1. Introduction
A vast body of literature exists on the effect of long chain omega-3 polyunsaturated fatty acids (
n-3 LC-PUFA) in the areas of infant development [
1], cardiovascular disease, platelet aggregation [
2], cancer [
3], dementia, Alzheimer’s disease, depression [
4,
5], and inflammation [
6,
7].
Recommendations for eicosapentaenoic acid (EPA; 20:5n-3) plus docosahexaenoic acid (DHA; 22:6n-3) intake have been put forth by several organizations globally, with ranges from 250 mg to 500 mg per day for adults with an additional 200 mg of DHA per day for pregnant and lactating women [
8]. The recommendation for patients with coronary heart disease generally is 1 g EPA plus DHA per day, and for patients with high triglycerides the recommendations range from 1.2 to 4 g EPA plus DHA per day [
8]. Some organisations, including the American Heart Association and Australian National Health and Medical Research Council, recommend foods (oily fish) once to twice per week [
9], whereas others like the National Heart Foundation of Australia deal with nutrients daily based on daily intake (250–500 mg per day of EPA plus DHA) [
10]. Accordingly, despite the actual quantity of recommended intake, it appears to be important to understand what feeding strategy, such as a constant daily dose or large doses fewer times per week, is more efficient to fulfil dietary requirements of EPA and DHA.
There are two human studies which have compared a constant daily dose of
n-3 LC-PUFA
vs. twice weekly doses of
n-3 LC-PUFA [
11,
12]. Harris
et al. [
11] compared the bioavailability of EPA and DHA from twice weekly consumption of oily fish (3.40 g/week EPA + DHA) with daily fish-oil capsule supplementation (3.37 g/week EPA + DHA) in a 16 week study in 23 women; they found that there were no treatment differences in the EPA and DHA content of red blood cell membranes or plasma phospholipids. Browning
et al. [
12] compared the bioavailability of EPA and DHA from fish oil capsules (twice per week) with daily fish oil consumption. Male and female participants (
n = 65) were given capsules (containing 6.54 g of EPA and DHA per week) either twice weekly or daily for one year. They found that there was no difference between treatments in the plasma phosphatidylcholine level of EPA and DHA, but that there were significantly higher levels of EPA and DHA in platelets and higher levels of EPA in mononuclear cells. Both studies used blood (plasma or red cell) levels of
n-3 LC-PUFA as a proxy for bioavailability, which as discussed previously has significant limitations [
13].
The aim of the present study was to compare the bioavailability and efficiency (as metabolic fate) of the same amount of dietary n-3 LC-PUFA administered either as a constant daily dose (Constant treatment)vs. a single weekly dose (Spike treatment). It was hypothesized that a single weekly dose of n-3 LC-PUFA would not be as bioavailable as a continuous daily dose of n-3 LC-PUFA.
4. Discussion
The present study sought to examine the whole body bioavailability and efficiency (as n-3 LC-PUFA metabolic fate) of the same overall dose of n-3 LC-PUFA, provided from the same source (fish oil), at the same overall weekly dose, but at different frequencies: dailyvs. weekly. It was found that the growth of the animals was not different between treatments and no mortalities were recorded amongst the three experimental treatments (Constant, Spike and Control). There were significant but small differences in the excretion of the n-3 LC-PUFA between the Spike and Constant treatment groups, which are unlikely to be nutritionally relevant.
The results obtained by simply comparing tissue FA concentrations (mg/g tissue) were interesting, but they are admittedly of limited value towards achieving a better understanding of n-3 LC-PUFA “bioavailability”. While the dietary intake of n-3 LC-PUFA provided by the two diets were similar, but not identical, there were some differences between treatments that might be independent of the difference in dietary intake. For example, the DPA concentration in liver and muscle was significantly greater in the Spike treatment than in the Constant treatment, despite the dietary DPA intake being significantly greater for the Constant treatment. This has no obvious explanation, but reveals that metabolic processing of dietary n-3 LC-PUFA is more complex than simply looking at dietary intake values or tissue levels.
A much greater and more accurate understanding of the actual metabolic fate of n-3 LC-PUFA provided by different oils can be achieved by observing the results of the Whole Body Fatty Acid Balance Method, which takes into account, and thus balances out, any differences in dietary intake.
This data showed that the major metabolic fate of the n-3 LC-PUFA (EPA + DPA + DHA) was towards catabolism (β-oxidation) accounting for over 70% of total dietary intake, whereas deposition accounted less than 25% of total dietary intake. It was found that significantly more n-3 LC-PUFA were β-oxidised when originating from the Constant treatment (84% of dose), compared with the Spike treatment (75% of dose). Conversely, it was found that significantly more n-3 LC-PUFA were deposited when originating from the Spike treatment (23% of dose), than from the Constant treatment (15% of dose). This result suggests that the n-3 LC-PUFA provided by the Spike treatment were more deposited (bioavailable), compared with those provided by the Constant treatment.
The differences in β-oxidation and deposition were not the same for each of EPA, DPA and DHA for either the Spike or Constant treatment. That is, EPA was more extensively β-oxidised than DPA and DHA on both treatments, but the differences between the 20 carbonand the 22 carbon PUFA were accentuated in the Spike treatment. In the case of β-oxidation, the Constant/Spike ratios were 1.7 for DPA and 1.4 for DHA, but only 1.0 for EPA. In terms of deposition, the Constant/Spike ratios were 0.5 for DPA, 0.6 for DHA, and 0.7 for EPA. This suggests that EPA is preferentially directed towards β-oxidation almost independent of whether the EPA is provided daily or once per week. This is consistent with data showing high affinity of EPA to catabolism (β-oxidation) in animal models [
23]. It has been reported using Wistar rats that EPA-CoA was a good substrate for mitochondrial carnitine acyl-transferase-I and DHA was a poor substrate for both mitochondrial and peroxisomal β-oxidation, which could explain the high rate of β-oxidation for EPA [
23].
