Similar eicosapentaenoic acid and docosahexaenoic acid plasma levels achieved with fish oil or krill oil in a randomized double-blind four-week bioavailability study.

BACKGROUND: Long-chain n-3 polyunsaturated fatty acids (LC n-3-PUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) provide multiple health benefits for heart, brain and eyes. However, consumption of fatty fish, the main source of LC n-3-PUFAs is low in Western countries. Intakes of LC n-3-PUFA can be increased by taking dietary supplements, such as fish oil, algal oil, or krill oil. Recently, conflicting information was published on the relative bioavailability of these omega-3 supplements. A few studies suggested that the phospholipid form (krill) is better absorbed than the fish oil ethyl ester (EE) or triglyceride (TG) forms. Yet studies did not match the doses administered nor the concentrations of DHA and EPA per supplement across such comparisons, leading to questionable conclusions. This study was designed to compare the oral bioavailability of the same dose of both EPA and DHA in fish oil-EE vs. fish oil-TG vs. krill oil in plasma at the end of a four-week supplementation. METHODS: Sixty-six healthy adults (n = 22/arm) were enrolled in a double blind, randomized, three-treatment, multi-dose, parallel study. Subjects were supplemented with a 1.3 g/d dose of EPA + DHA (approximately 816 mg/d EPA + 522 mg/d DHA, regardless of formulation) for 28 consecutive days, as either fish oil-EE, fish oil-TG or krill oil capsules (6 caps/day). Plasma and red blood cell (RBC) samples were collected at baseline (pre-dose on Day 1) and at 4, 8, 12, 48, 72, 336, and 672 h. Total plasma EPA + DHA levels at Week 4 (Hour 672) were measured as the primary endpoint. RESULTS: No significant differences in total plasma EPA + DHA at 672 h were observed between fish oil-EE (mean = 90.9 ± 41 ug/mL), fish oil-TG (mean = 108 ± 40 ug/mL), and krill oil (mean = 118.5 ± 48 ug/mL), p = 0.052 and bioavailability differed by < 24 % between the groups. Additionally, DHA + EPA levels were not significantly different in RBCs among the 3 formulations, p = 0.19, providing comparable omega-3 indexes. CONCLUSIONS: Similar plasma and RBC levels of EPA + DHA were achieved across fish oil and krill oil products when matched for dose, EPA, and DHA concentrations in this four week study, indicating comparable oral bioavailability irrespective of formulation. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT02427373.

Controlled formation of mono- and dihydroxy-resolvins from EPA and DHA using soybean 15-lipoxygenase.

Resolvins and protectins are important anti-inflammatory and pro-resolution compounds derived from the enzymatic oxidation of omega-3 fatty acids all-cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and all-cis-4,7,10,13,16,19-docosahexaenoic acid (DHA). We have developed a simple, controlled method to synthesize an array of resolvin and protectin analogs from fatty acid starting materials using soybean 15-lipoxygenase. The conditions were optimized for the production of both mono- and dihydroxy derivatives, with enzyme concentration and pH found to have a significant effect on the reaction products. The methods were applied to five biologically important omega-3 and omega-6 fatty acid substrates. Mono- and dihydroxy compounds were successfully synthesized from all substrates and the products were characterized by normal phase (NP) HPLC, GC-MS, TOF-MS, UV-visible (UV-vis) spectroscopy, and NMR spectroscopy. The methods could be further applied to any polyunsaturated fatty acids containing the cis-1,4,7,10-undecatetraene moiety to produce a range of novel compounds with potential biological activity.

First isolation and structural determination of cyclic beta-(1-->2)-glucans from an alga, Chlorella pyrenoidosa.

