Michael L.
Kagan
*a,
Aharon
Levy
b and
Alicia
Leikin-Frenkel
c
aQualitas Health Ltd, 19 Hartom Street, P.O. Box 45423, Jerusalem 91450, Israel. E-mail: mkagan@qualitas-health.com
bPharmaseed Ltd, 9 Hamazmera St., Ness Ziona 74047, Israel
cSackler School of Medicine, Tel Aviv University and The Bert W. Strassburger Lipid Center, Sheba Medical Center, Tel Hashomer, 52621, Israel
First published on 20th October 2014
Long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) exert health benefits which are dependent upon their incorporation into blood, cells and tissues. Plasma and tissue deposition of LC n-3 PUFA from oils extracted from the micro-algae Nannochloropsis oculata and from krill were compared in rats. The algal oil provides eicosapentaenoic acid (EPA) partly conjugated (15%) to phospholipids and glycolipids but no docosahexaenoic acid (DHA), whereas krill oil provides both EPA and DHA conjugated in part (40%) to phospholipids. Rats fed a standard diet received either krill oil or polar-lipid rich algal oil by gavage daily for 7 days (5 ml oil per kg body weight each day). Fatty acid concentrations were analyzed in plasma, brain and liver, and two adipose depots since these represent transport, functional and storage pools of fatty acids, respectively. When measuring total LC n-3 PUFA (sum of EPA, docosapentaenoic acid (DPA) and DHA), there was no statistically significant difference between the algal oil and krill oil for plasma, brain, liver and gonadal adipose tissue. Concentrations of LC n-3 PUFA were higher in the retroperitoneal adipose tissue from the algal oil group. Tissue uptake of LC n-3 PUFA from an algal oil containing 15% polar lipids (glycolipids and phospholipids) was found to be equivalent to krill oil containing 40% phospholipids. This may be due to glycolipids forming smaller micelles during ingestive hydrolysis than phospholipids. Ingestion of fatty acids with glycolipids may improve bioavailability, but this needs to be further explored.
Various species of the algal genus Nannochloropsis have been found to contain high concentrations of EPA with no DHA21 and to present the LC n-3 PUFA as a mixture of phospholipids and glycoplipids (polar-lipids).22 We recently compared the appearance of EPA and DHA in plasma of healthy humans taking krill oil or polar-lipid rich oil from Nannochloropsis oculata over 10 hours following the oil consumption as part of a high fat meal.23 We found that when the subjects consumed the polar-rich algal oil they had higher post-prandial EPA concentrations in their plasma than when they consumed the krill oil. When comparing the content of phospholipids in krill oil (∼40%) to the polar-lipids in algal oil (∼15%), where the main difference is the presence of glycolipids, it may be inferred from the results of this study that LC n-3 PUFA, and EPA specifically, when conjugated to glycolipids, may be more efficiently handled in the gastrointestinal tract; this may relate to enhanced digestion or absorption. This suggests that the glycolipids in algal oil may offer an advantage in delivering EPA to blood plasma and thus in influencing those biological functions where EPA is important.
