Download ANALYSIS OF ω-3 FATTY ACID CONTENT OF POLISH FISH OIL

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 4 pp. 875ñ883, 2016
ISSN 0001-6837
Polish Pharmaceutical Society
ANALYSIS OF ω-3 FATTY ACID CONTENT OF POLISH FISH OIL DRUG
AND DIETARY SUPPLEMENTS
KAMILA OSADNIK1* and JOANNA JAWORSKAb
School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University
of Silesia, Katowice, Poland, Department of Biopharmacy, Jednoúci 8, Sosnowiec, Poland
2
Centre of Polymer and Carbon Materials, Polish Academy of Sciences,
34 M. Curie-Sk≥odowska St., 41-819 Zabrze, Poland
1
Abstract: Study results indicate that a diet rich in polyunsaturated fatty acids ω-3 (PUFA n-3) exerts favorable
effect on human health, accounting for reduced cardiovascular morbidity and mortality. PUFA n-3 contained in
marine fish oils, particularly eicosapentaenoic (EPA, 20:5 n-3) and docosahexaenoic (DHA, 22:6 n-3) acids, are
attributed antithrombotic, anti-inflammatory, anti-atherosclerotic and anti-arrhythmic effects. They have also
beneficial effects on cognitive functions and immunological mechanisms of an organism. Considering the fact
that marine fish are not abundant in Western diet, the pharmaceutical industry reacts with a broad selection of
PUFA n-3 containing dietary supplements and drugs. Increased consumersí interest with those products has
been observed recently. Therefore, their quality, understood as reliability of manufacturerís declaration of composition of offered dietary supplements, is highly important. We have tested 22 products available in pharmacies and supermarkets, manufacturers of which declared content of n-3 fatty acids (21 dietary supplements and
1 drug). Identity and content of DHA and EPA were assessed using 1H NMR spectroscopy, based on characteristic signals from protons in methylene groups. Almost one in five of the examined dietary supplements contains < 89% of the PUFA n-3 amount declared by its manufacturer. For a majority of tested products the manufacturer-declared information regarding DHA (58%) and EPA (74%) content was consistent with the actual
composition. It is notable that more cases of discrepancy between the declared and the actual content regarded
DHA than EPA, which indicates a less favorable balance, considering the pro-health effect of those acids. Over
a half of tested products provides the supplementary dose (250 mg/day) with one capsule taken daily, and in
27% of cases the daily dosage should be doubled. Only 10% of those products ensure the appropriate dose for
cardiovascular patients (1 g/day) with the use of 1 capsule a day. Correct information provided by a manufacturer on a label regarding the total amount of DHA and EPA is a basis for selection of an appropriate dosage.
Keywords: dietary supplements, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), fish oil
Polyunsaturated fatty acids ω-3 (PUFA n-3)
play numerous roles in the human organism, therefore they are an important compound of healthy diet.
PUFA n-3 belong to exogenous compounds, and
their availability is limited to fatty marine fish
(salmon, mackerel, tuna, sardines, herring). Two
most important acids belonging to the n-3 family
are: eicosapentaenoic acid (EPA, 20:5 n-3) and
docosahexaenoic acid (DHA, 22:6 n-3). DHA and
EPA may be also produced by conversion of the 18carbon α-linolenic acid (ALA), present in vegetable
products including soya beans, linen and nuts.
Conversion of ALA into EPA and DHA remains at
a very low level in humans, and therefore, those
fatty acids should be supplied with a diet, mostly by
consumption of fish (1-4).
PUFA n-3 (especially DHA and EPA) contained in marine fish oils are attributed numerous
favorable effects associated with reduction of cardiovascular morbidity and mortality. Studies indicate that PUFA n-3 rich diet, or a regular supplementation of those fatty acids, has an antithrombotic, anti-inflammatory, anti-atherosclerotic and antiarrhythmic effects (5-9). Scientific data indicate that
the use of PUFA n-3 by type 2 diabetes patients
largely supports the therapy aimed at reduction of
VLDL and triglyceride levels (10).