The aqueous extract of the edible green microalgae Chlorella pyrenoidosa is of interest because of its immunostimulatory activity. Some components in the extract have been identified previously, namely a unique type of arabinogalactan and a galactofuran. Further fractionation of this extract was accomplished by treating the aqueous solution of the fraction precipitated by addition of 1.5vol of 95% ethanol with cetyltrimethylammonium bromide. The residue obtained by concentration of the supernatant was fractionated further by anion-exchange chromatography and size-exclusion chromatography on Sephadex G-100. Two fractions from the latter column were retained, of which one was a starch-like alpha-(1-->4)-linked d-glucan with some alpha-(1-->6) branches, and the other contained a starch plus a mixture of beta-(1-->2)-d-glucans. ESI mass spectrometry was used to show that the mixture contained both cyclic and linear beta-(1-->2)-d-glucans in a cyclic:linear ratio of 64:36, based on intensities of mass spectral peaks. For the cyclic beta-(1-->2)-d-glucans, ring sizes ranged from 18 to 35 monosaccharides with the ring containing 21 glucose units (54% of the cyclic glucans) being greater than three times more abundant than the next most abundant component, the ring containing 22 glucose units (15%). No rings containing 20 glucose units were present. This is the first observation of cyclic beta-(1-->2)-d-glucans in algae, as far as we are aware. For the linear beta-(1-->2)-d-glucans, the component containing 20 glucoses was most abundant (35% of the linear glucans), while the component containing 21 glucose units was the next most abundant (17%). These relatively low-molecular-weight glucans had low immunostimulatory activity.

Isolation, characterization and structural determination of a unique type of arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa.

An arabinogalactan was isolated from a hot water extract of freeze-dried cells of the green microalga, Chlorella pyrenoidosa. This hot water extract is a proprietary immunomodulator, with the trademark Respondintrade mark (ONC-107). The arabinogalactan was recovered from the ethanol-soluble fraction of the supernatant resulting from a process that involved controlled ethanol precipitation followed by size exclusion chromatography on Sephadex G-100, then Cetavlon precipitation. Sugar analyses, GC-MS data for (S)-2-octyl glycosides, and 1D and 2D NMR experiments established unambiguously that the repeating unit was -->2)-alpha-L-Araf-(1-->3)-[alpha-L-Araf-(1-->4)]-beta-D-Galp-(1-->. This structure does not fit into any of the known classes of arabinogalactans. SEC/MALS experiments gave a molecular mass for the arabinogalactan isolated as 47 +/- 4 kDa but the original structure was probably larger.

Improvement of vascular dysfunction and blood lipids of insulin-resistant rats by a marine oil-based phytosterol compound.

The syndrome that is characterized by obesity, insulin resistance, and hyperlipidemia is increasingly prevalent in all prosperous societies. It is now recognized as a major contributor to cardiovascular disease. Vascular dysfunction in the form of hypercontractility and impaired nitric oxide-mediated relaxation is a significant component of cardiovascular disease, predisposing to ischemic events. The JCR:LA-cp strain of rats exhibits all major aspects of the obesity/insulin resistance syndrome, including vascular dysfunction and ischemic lesions of the heart. Dietary lipid intake may have a marked effect on plasma lipid levels and, potentially, on vascular disease. We have investigated the effects of a novel preparation, ONC101 (a phytosterol esterified with fish oil), on plasma lipids and vascular function in the insulin-resistant JCR:LA-cp rat. Treatment of obese male rats with ONC101 from 8 to 12 wk of age resulted in no change in plasma lipid concentrations at 0.5 g/kg body weight. At the higher dose of 2.6 g/kg, plasma TG fell 50% (1.26 vs. 2.59 mmol/L, P < 0.002) and cholesterol esters were significantly reduced (1.34 vs. 1.61 mmol/L, P < 0.002). Food intake and body weights were unaffected by ONC101 treatment. At the low dose of 86 mg/kg, the hypercontractility of aortic rings in response to phenylephrine was normalized and the relaxant response to acetylcholine was significantly improved. The results indicate that ONC101 at high doses has significant hypolipidemic effects and, at very low doses, has beneficial effects on endothelial and vascular smooth muscle cell function.

Quantitative determination of fatty acid compositions in micro-encapsulated fish-oil supplements using Fourier transform infrared (FTIR) spectroscopy.