Thus far, the appearance of LC n-3 PUFA from the novel algal oil has only been examined acutely (i.e. over 10 hours following consumption by healthy human volunteers).23 In the current study, we examined the incorporation of LC n-3 PUFA not only into plasma but further into several tissues in the rat. Thus this rat study represents a natural extension of our earlier human study. We set out to compare krill oil and polar-lipid rich oil from Nannochloropsis oculata by providing these two oils to rats daily for seven days. We analyzed the EPA, docosapentaenoic acid (DPA; 22:5n-3) and DHA concentrations of plasma, brain and liver, and two adipose depots. These sites were selected because they represent transport, functional and storage pools of fatty acids,24 because liver and brain represent key targets for functional activity of LC n-3 PUFA,25–29 and because these sites have all been studied in earlier research evaluating incorporation patterns of LC n-3 PUFA in rats.30–32
Algal oil | Krill oil | |
---|---|---|
Plasma | ||
EPA | 9.8** (6.25, 13.34) | 5.22 (3.89, 6.55) |
DPA | 0.86 (0.02, 1.7) | 1.24 (−0.19, 2.67) |
DHA | 1.31** (0.81, 1.81) | 3.48 (2.46, 7.99) |
Total EPA + DPA | 10.66* (6.52, 14.79) | 6.46 (4.94, 7.99) |
Total LC n-3 PUFA (EPA + DPA + DHA) | 11.97 (7.42, 16.51) | 9.95 (8.12, 11.78) |
Algal oil | Krill oil | |
---|---|---|
Liver | ||
EPA | 116.1 (90.53, 141.7) | 95.79 (64.3, 127.3) |
DPA | 116.2** (88.79, 143.6) | 73.37 (55.3, 91.44) |
DHA | 112.8** (58.8, 166.7) | 297.0 (209.9, 384.1) |
Total EPA + DPA | 232.3* (185.7, 279.0) | 169.2 (120.9, 217.4) |
Total LC n-3 PUFA (EPA + DPA + DHA) | 345.1 (257.8, 432.3) | 466.2 (340.6, 591.8) |
Brain | ||
EPA | 3.17 (2.09, 4.24) | 2.06 (1.19, 2.94) |
DPA | 7.93 (6.25, 9.61) | 10.77 (2.68, 18.87) |
DHA | 210.2 (161.9, 258.6) | 213.1 (147.6, 278.6) |
Total EPA + DPA | 11.1 (8.82, 13.37) | 12.84 (4.43, 21.25) |
Total LC n-3 PUFA (EPA + DPA + DHA) | 221.3 (171.0, 271.5) | 225.9 (159.1, 292.7) |
Gonadal adipose tissue | ||
EPA | 74.08* (39.38, 108.8) | 38.87 (25.59, 52.15) |
DPA | 29.66 (14.91, 44.4) | 21.02 (14.06, 27.98) |
DHA | 21.78** (12.1, 31.46) | 47.38 (40.25, 79.53) |
Total EPA + DPA | 103.7* (54.44, 153.0) | 59.89 (40.25, 79.53) |
Total LC n-3 PUFA (EPA + DPA + DHA) | 125.5 (67.23, 183.8) | 107.3 (73.75, 140.8) |
Retroperitoneal adipose tissue | ||
EPA | 387.1** (231.0, 543.2) | 125.8 (26.49, 225.1) |
DPA | 111.7** (79.84, 143.5) | 53.51 (12.73, 94.29) |
DHA | 56.61 (39.65, 73.56) | 158.9 (17.63, 300.2) |
Total EPA + DPA | 498.8** (312.2, 685.4) | 179.3 (39.67, 318.9) |
Total LC n-3 PUFA (EPA + DPA + DHA) | 555.4* (368.9, 741.9) | 338.2 (57.33, 619.1) |
The focus of the current study was the longer-term appearance of EPA and DHA from krill oil and polar-lipid rich oil from Nannochloropsis oculata in tissues of rats. This is important as an extension of the previous human study because it is not generally feasible to biopsy tissues from humans. The fatty acids were measured in plasma, brain and liver, and two adipose depots since these represent transport, functional and storage pools of fatty acids, respectively.24 Krill oil contains both EPA and DHA and 40% phospholipids while the algal oil contains only EPA and 6% phospholipids and 9% glycolipids. When measuring total LC n-3 PUFA, there was no difference in plasma, brain, liver or gonadal adipose tissue between the two oils. Polar-lipid rich algal oil resulted in a significantly higher level of LC n-3 PUFA (as EPA) in retroperitoneal adipose tissue. There was an average 3-fold differential in EPA content of retroperitoneal adipose tissue between groups which is much greater than the difference in EPA content of the two oils.