The growing awareness of health benefits associated with eating marine fish and substitution of
saturated fats with unsaturated ones in the daily diet
becomes more and more noticeable in the Polish
society (11). Unfortunately, statistics show that
* Corresponding author: e-mail: [email protected]
875
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KAMILA OSADNIK and JOANNA JAWORSKA
Poles, in general, do not comply with the guidelines
of the Polish Society of Family Medicine, and the
European Society of Cardiology that recommend
increasing quantity of fish in the weekly diet. This
resulted with a response of the pharmaceutical and
food industry and introducing multiple dietary supplements containing PUFA n-3 acids on the market
(12, 13). Availability of dietary supplements in various points of sale (supermarkets, drugstores, newsstands) beyond pharmacies causes a systematic
expansion of the group of consumers of that kind of
products, who want to completely replace a seabased fish diet by dietary supplements of n-3 fatty
acids. Currently, n-3 acids containing products
designed for all age groups are available in the
Polish market. They are recommended for healthy
individuals as supplements (for vegetarians, pregnant women, elderly) and for patients with cardiovascular diseases, vision problems, impaired immunity and memory disorders.
Taking PUFA n-3 in the form of dietary supplements seems to be equivalent to consumption of
fish. That assumption is supported by the fact that
dietary supplements contain fish oil from the same
natural source. It should be noted, however, that not
all products are marked with the information concerning fish species or fishing area. Question of the
supplementary dose of PUFA n-3 determined by the
European Food Safety Authority and the Food and
Agriculture Organization of the United Nations at
250 mg/day for a healthy adult, and of therapeutic
doses recommended by scientific societies to
patients with various conditions, used to be treated
rather subjectively by some manufacturers (14, 15).
Considering a very low consumption of marine fish
in the Polish population, the Polish Society of
Family Medicine stresses the urgent need for intensification of educational efforts aimed at increased
consumption of EPA and DHA in everyday diet, up
to the optimum level of 1 g a day. In risk groups of
cardiovascular diseases, cancer, rheumatoid and
neurodegenerative diseases the EPA and DHA content should be increased to 1.5 g a day, if possible.
Experts state that the basic source of both acids
should be marine fish: herring, mackerel, salmon,
cod, flounder, halibut, served twice a week. If meeting those recommendations proves impractical, EPA
and DHA supplements should be used, containing
the amount of acids consistent with the declaration
(12).
According to the Polish law, dietary supplements are treated as food, and thus they are exempt
from rigorous control applicable to drugs. As a
result, in terms of quality and purity of those products, consumers of dietary supplements have to
depend on declarations made by their manufactures.
Figure 1. Study design
Analysis of ω-3 fatty acid content of Polish fish oil drug a dietary supplements
877
Figure 2. Standard curves: a) DHA, b) EPA
Reliable label information is therefore of exceptionally high importance.
This paper presents results of identity and content analyses of two principal PUFA n-3: DHA and
EPA in selected dietary supplements available in the
Polish market, and in one registered medicinal product. Quality of selected products and correctness of
manufacturersí declarations were also assessed by
comparison of label information with results of the
analysis of liquid content of capsules. The paper
confronts also manufacturersí recommendations
regarding dosage of PUFA n-3 contained in their
products and recommendations of scientific societies issued in Poland and worldwide (15-17).
MATERIAL AND METHOD
Material
Study material was dietary supplements (n =
21) available in pharmacies and markets, and the
only one registered in Poland as a drug, available
only in pharmacies. Each product was accompanied
by a leaflet stating the declared n-3 acid content.
Among the selected product, three were tested only
for α-linolenic acid content, as they contained no
DHA and EPA (Fig. 1)
Methods
Total 19 products were tested using high definition nuclear magnetic resonance (NMR) for determination of identity and DHA and EPA content. The
testing procedure was developed and performed
according to methodology accepted for the given
drug form (18). In case of products containing any
additional ingredients (herbal extracts, vitamins) 0.5
mL of chloroform was added to 1 mL of liquid capsule content, and centrifuged. Identity and content of
capsules filled with liquid oil was tested directly.