The research describes a rapid method for the determination of fatty acid (FA) contents in a micro-encapsulated fish-oil (µEFO) supplement by using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic technique and partial least square regression (PLSR) analysis. Using the ATR-FTIR technique, the µEFO powder samples can be directly analysed without any pre-treatment required, and our developed PLSR strategic approach based on the acquired spectral data led to production of a good linear calibration with R(2)=0.99. In addition, the subsequent predictions acquired from an independent validation set for the target FA compositions (i.e., total oil, total omega-3 fatty acids, EPA and DHA) were highly accurate when compared to the actual values obtained from standard GC-based technique, with plots between predicted versus actual values resulting in excellent linear fitting (R(2)≥0.96) in all cases. The study therefore demonstrated not only the substantial advantage of the ATR-FTIR technique in terms of rapidness and cost effectiveness, but also its potential application as a rapid, potentially automated, online monitoring technique for the routine analysis of FA composition in industrial processes when used together with the multivariate data analysis modelling.

Effect of chemical randomization on positional distribution and stability of omega-3 oil triacylglycerols.

Randomization has been commonly used to modify the chemical and physical properties of natural fats and oils. In this study, seal blubber oil (SBO) and menhaden oil (MHO) were modified through chemical randomization using sodium methoxide, and the effect on positional distribution of fatty acids was investigated using gas chromatography (GC) and (13)C nuclear magnetic resonance (NMR) spectroscopy. The effect of randomization on the stability of the original oils and their randomized counterparts was analyzed by comparing conjugated dienes and thiobarbituric acid reactive substances (TBARS) values after accelerated oxidation at 60 degrees C for 4 days. The omega-3 polyunsaturated fatty acids (PUFA) were distributed more evenly among the terminal sn-1,3 positions and the middle sn-2 position in chemically randomized oils when compared to the starting oils. The effect was more pronounced for SBO with omega-3 PUFA attached preferentially to sn-1,3 positions of triacylglycerols before randomization, and it was less pronounced for MHO, which contained omega-3 PUFA more evenly distributed before randomization. However, different levels of commonly known omega-3 fatty acids, namely, docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), and stearidonic acid (STA), were obtained in both original and randomized oils from GC and (13)C NMR spectroscopy. The stability of the randomized oils was also affected to different degrees, depending on the storage time.

Isolation of phosphorylated polysaccharides from algae: the immunostimulatory principle of Chlorella pyrenoidosa.

The major immunostimulatory principle in the hot aqueous extract of Chlorella pyrenoidosa has been isolated by a sequence of ethanol precipitation, precipitation with a cationic surfactant (CTAB), size exclusion chromatography, and anion exchange chromatography. A series of phosphorylated polysaccharides were obtained having different molecular masses but with similar structures. The higher molecular mass fractions showed considerable activity in the stimulation of mouse peritoneal macrophages to synthesize nitric oxide. The structure of the major polysaccharide was established by sugar analysis, configurational analysis, and 1D and 2D NMR experiments at 500 and 800 MHz on the parent polysaccharide, the de-O-acetylated polysaccharide, and on the components obtained after hydrolysis of the phosphate diesters. It had a beta-D-Galp-(1-->3)-beta-D-Galp-(1-->3)-backbone with half of the Galp units substituted at O-6 by terminal beta-D-Glcp units. The remaining Galp units were substituted on O-6 by about equal amounts of alpha-D-Manp-1-phosphate and 3-O-Me-alpha-Manp-1-phosphate diesters. The substituents were not located in a regularly alternating fashion on the backbone. The O-acetyl groups were largely located on O-2 and O-4 of Galp and 35% of the Galp residues were O-acetylated. This is the second observation of a phosphorylated polysaccharide in an alga and the first where it is present to a significant extent.

Stimulation of cytokine production in human peripheral blood mononuclear cells by an aqueous Chlorella extract.

