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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] 875 876 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. 878 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 880 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. 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