Identity and content of manufacturer-declared DHA
and EPA were assayed for all products.
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KAMILA OSADNIK and JOANNA JAWORSKA
Figure 3. The percentage of the declared content of DHA and EPA
n = 19
n = 19
Figure 4. Necessary number of capsules of a PUFA n-3 product
ensuring a daily supplementation dose (250 mg/day)
Figure 5. Necessary number of capsules of a PUFA n-3 product
ensuring the daily therapeutic dose (1 g/day)
Identity testing
The identity was tested in order to confirm that
the product corresponds to its label description. The
test involved the comparison of characteristic signals of fatty acids present in 1H NMR proton spectra
of a tested product (dietary supplement or drug) with
1
H NMR spectra of purchased analytical standards:
docosahexaenoic acid analytical standard (SigmaAldrich), eicosapentaenoic acid analytical standard
(Sigma-Aldrich).
Chemical shifts characteristic for DHA and
EPA are related to protons of β-methylene groups,
present within the range of 2.30-2.34 ppm for DHA,
and 1.61-1.72 ppm for EPA.
Content verification
Verification of content consisted in quantitative determination of DHA and EPA in each tested
product. Calibration curves for each determined
fatty acid were developed to that purpose.
Standard curves
Contents of individual fatty acids in each product were calculated based on the measurement of
intensity of characteristic signals emitted by both n3 acids, based on the equation curve of relative
intensity of those groups in relation to the intensity
of the internal standard (methanol). Standard curves
for DHA and EPA are presented in Figure 2.
Analysis of ω-3 fatty acid content of Polish fish oil drug a dietary supplements
879
should fit within the ±5% range for capsules with
the declared active substance content of over 100
mg, and for capsules with the declared acid content
below 100 mg the range was ± 10% (18).
Solutions for each calibration curve were prepared by dilution of known amounts of DHA and
EPA analytic standards in sunflower oil. Prepared
solutions were mixed with the internal standard in
the amount corresponding to 1% concentration in
prepared dilutions. Obtained solutions were assayed
using NMR spectroscopy, according to the method
developed by Igarashi et al. (19).
1
H NMR spectroscopy
1
H NMR spectra of solutions prepared for
development of standard curves and of tested products were acquired from the Bruker-Avance II
Ultrashield Plus spectrometer, with frequency of
600 MHz, using deuterated chloroform as a diluent
and methanol as the internal standard. All spectra
were acquired after 64 passages, with pulse length of
11 µs and the acquisition time of 2.65 s. All determinations were performed at 23OC, considering perfect detection of fatty acids at higher temperatures
and storage conditions recommended by manufacturers.
Determination of DHA and EPA content in tested
products
Tested sample was drawn from a mixed content of 10 capsules of the particular product. Liquid
filling was collected with a syringe and needle into
a beaker. Representative sample (200 µL) of the tested product was transferred into a 2 mL disposable
test tube. Seven hundred fifty microliters of deuterated chloroform and 7.5 µL of methanol were added.
Content of the disposable test tube was mixed, and
300 µL of the solution was collected and transferred
into the NMR test tube.
All samples of tested products were assayed
twice. Mean values and deviations from the declared
n-3 acid content defined in requirements for that
form of drug were calculated. The admissible deviation from the declared DHA and EPA content
RESULTS
Comparison of identity of n-3 acids contained in
dietary supplements and a drug with manufacturersí declarations
The comparative analysis of 1H NMR spectra
of tested products with analytical standards demon-
Table 1. EPA to DHA ratio in Polish n-3 fish oil drug and dietary supplements.
Ratio
EPA:DHA (n)
EPA:DHA (%)
≤ 0.5 : 1
5
26
1.5 : 1
10
53
1.51-2.0 : 1
2
11
2.1-2.5 : 1
1
5
2.6-3.9 : 1
0
0
≥4:1
1
5
Figure 6. Cost of supplementation with PUFA n-3 products to achieve daily intake of 1 g/day
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KAMILA OSADNIK and JOANNA JAWORSKA
strated a complete consistency of DHA and EPA
identity from both the tested drug, and all dietary
supplements. It is worth noting that, regardless the
price, each product contained those n-3 acids that
had been declared on the information leaflet by a
manufacturer.