CPE is an aqueous extract of the edible micro alga Chlorella pyrenoidosa, which has been shown to have immunostimulatory effects in vivo. In the present study, CPE was evaluated for an ability to stimulate cytokine production by human peripheral blood mononuclear cells (PBMC). PBMC from healthy individuals were treated ex vivo for 24 hours with 1, 10 and 100 microg/mL CPE. This resulted in a marked increase in the level of IL-10, a regulatory cytokine, and strong stimulation of the T-helper-1 (Th1) cell cytokines, IFN-gamma and TNF-alpha. In contrast, stimulation of representative T-helper-2 (Th2) cell cytokines, IL-4 and IL-13, was minor. CPE (1, 10 or 100 microg/mL) did not cause a proliferation of human PBMC suggesting that enhanced secretion of cytokines was not secondary to an increase in cell number. We conclude that CPE stimulation of human PBMC induces a Th1-patterned cytokine response and a strong anti-inflammatory regulatory cytokine response, observations that await confirmation in vivo.

Biosynthetic production of universally (13)C-labelled polyunsaturated fatty acids as reference materials for natural health product research.

Long-chain polyunsaturated fatty acids (LCPUFA) including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have become important natural health products with numerous proven benefits related to brain function and cardiovascular health. Not only are omega-3 fatty acids available in a plethora of dietary supplements, but they are also increasingly being incorporated as triglycerides into conventional foods, including bread, milk, yoghurt and confectionaries. Recently, transgenic oil seed crops and livestock have been developed that enhance omega-3 fatty acid content. This diverse array of matrices presents a difficult analytical challenge and is compounded further by samples generated through clinical research. Stable isotope (13)C-labelled LCPUFA standards offer many advantages as research tools because they may be distinguished from their naturally abundant counterparts by mass spectrometry and directly incorporated as internal standards into analytical procedures. Further, (13)C-labelled LCPUFAs are safe to use as metabolic tracers to study uptake and metabolism in humans. Currently, (13)C-labelled LCPUFAs are expensive, available in limited supply and not in triglyceride form. To resolve these issues, marine heterotrophic microorganisms are being isolated and screened for LCPUFA production with a view to the efficient biosynthetic production of U-(13)C-labelled fatty acids using U-(13)C glucose as a carbon source. Of 37 isolates obtained, most were thraustochytrids, and either DHA or omega-6 docosapentaenoic acid (22:5n-6) were produced as the major LCPUFA. The marine protist Hyalochlorella marina was identified as a novel source of EPA and omega-3 docosapentaenoic acid (22:5n-3). As proof of principle, gram-level production of (13)C-labelled DHA has been achieved with high chemical purity ( >99%) and high (13)C incorporation levels (>90%), as confirmed by NMR and MS analyses. Finally, U-(13)C-DHA was enzymatically re-esterified to glycerol to yield a (13)C-labelled tridocosahexaenoin.

Immunostimulatory polysaccharides from Chlorella pyrenoidosa. A new galactofuranan. measurement of molecular weight and molecular weight dispersion by DOSY NMR.

Fractionation of the hot water extract of Chlorella pyrenoidosa was performed using a combination of ethanol precipitation, size exclusion chromatography, and anion exchange chromatography. One fraction contained a new polysaccharide, and this compound was shown to be a 1-->2-linked beta-d-galactofuranan from its 1D and 2D (1)H and (13)C NMR spectra, with a molecular weight of 15 kDa from DOSY NMR measurements. A number of other fractions were shown to have the same repeating unit as the previously identified arabinogalactan. However, arabinogalactans from different fractions were shown by DOSY NMR to have different molecular weights, which ranged from 27 to 1020 kDa. Agreement with molecular weights measured for some of these fractions by SEC-MALS was very good, further confirming the relationship established by Viel et al. between molecular weights of neutral polysaccharides and self-diffusion coefficients. The smaller molecular weight polysaccharides, the galactofuranan and the 27 and 50 kDa arabinogalactans, were shown to be close to monodisperse by analysis of the distributions of the self-diffusion coefficients for the polymers. The larger arabinogalactans had considerable variation in their molecular weights (188 +/- 109 kDa and 1020 +/- 370 kDa). Only the two larger arabinogalactans showed immunostimulatory activity.