In contrast to EPA, it would appear that DHA and DPA are somewhat spared from β-oxidation when consumed, as observed previously [
24] but especially when a large dose of dietary
n-3 LC-PUFA is provided weekly. This is consistent with the finding of Kaur
et al. [
24] in rodents who showed, using radiolabelled EPA, DPA and DHA in rats, that six hours after dosing 19% of the EPA was β-oxidized and expired as CO
2 compared with 5% in case of DPA and 7% of DHA. However, these data do not shed any light on why providing a bolus dose of
n-3 LC-PUFA leads to a greater partitioning of DPA and DHA towards deposition. With the Spike treatment, it is therefore possible to speculate that this “flood” of
n-3 LC-PUFA could have saturated the capacity of the mitochondria to β-oxidise any extra DPA and DHA, resulting in a greater retention and deposition of these fatty acids in tissues.
To our knowledge, this is the first study to compare the whole body bioavailability of
n-3 LC-PUFA in rats fed a constant daily dose
vs. a larger and less frequent dose of the same dietary source of
n-3 LC-PUFA. From the two available human studies comparing a weekly dose of
n-3 LC-PUFA with daily dose, only one study used the same source of
n-3 LC-PUFA (capsules not fish meal) [
12]. Both studies used blood levels (plasma, platelets or mononuclear cells) as a proxy for bioavailability. The limitations of these studies include a failure to provide the dose adjusted on a body weight basis, the failure to measure excretion and the failure to measure the EPA and DHA levels in the red blood cells (which are widely regarded as the best measure of EPA + DHA tissue status). Furthermore, because of the known high level of variability in the response of subjects to the same dose of fish oil (as noted by Kohler
et al. [
25]), these studies have limitations because the data was not adjusted for by gender, body weight or exercise level [
26]. In the present rat study, rats were fed to fixed predetermined ration, which was adjusted weekly relative to body weight, for 6 weeks which helped to reduce the variability of the results achieved, increased the statistical power of the test (greater than 80% for the vast majority of data recorded), and ultimately contributed to obtaining more robust, substantiated and more easily interpretable findings.
The possible difference in bioavailability of
n-3 LC-PUFA when provided in different edible sources has received some research attention. Specifically, the blood levels of
n-3 LC-PUFA derived from daily fish oil capsules compared with either adaily fish meal or daily fish oil enriched food have been reported in a few studies [
27,
28,
29,
30].
In the current study, the Constant and the Spike treatment showed lower levels of apparent
in vivo enzyme activity for elongase, Δ6 and Δ5 desaturase compared with the Control treatment. Lower Δ6 desaturase activity with fish oil feeding has been previously reported [
31]; however, there is no data looking at a weekly
vs. daily dose to compare with. These desaturases and elongases are required for the biosynthesis of LC-PUFA and their inter-conversion. High availability of these fatty acids in Constant and the Spike treatment can act via a negative feedback control mechanism and reduce the gene transcription rate and the actual activity of the desaturase and elongase enzymes in these treatments, possibly via sterol regulatory element binding protein (SREBP-1c) [
32,
33]. On the other hand, the lack of
n-3 LC-PUFA intake in the diet of the Control rats has likely increased their elongase and desaturase enzyme activities in order to increase endogenous
n-3 LC-PUFA synthesis. Dietary
n-3 LC-PUFA deprivation has previously been shown to upregulate liver mRNA levels of Δ6 and Δ5 desaturases as well as activities of Δ6 and Δ5 desaturases [
34,
35].
Overall, there were no significant differences in the assessed enzyme activities between the Spike and Constant treatments, with the exception of Δ9 desaturase (required for the biosynthesis of monounsaturated fatty acids), which was significantly higher in the Spike treatment.
Admittedly, one of the limitations of the present study includes being an animal study in male rats. Nevertheless, these preliminary, novel and highly interesting findings warrant further investigations, and in particular the need to be substantiated by conducting appropriate trials in humans. It is worth noting that the dose of
n-3 LC-PUFA used in the Constant treatment equates to 1012 mg/day for a 70 kg human [
36], which is in the range of human recommendations for these fatty acids. Another limitation, as previously mentioned, was that the slightly different total amounts of
n-3 LC-PUFA administered by the two
n-3 LC-PUFA enriched dietary treatments used in this study. However, these differences are relatively minimal, and unlikely to be responsible of any major modification in the overall
n-3 LC-PUFA metabolism, and have been accounted for in the Whole Body Fatty Acid Balance Method. A third limitation is that these results do not apply to comparative effects of consumption of daily fish oil capsules
vs. a sporadic meal with fish or seafood, since the study investigated the bioavailability of the same food source of
n-3 LC-PUFA; but this was intentional to exclude the possible effect of the matrix (food source) of the dietary
n-3 LC-PUFA.
In conclusion, our data show that there was a significantly greater deposition of the n-3 LC-PUFA associated with a single large dose of dietary n-3 LC-PUFA compared with the smaller daily doses in rats, due to less β-oxidation and greater deposition, and not due to differences in excretion (digestibility). The results from this animal study provide a suitable platform for future human studies aimed at developing substantiated evidence for advising consumers on the most efficient way to increase their n-3 LC-PUFA status.These findings suggests that a large dose of n-3 LC-PUFA once per week is more effective in increasing whole body n-3 LC-PUFA content compared to a smaller dose delivered daily. This observation, if validated in humans, could have remarkable effects on the possible development of more effective and sustainable utilisation strategies of these limited and metabolically important nutrients, currently derived primarily from the dwindling oceanic fish stocks.