Comparison of n-3 acid content in dietary supplements and a medicinal product with the manufacturerís declaration
Based on the NMR spectroscopy it was found
that: in case of DHA 42% of products (n = 8) the
result of analysis was different from the declared
one; in case of EPA a discrepancy was found in 26%
of products (n = 5).
It is worth noting that in case of the medicinal
product registered as a drug and available in the
Polish market, the NMR analysis of DHA and EPA
content demonstrated a complete consistency with
the manufacturerís declarations.
In case of 21% (n = 4) of tested products the
amount of DHA and/or EPA determined experimentally was higher than declared by the manufacturer
(Fig. 3).
Necessary number of capsules of a PUFA n-3
product ensuring a daily supplementation dose
Sixty three percent of tested products (n = 12)
satisfy the dietetic demand with one capsule taken a
day, as they contain overall amount of 250 mg of
DHA and EPA. Twenty seven percent (n = 5) of
them require ordination of two capsules a day (Fig.
4).
Necessary number of capsules of a PUFA n-3
products allow the daily therapeutic dose
The number of capsules necessary to reach the
daily EPA + DHA consumption of 1 g/day recommended by the European Society of Cardiology
(ESC) and the European Athercoslerosis Society
(EAS) was summed up for tested products (16).
Only 21% of products allows realization of ESC recommendations with the use of 2 capsules a day, and
only 10% meet those requirements with the use of 1
capsule a day. Over a half of tested products require
taking at least 4 capsules a day. Results of the analysis are presented graphically in Figure 5.
Cost of supplementation with PUFA n-3 products
A majority of PUFA n-3 containing products
available in the Polish market is priced below 20
PLN per pack. However, the number of capsules in
a single pack, and n-3 acid content are highly variable. The analysis indicated that in one of three
cases a consumer who had bought a relatively cheap
product has to consider the necessity of taking more
capsules a day to meet the ESC guidelines regarding
consumption of 1 g of PUFA n-3/day. Increasing
price (as per 1 capsule) reduces the necessity of taking multiple daily doses, which accounts for convenient use of a product.
It is surprising that in the group of the most
expensive products there are dietary supplements
containing low amount of PUFA n-3. Figure 6 presents the listing.
EPA : DHA index
The relative EPA and DHA content in each
product was calculated based on experimental data.
In half of them (n = 10) the EPA : DHA index was
1.5 : 1. Every fifth product is characterized by the
EPA : DHA index higher than 1.5 : 1, but as much
as 26% of products contain more DHA than EPA
(Table 1).
DICUSSION
Nutritional habits of Polish people do not follow dietary recommendations of the Food and
Nutrition Institute regarding fish consumption.
According to recommendations, fish should be
served 2-3 times a week, whereas just over 40% of
Poles eat fish once a week, and 60% consumed fish
hardly ever (20). The consequence of depletion of
PUFA n-3 containing foods is abnormal proportion
of n-6 to n-3 acids of 10-20 : 1, whereas a correctly
balanced diet should provide the ratio at the level of
4 : 1 - 5 : 1. Increased cardiovascular morbidity and
mortality may be an effect of that nutritional error
(21).
Increasing awareness of healthy lifestyle is
reflected rather by growing demand on dietary supplements with PUFA n-3, than in changes of diet
(12, 13). Fortunately, a complete identity consistence of those acids was demonstrated in all tested
products, which means that oil contained in capsules
is of marine fish origin, as declared by their manufacturers.