Safety and immunoenhancing effect of a Chlorella-derived dietary supplement in healthy adults undergoing influenza vaccination: randomized, double-blind, placebo-controlled trial.

BACKGROUND: Enhancement of immune function has been claimed as a benefit of some natural health products, although few have been subjected to randomized clinical trials. We evaluated the effect of an oral dietary supplement derived from the edible microalga Chlorella pyrenoidosa on immune response after influenza vaccination. METHODS: We conducted a randomized, double-blind, placebo-controlled community-based clinical trial in a convenience sample of 124 healthy adults at least 50 years of age randomly assigned to receive the study product (200 or 400 mg of a Chlorella-derived dietary supplement) or placebo. Participants took the study product or placebo once daily for 28 days. On day 21, we administered a single dose of a licensed trivalent, inactivated influenza vaccine. We obtained serum specimens to measure hemagglutination inhibition titres before and 7 and 21 days after vaccination. The primary immunological outcomes were the proportion of participants with a 4-fold or greater increase in antibodies and geometric mean antibody titres after vaccination; the proportion of participants reporting adverse events during therapy was the safety outcome. RESULTS: A total of 117 (94%) participants completed all aspects of the study. There were no differences in the proportions of recipients of 200 or 400 mg of the Chlorella-derived dietary supplement or placebo who achieved at least a 4-fold increase in antibodies (proportions for the 3 virus strains ranged from 17.9% to 28.2% for the 200-mg group, from 11.1% to 22.2% for the 400-mg group and from 19.0% to 21.4% for the placebo group; p > 0.05 for all comparisons). Reports of adverse events were similar for recipients of the supplement and placebo, except with regard to fatigue, which was reported more frequently by recipients of 200 mg of the supplement (18/41 or 44%) than by those who received 400 mg of the supplement (8/40 or 20%; p = 0.032) or placebo (8/42 or 19%; p = 0.019). Recipients of 400 mg of the supplement who were 55 years of age or younger had significantly higher geometric mean antibody titres against influenza A/New Caledonia 21 days after vaccination (p = 0.047) and against B/Yamanashi 7 days after vaccination (p = 0.034); the trends were nonsignificant for titres against A/Panama. We also observed similar increases for the proportions of subjects with a 2-fold or greater or a 4-fold or greater increase in antibodies. INTERPRETATION: The Chlorella-derived dietary supplement did not have any effect in increasing the antibody response to influenza vaccine in the overall study population, although there was an increase in antibody response among participants aged 50-55 years. Adverse events were similar among those receiving the supplement and the placebo. Further studies are warranted to explore the range of clinical effects resulting from ingestion of this dietary supplement.

Fish oil containing phytosterol esters alters blood lipid profiles and left ventricle generation of thromboxane a(2) in adult guinea pigs.

This study was designed to investigate the lipid-lowering ability of a novel dietary ingredient composed of phytosterols esterified to (n-3) polyunsaturated fatty acids (PUFA) [PS(n-3)]. Adult guinea pigs were fed a test diet supplemented with PS(n-3) (25 g/kg) and corn oil (CO, 5 g/kg), whereas the diet fed to control guinea pigs was supplemented with CO only (30 g/kg). Cholesterol was added to both diets (0.8 g/kg). After 3-4 wk of consuming the diets, serum total cholesterol (TC) and triacylglycerol (TAG) in the PS(n-3) group were 36 and 29% lower, respectively, than levels in controls (P < 0.05). The lower TC levels in the PS(n-3) group reflected a 38% reduction in non-HDL cholesterol (non-HDL-C), whereas the HDL-C concentration was unaffected. Analysis of cardiac left ventricle indicated that generation of the proaggregatory, arrhythmic eicosanoid, thromboxane A(2), was >60% lower in the PS(n-3)-supplemented guinea pigs than in CO controls (P < 0.001). This study demonstrates that the TAG-lowering and eicosanoid-modifying properties of the fish oil (n-3) PUFA are retained when they are provided in the diet in ester linkage with hypocholesterolemic phytosterols.