Glycolipids are a class of compounds containing one or more monosaccharides bound by a glycosidic linkage to a hydrophobic membrane-anchoring compound such as an acylglycerol or a sphingoid. Galactolipids are a type of glycolipid whose sugar group is galactose and in plants consist mainly of monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG) (Fig. 1) containing one or two saturated and/or unsaturated fatty acids linked to the glycerol moiety.35,36 Galactolipids are important food constituents in both animals and humans and are an important source of essential fatty acids.37 Both macro-algae38 and micro-algae22 contain glycolipids. MGDG and DGDG levels have been measured by 13C NMR (Fig. 2) in Nannochloropsis and found to be conjugated across the fatty acid spectrum.22 The role of galactolipids as intracellular messengers has been investigated by Wakelam39 and as anti-inflammatory agents by Lenti et al.40 and Bruno et al.41
In a study on the bioavailability and accumulation of lutein in mice, Gorusupudi and Vallikannan42 found that the percent of micellarization of lutein was higher with glycolipids than phospholipids and neutral lipids. Likewise, the mean plasma lutein response was higher for glycolipids than for phospholipids and neutral lipids. The authors postulated that these differences might be due to smaller micellar size with glycolipids that would favour absorption.
In this study, the presence of glycolipids in the polar-lipid rich algal oil and their different digestion and metabolism might explain the tissue uptake of the LC n-3 PUFA (EPA). While the total amount of polar lipids was lower in algal oil compared to krill oil (15% vs. 40%, respectively), tissue uptake was similar, and EPA uptake in retroperitoneal adipose tissue was higher with algal oil. Further research is needed to understand the specific function and mechanism of glycolipids in LC n-3 PUFA digestion, absorption and metabolism.
EPA, DPA and DHA concentrations did not differ between the brains of rats receiving the two oils. The feeding time used here was short (7 days) and the lack of effect on brain fatty acids reflects the relative insensitivity of the brain to dietary fatty acid modification.
Although the total LC n-3 PUFA content of the liver was not different between groups, animals in the algal oil group had a higher hepatic DPA concentration than those in the krill oil group. The sum of EPA plus DPA did show a significant difference between groups. This suggests some elongation of EPA to DPA occurs in the liver of rats in the algal oil group. This elongation would use EPA and may explain why hepatic EPA did not differ between the two groups of rats.
Total EPA concentration in retroperitoneal adipose tissue was higher in rats in the algal oil group compared with those in the krill oil group. Conversely DHA concentration was higher in gonadal adipose tissue of rats in the krill oil group. These differences reflect the differences in fatty acid content of the two oils.
One interesting observation made in the current study is that the EPA, DPA and DHA contents were higher in retroperitoneal than in gonadal adipose tissue in the rats in the algal oil group, although this was not seen in those in the krill oil group. The higher DPA in retroperitoneal adipose tissue of rats receiving algal oil may reflect local synthesis of DPA from EPA or may reflect that DPA (resulting from hepatic synthesis) is readily taken up by this adipose tissue store. Nevertheless some of the rats in the krill oil group did show high EPA, DPA and DHA contents in their retroperitoneal adipose tissue. These findings suggest that different adipose depots may take up and store LC n-3 PUFA differentially. There is support for this suggestion from the literature.43,45 First, de Heredia et al.43 reported much higher DHA in the mesenteric adipose tissue than in gonadal or subcutaneous adipose tissue of female rats fed a high fat diet containing some EPA and DHA. Secondly, Tou et al.44 reported higher (on average about 2-fold higher) EPA and DHA in retroperitoneal adipose tissue than in gonadal adipose tissue from female rats fed a high fat diet with various sources of preformed EPA and DHA. It is not clear what the mechanism underlying the differential enrichment of adipose tissue with LC n-3 PUFA is, but this may be important if dietary fatty acid interventions are to be used to influence adipose tissue biology. The current findings alongside those in the literature43,44 indicate that some adipose depots may be more sensitive than others to the influence of dietary LC n-3 PUFA.
One limitation of the current study is that there was no group that did not receive a LC n-3 PUFA rich oil. However, it is known that the EPA and DPA contents of most rat tissues are very low if the animals do not receive preformed EPA.43–45 Conversely the brain, and some other tissues like the heart, contain significant amounts of DHA even when the diet is very low in LC n-3 PUFA.44,45 This limitation does not detract from the main focus of this study, which was to observe whether the LC n-3 PUFA concentration of selected tissues would be higher in rats receiving polar-lipid rich oil from Nannochloropsis oculata than in those receiving krill oil.
