
250
Antioxidant cellular response
stressor independent
% Protection
60
50
40
30
20
In order to assess that Algatrium Plus antioxidant protection was a general
10
process, we changed the oxidative stress inducer to X/XO system or endog-
0
retinal cells
AAPH
ROT
ANT A
X/XO
50
100 150
Time (min.)
200
Rise % Protection
-49%
0
retinal cells with respect to the AAPH inducer.
2,1
1,8
1,5
1,2
The antioxidant protection of Algatrium Plus depends on the doses,
showing a doses-dependent inversely behaviour. The major effect was
0,9
observed at low DHA concentration, whereas at high concentration, the ef-
0,6
0,3
fect decreased independently of the oxidative stress strength.
0
250
0,5
Algatrium Plus
5
dose dependent
60
% Protection
Fluorescence (UA)
additive effect
enous inducers (ROT and ANT A). The same behaviour was observed in
Also, the antioxidant effect of Algatrium Plus shows an additive administration frequency dependent effect. The administration of a daily doses
increments the antioxidant effect to a singular dose.
50
40
30
20
10
0
0,5
5
50
Algatrium Plus
Daily doses
Singular dose
Antioxidant cellular response
CONTROL
ALGATRIUM PLUS 0,5 μM
T= 0 min.
The free radicals intracellular generation was confirmed using fluorescent microscopy. These micrographs show a time progressive accumulation of free radicals in the control cells without DHA, while in the
T= 15 min.
one with Algatrium Plus in the membrane, the intracellular level of
free radicals has not increased.
T= 30 min.
SKIN CELLS
22
Antioxidant cellular response
60
At a cellular level, the free radicals generation seems dependent on the cell
fatty acid composition, but the specific influence of each class of fatty acid
SKIN
CELLS
% PROTECTION
40
remains ignored.
20
Moreover, at the industrial production level, the synthetic method used to
0
obtain enriched oil DHA could be very important in the final quality of
-20
the DHA. In this sense we have studied the importance of the DHA content
and synthetic origin in the antioxidant cellular response.
-40
FISH
OIL
DHA TG50 ALGATRIUM
PLUS
CHEMICAL
As we can see in the figure, a decrease in the DHA content to 50% (chemic
origin) or 20% ( fish oil) of total fatty acid, induces a decrease in the antioxi-
60
dant effect in retinal cells, although in any case shows a pro-oxidative activity.
RETINAL
CELLS
% PROTECTION
Whereas, in foreskin cells, this decrease is so high that only Algatrium Plus is
antioxidant, being the other two left pro-oxidative.
40
0,5 μM
20
5 μM
50 μM
0
FISH
OIL
DHA chemical vs. Algatrium Plus.
DHA TG50 ALGATRIUM
PLUS
CHEMICAL
Given that the multifactorial temper of a nutritional treatment, it’s not convenient the administration of a product that shows divergent activities in function to the tissue that is incorporated.
Antioxidant cellular response
RETINAL CELLS
60
If low activity of chemic oil (50% in DHA), would be related with the pres-
% PROTECTION
50
ence of polimerics structures, its elimination by column chromatogra-
40
phy would permitt to bring back it antixocidant activity. As the figure
30
shows, an excessive manipulation of the original product to elimi-
20
nate contaminants or to improve its organoleptics characteristics
10
means a lost of bioactivity.
0
-10
ALGATRIUM
PLUS
DHA TG50
CHEMICAL
(DHA)
DHA TG50
CHEMICAL PURIFIED
0,5 μM
5 μM
50 μM
DHA chemical vs. Algatrium Plus.
24
Antioxidant cellular response
RETINAL CELLS
% PROTECTION
70
60
The antioxidant activity of Algatrium Plus, it is also related with its great-
50
er bioavailability since an ethyl esther or a free fatty acis with the same
content in DHA, present an antioxidant activity as maximum half of the
40
Algatrium Plus.