Unfortunately, information regarding DHA
and EPA content provided for some products are
unreliable and misleading, which may lead to
improper PUFA n-3 levels in consumersí organisms. Kris-Etherton et al. and Piecyk et al. compared
EPA : DHA proportions declared by manufacturers
of dietary supplements. Their results indicate the
unfavorable prevalence of EPA over DHA in a
majority of tested products (22, 23). The problem
has been also discussed by Gorjão et al., who indi-
Analysis of ω-3 fatty acid content of Polish fish oil drug a dietary supplements
cated a higher DHA content compared to EPA in
cold-water fish, and the opposite relation of acids in
available dietary supplements (24). It has not been
determined which of n-3 acids is of a greater therapeutic value, because a majority of clinical trials use
a mixture of both acids. There are some single
reports indicating that only DHA is able to reduce
the arrhythmia-induced mortality (the risk reduced
by 45%), effectively reduce the total cholesterol
level, increase the HDL level, inhibit dementia and
depression (25-29). On the other hand, EPA is of
significance for reduction of myocardial infarctioninduced mortality (the risk reduced by 28%) (25).
Results of the EPA : DHA proportion analysis in 19
Polish products containing both acids are comparable to results reported by other authors. In case of
over a half of them the ratio is 1.5. Each fourth
product contains more DHA than EPA, which
stands the Polish market out in comparison to other
ones (22, 23, 30). Unfortunately, it is notable that a
more common discrepancy is related to the difference between the declared and the actual content of
DHA, than of EPA, which in consequence maintains
a less favorable balance, considering the pro-health
effects of those acids. Results of this study indicate
that not only the manufacturer-declared EPA : DHA
ratio is unfavorable, but also in 21% (n = 4) of tested product the situation is additionally made worse
by lower than declared actual DHA level.
Those fluctuations may be explained by a variable DHA and EPA content in marine fish, depending on their physiological condition, season of the
year, and especially the fishing area (31, 32). Fish
from northern seas are characterized by a higher
EPA content, while DHA content is higher in southern sea fish. The observation has been confirmed by
Opperman et al. for dietary supplements available in
the South African markets. Contrary to European
researchers, the authors indicate reduced EPA content in products available in South Africa (30).
Manufacturers of dietary supplements available in
the Polish market base on salmon, sardine,
anchovies and cod fished in the Atlantic Ocean, the
Northern Sea and the Baltic Sea, which could
explain inconsistent DHA contents in their products
(32).
There are also products available in the Polish
market, with α-linolenic acid (ALA) as the only
declared PUFA n-3. Three dietary supplements of
that kind were also analyzed for identity of DHA
and EPA. In case of two product, the 1H NMR spectrum demonstrated no signals characteristic for
DHA and/or EPA. One supplement contained minor
(at the border of the tolerated content deviation)
881
DHA content. According to scientific data, adult
human organism is able to convert ALA into longchain n-3 fatty acids. However, the process is inefficient and in men constitutes only a fraction of percent. Organism of a young healthy male converts
8% of dietary ALA into EPA, and only 0-4% of
ALA into DHA (3). In young healthy females
approx. 21% of dietary ALA is converted into EPA,
and 9% into DHA (4). The study by Plourde et al.
demonstrated that consumption of ALA has only a
minor effect on increased EPA and DHA levels in
serum or red blood cells. Moreover, it was demonstrated that increased consumption of EPA leads to
its increased serum level, but fails to increase the
DHA level, whereas the increased consumption of
DHA results in increased levels of DHA and EPA
(although to a lesser extent). The fact is a result of a
partial reconversion of DHA into EPA (33).
Considering a relatively low conversion of ALA
into long-chain n-3 fatty acids, especially DHA,
those fatty acids have to be supplied. A sale of
dietary supplements under the name of Omega-3,
although correct from the chemical point of view,
may have some measureable consequences for consumers. Therefore, precise information concerning
composition of dietary supplements is so important.
The importance of dietary supplements containing oil from crustaceans has been growing lately. One of the tested products contained PUFA n-3
from krill. It may be surprising that the supplement
is one of the most expensive ones, and its n-3 acid
content is low (32.5 mg DHA and 70 mg EPA).
High price of some products may be explained by its
association with a particular fish species, from
which the oil is pressed, extraordinary purity of
some water, where animals were fished, and a timeconsuming and expensive process of production
based on small crustaceans. One of the recent studies on bioavailability index of n-3 acids indicates,
that the highest index if found in products combining krill oil with phospholipids (34).