Animals were housed under standard laboratory conditions, air conditioned and filtered with adequate fresh air supply (minimum 15 air changes per hour). Animals were kept in a climate controlled environment: the temperature range was between 20 and 24 °C and the relative humidity range was between 30 and 70% with a 12 hours light and 12 hours dark cycle. Animals were housed in polyethylene cages (3 rats per cage) measuring 35 × 30 × 15 cm, with a stainless steel top grill facilitating pelleted food and drinking water in a plastic bottle. Bedding was steam sterilized clean paddy husk (Harlan, Sani-chip) and was changed along with the cage at least twice a week.
Animals were fed ad libitum a commercial rodent diet (Certified Global 18% Protein Diet; Teklad, Madison, WI, USA). The diet contained (per kg diet) 180 g protein, 60 g fat (as soybean oil) and 440 g carbohydrate. Contributions to energy intake for protein, fat and carbohydrate were 24%, 18% and 58%, respectively. The fatty acid composition of the diet was as follows (g/100 g total fatty acid): palmitic acid (16:0): 11.7; stearic acid (18:0): 3.3; oleic acid (18:1n-9): 20; linoleic acid (18:2n-6): 51.7; α-linolenic acid (18:3n-3): 5.0.
Each day for 7 days the animals received 5 ml of supplement oil homogenized with 5 ml olive oil per kg body weight by oral gavage. Dilution and warming in a water bath to 35 °C before gavage was necessary because of the high viscosity of both the krill oil and the algal oil. Krill oil contained 23% EPA + DHA and 41% phospholipids (2:1 EPA–DHA; Neptune Technologies) and algal oil 25% EPA and no DHA (Qualitas Health) with 6% phospholipids and 9% glycolipids. Therefore, over the course of the study, the animals were fed a total of 7.245 g kg−1 body weight EPA + DHA fatty acids from krill oil and 7.315 g kg−1 body weight EPA from algal oil. To put these amounts of oil and of LC n-3 PUFA into context, rats weighing 250 g eat about 25 g of food daily. In the current study, the diet contained about 60 g of fat per kg. Thus, these rats were eating about 2 g of fat from their diet each day. The amount of oil provided by gavage (10 ml kg−1 body weight each day) was 2.5 g each day for a 250 g rat. Thus the gavage slightly more doubled daily fat intake. As far as LC n-3 PUFA are concerned, a 250 g rat received about 0.26 g per day. Thus, LC n-3 PUFA contributed approximately 5.8% of total fat intake. This is higher than minimum recommendations made for humans which equate to about 0.5 to 1% of dietary fatty acids; for example intake of LC n-3 PUFA at the level of the minimum UK recommendation (0.45 g per day)12 by a woman or man consuming the average amount of fat for UK adults (60 and 80 g per day, respectively) would equate to an intake of about 0.8 and 0.6% of total dietary fatty acids, respectively. Contributions of LC n-3 PUFA from concentrated supplements, from prescription preparations and from fatty fish to fat intake would be greater than this. For example, the maximum prescribable dose of LC n-3 PUFA (4 g product providing 3.6 g EPA + DHA) equates to an LC n-3 PUFA contribution of 4.5% of total dietary fatty acids in a person consuming 80 g fat per day, and even more if that person is consuming a low fat diet. Finally, it is worth noting that in many experiments rodents are fed diets providing much more LC n-3 PUFA than used in the current study. For example, Yaqoob et al.45 fed rats diets providing 200 g fish oil per kg diet, 20% of which was EPA + DHA, resulting in EPA + DHA intakes of 1 g per day for a 250 g rat.
Animals were sacrificed after 8 days. Blood was collected into EDTA as anticoagulant by cardiac puncture and plasma was prepared by centrifugation. Brain, whole liver, and retroperitoneal and gonadal adipose tissues were collected, weighed and snap frozen for further analysis.
This journal is © The Royal Society of Chemistry 2015 |