30
20
10
0
0,5 μM
5 μM
(DHA)
Efecto de la estructura química Fa vs. Ee vs. Tg.
FA-BSA
FA: Free fatty acid
Algatrium Plus
EE: Ethyl esther
TG 50 CHEM
TG: Triglyceride
EE 70
Antioxidant cellular response
RETINAL CELLS
60
% PROTECTION
50
Compared with the antioxidant normally used in ocular treatments to
the very best concentrations described, Algatrium Plus shows in all stud-
40
ied doses a higher effect.
30
20
10
0
s
um
ri
at
g
Al
u
Pl
M
C
NA
m
Algatrium Plus vs. antioxidants.
26
s1
Cy
M
m
0
Co
Q
10
μM
0
t
Vi
0
E1
μM
0
t
Vi
C
10
μM
Antioxidant cellular response
RETINAL CELLS
60
% PROTECTION
50
Even if these antioxidants are combined or increases its concentration
10 or 100 times.
40
30
20
10
0
s
um
tri
ga
u
Pl
T1
AN
Al
Algatrium Plus vs. antioxidants.
AN
T1
0
x1
0
T1
AN
0
x1
T2
AN
Bioactivity
At this point, the question is: What is the mechanism by which DHA performed its
antioxidative activity?
Omega-3 fatty acids can act at very different levels of the cellular physiology.
Defense against free radicals is provided by a number of anti-oxidant enzymes,
coupled with other chemical anti-oxidants that prevent lipid, protein and DNA
damage. We have studied the effect of DHA on different pathways of the cellular
antioxidant system:
· Anion superoxide generation (O2-).
· Superoxide dismutase (SOD) and glutathione peroxidase activities (GPx).
· Intracellular glutathione concentration (GSH).
· Lipid peroxidation.
· DNA damage.
28
Bioactivity
1.
Cellular
Respiration
Oxidative
Burst
O2-
2.
SOD
e
s
ala
t
Ca
H2O2
H2O + O2
GPx
H2O
GSH
Environmental
Factors
Protein
Peroxidation
mechanism of antioxidant activity.
3.
GSSG
GRed
OH
4.
DNA Damage
Lipid
Peroxidation
Bioactivity
1.
Cellular
Respiration
Oxidative
Burst
O2-
2.
SOD
e
s
ala
t
Ca
H2O2
H2O + O2
GPx =
GSH
+
Environmental
Factors
Protein
Peroxidation
Mechanism of antioxidant activity.
30
3.
H2O
GSSG
GRed
OH
4.
DNA Damage
Lipid
Peroxidation
Bioactivity
We have found that the previous incorporation of Algatrium Plus in the cells decreases the generation of superoxide in an inverse dose-dependent manner and DNA damage.
The relatively lower levels of superoxide anion in cells after Algatrium Plus treatment might be
due to the higher activities of SOD and GPx.
The PGx activity in the treated cells did not differ from that in the control cells with or without
oxidative stress.
The most significant change was the increase (300%) of the GSH intracellular concentration,
related with and increment of the novo synthesis.
These results shows us that Algatrium Plus does its antioxidant effect by an increment of
rge antioxidant cellular system response, without a direct participation in the same that
differs it from the conventional antioxidant chemicals.
This suggests that the cells tolerate small quantities of DHA and that this acts as a stimulus to
avoid any accumulation of free radicals, the incorporation of Algatrium Plus in the biomembranes provokes an adaptive antioxidant cellular response.
In vivo studies
In order to give credibility to the results obtained in vitro, it is necessary
To valuate the action of Algatrium Plus, our model of oxidative stress
to verify the same in vivo. In this sense, a clinical test doble-blind con-
was the induced one by the aerobic physical exercise.
trolled of 4 months duration (N=40 sportsmen), to a doses of 2g/day of
Algatrium Plus.
We put the sportsmen to an effort continued to 75% of its maximum
oxygen consumption during 90 minutes, which supposes a sufficient
32
The variables to study were DNA damage and serum antioxidant param-
production of free radicals and oxidative stress in order to determine
eters.
the antioxidant capacity.
