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- 0 e os ip Ad t pi e al ct Re m iu l he ek e Ch ar He t cle M us er Liv n ee l Sp C RB r Ce a br or lc x te em Sp a in t Re 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: [email protected] Made in España by: Brudy Technology - C/Riera de Sant Miquel, 3 - 2º 4ª - 08006 Barcelona - Tel. 93 217 03 66
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