The question of consumption of PUFA n-3 is
regulated in Poland by recommendations of several
scientific and research organisations, including the
European Food Safety Authority and the Food and
Agriculture Organization of the United Nations, that
determine the minimum daily intake of DHA + EPA
by healthy adults at 250 mg (13, 14). The World
Health Organization recommends daily intake of
both EPA and DHA in amount of 250-300 mg plus
800-1100 mg of ALA (35). That problem seems to
be favorably reflected by dietary supplements present in the Polish market. A majority of tested products contain an appropriate amount of n-3 acids,
882
KAMILA OSADNIK and JOANNA JAWORSKA
allowing provision of the supplementation dose by
consumption of 1 or 2 capsules a day. It should be
noted that the dose corresponds to an amount preventing PUFA n-3 deficiency in a healthy adult,
which means that 250 mg/day is a necessary dietetic minimum. It should be also noted that the supplementation dose is not a synonym of the therapeutic
dose ñ a dose appropriate for treatment of diseases
in which long-chain PUFA may lead to improvement of the health condition.
However, some producers of n-3 containing
dietary supplements offer much more than just a
nutritional supplementation. Approx. 63% (n = 13)
of tested dietary supplements (n = 22) clearly states
on the label or in the product leaflet (as a purchase
promoting campaign) that taking the product will
result in ìreduced cholesterol levelî, ìimprove the
function of vascular endotheliumî, ìreduce blood
pressureî, and ìreduce inflammationî. It should be
stated that, according to scientific research, those
effects are possible, but achievable only with doses
much higher than 250 mg/day (10, 35, 36).
Current guidelines of the European Society of
Cardiology (ESC) and the European Atherosclerosis
Society (EAS) recommend 1 g of PUFA n-3/day for
all patients with a history of myocardial infarction.
That dosage is difficult to achieve based on natural
food sources, but achievable with dietary supplements, nutraceuticals and/or medicinal products
(16). Polish Diabetes Association recommends type
1 diabetes patients using 2-4 g of n-3 acids/day (17).
Despite clear scientific recommendations, producers
of n-3 containing dietary supplements often suggest
the number of capsules to be taken daily, with no
information concerning the basis for that recommendation.
A majority of tested products requires taking at
least 4 capsules a day to reach the ESC-recommended therapeutic dose of 1 g and to allow health effects
promised in information materials. Unfortunately,
only 31% of dietary supplements dedicated to cardiovascular patients allows intake of the dose of 1
g/day with not more than 2 capsules a day. The
problem of incorrect dietary supplement dosage recommendations has been also described by Belgian
scientists. For a majority of dietary supplements
tested by them (n = 9) reaching the dose of 1 g
proved impossible, even with the use of the highest
dosage recommended by a manufacturer (37).
Unreliable label information are misleading for consumers, with a possible consequence of deterioration of their heath condition.
One have to remember that according to medical recommendation and legislation dietary supple-
ments should be used only to supplement but not
substitute a healthy diet that is recommended as a
base of cardiovascular disease prevention (class I,
ESC recommendation) (38).
CONCLUSIONS
1. All tested dietary supplements and the single
tested drug available in the Polish market demonstrate consistency in terms of identity of DHA and
EPA fatty acids, which means that they are based on
raw material obtained from marine fish.
2. For a majority of tested products, the information provided by a manufacturer on a label
regarding the total amount of DHA and EPA was
reliable. Correct information in that respect is a basis
for selection of an appropriate dosage.
3. Choosing n-3 acid containing products it is
reasonable to consider the content of individual
acids and their ratio, as those parameters decide on
pro-health effects of a product.
4. Patients with cardiovascular conditions or
diabetes should pay special attention to the number
of capsules to be taken to meet the DHA + EPA dose
recommended by scientific societies.
Acknowledgment
Kamila Osadnik is a scholarship holder within
the DoktoRIS project - Scholarship program for the
innovative Silesia, supported by the European
Community from the European Social Fund.
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Received: 30. 07. 2015