In vivo studies
antioxidant total activity
An statistically significant increase on serum antioxidant total activity
2,00
was observed (p<0,05) during and after the accomplishment of the rectH2O2 (nmol/L min.)
1,90
angular tests in the sportsmen after Algatrium Plus intake during a treatment of 3 weeks to 4 months.
1,80
1.70
Algatrium Plus
1,60
Control
1,50
BASAL
In vivo antioxidant parameters.
20 DAYS
4 MONTHS
In vivo studies
lipid peroxidation
60
The plasmatic MDA level as lipid peroxidation index was increased dur-
50
ing the accomplishment of all the effort tests (p<0,035). After the intake
MDA (nmol/L)
40
of Algatrium Plus for 3 weeks, the lipid oxidative damage in the same
30
conditions was decreased practically to basal level (p<0,05); observed
20
after 4 months of treatment a protective effect in this variable.
10
0
-10
BASAL
In vivo antioxidant parameters.
34
20 DAYS
4 MONTHS
In vivo studies
DNA damage
900
8-oxo-dG (pmol/Kgx24h.)
800
There is an increase in the DNA oxidative damage during the accom-
700
600
plishment of the rectangular effort tests measured as 8-oxo.dG level in
500
urine (p<0,011). The intake of Algatrium Plus during 3 weeks and until 4
400
moths protects the DNA of the oxidative damage induced by the effort
300
in a significant manner (p<0,035).
200
100
0
BASAL
In vivo antioxidant parameters.
20 DAYS
4 MONTHS
In vivo studies
variation in the glucose plasmatic concentration
4
2
As collateral effects in the sport yield output it has been observed that
the glycaemia levels decrease during the accomplishment of the rect-
mg/dL
0
angular effort test (p<0,0009). This reduction in the glycaemia was less
after 3 weeks of Algatrium Plus intake what meant an increase in the
-2
glycaemia levels after 4 months of treatment with Algatrium Plus.
-4
-6
-8
BASAL
3 WEEKS
Physiological variables of sport yield.
36
4 MONTHS
In vivo studies
oxygem consumption
2900
2850
It is also been observed a significant increase of the absolute (p<0,019)
and relative (p<0,036) oxygen consumption obtained in the threshold
2800
mL/min.
of the triangular effort test with the intake of Algatrium Plus up to 4
2750
months.
2700
These results seem to indicate that the intake of Algatrium Plus can
increment the output in sport yield.
2650
2600
2550
BASAL
3 WEEKS
Physiological variables of sport yield.
4 MONTHS
In summary
The Omega-3 fatty acids are a nutritional and therapeutic concept with a high
potential of development.
The application of the Omega-3 fatty acids in these fields requires a manufactured products according to pharmaceutical standards that include quality
control steps to insure purity and structural quality that it can condition it effectiveness. As an standard is demandable a minimum purity major than 65% in a
chemical structure very bioavailable as a triglyceride.
The incorporation of Algatrium Plus in the cellular membrane not only does not
promote the lipid peroxidation but that acts as an activator of the cellular antioxidant network probably as a result of an adaptive response of the cell.
38
Legal Information
ALGATRIUM PLUS is an International Brand property of BRUDY TECHNOLOGY.
The use of high concentration of DHA for the tumoural diseases treatment
is protected by the International Patent num.200501141 Property of Brudy
Technology.
The commercialization and use of Omega-3 Antioxidants are protected by the
International Patent num. 200503202 property of Brudy Technology.
Dr. Joan C. Domingo Pedrol - Departamento de Bioquímica y Biología Molecular - Universidad de Barcelona - Telf. 934 021 214 - e-mail: jcdomingo@ub.edu
Made in España by: Brudy Technology - C/Riera de Sant Miquel, 3 - 2º 4ª - 08006 Barcelona - Tel. 93 217 03 66