The new PMC design is here! Learn more about navigating our updated article layout. The PMC legacy view will also be available for a limited time. Federal government websites often end in. The site is secure. Both trials were double blinded, randomized, placebo-controlled with cross-over. Participants were randomly allocated to groups A placebo then supplement or B supplement then placebo.
Both included a washout. Outcome measures were analyzed using generalized linear mixed models, including terms for treatment, period, and a treatment-by-period interaction. CRP and calprotectin measures showed no evidence of a treatment effect on inflammation; however, model estimation was imprecise for both outcomes, hence further research is required to elucidate potential inflammation effects. The nutrient supplement increased serum levels of key N -3 PUFAs and vitamin D in both populations, showing the preparation was readily bioavailable.
Diet is a key component in the disease susceptibility of individuals. Diets enriched in Eicosapentaenoic acid EPA and Docosahexaenoic acid DHA in animal models have shown positive effects for chronic conditions [ 1 , 2 , 3 ]. Studies in humans have shown these fatty acids are a component of optimal diets for reducing the risks associated with cancer and cardiovascular disease [ 4 , 5 , 6 ].
N -3 PUFA have also been shown to be beneficial in reducing inflammation, especially in people with inflammatory disorders [ 7 , 8 , 9 , 10 , 11 ]. These fatty acids have been used as dietary supplements for some chronic conditions [ 1 , 12 , 13 , 14 ]. N -3 PUFA have furthermore been identified as precursors of mediators such as Resolvins, Protectins, and Maresins, which stimulate anti-inflammatory and pro-resolving mechanisms [ 15 ]. In addition, the sum of EPA and DHA in erythrocyte membranes and expressed as a percentage of total erythrocyte fatty acids the omega-3 index , is used as a risk factor measure diseases such as coronary heart disease [ 16 , 17 ].
Vitamin D is associated with many immune regulatory functions [ 18 , 19 , 20 ]. Vitamin D deficiency has been associated with increased risk of diseases in adults such as osteomalacia, inflammatory bowel disease IBD , hypertension, heart disease, and multiple sclerosis [ 21 , 22 , 23 ].
Furthermore, vitamin D has been implicated in seventeen varieties of cancer through its influence on signaling pathways [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. C-reactive protein CRP is a measure of acute inflammation or infection [ 33 ]. Calprotectin is a key protein found in the intracellular fluid of inflammatory cells and can be measured in the feces as an indicator of the migration of neutrophils through the bowel wall to the fecal material, a measure of bowel inflammation [ 34 ].
Fecal calprotectin scores for adults have been developed and compared to endoscopy results, CRP, blood leukocytes, and the CD activity index, and found to be reliable marker compared to these and is useful in discerning between mild, moderate, and highly active CD [ 35 ]. This was required by the NZ Health and Disability ethics committee before a trial could be undertaken to measure these effects of the supplement in people with CD.
In the trial with healthy people, the placebo was an encapsulated medium chain triglyceride MCT [ 37 ]. MCTs have been long used in clinical nutrition for the dietary management of malabsorption syndromes [ 38 ], and have an appropriate safety profile that has led to extensive use in clinical trials [ 39 , 40 , 41 , 42 , 43 ]. However, an early analysis of the data for the healthy participant trial indicated that the four week washout period was not sufficient, suggesting a carry-over effect of MCT on the vitamin D and fatty acid results [ 37 ].
For the CD trial, therefore, each of the intervention and washout periods were extended to six weeks, and the formulation for the placebo was changed from MCT to one containing long chain n -3 PUFA only. The study design for both trials was double blinded, randomized, placebo-controlled with cross-over. The study population for each group was recruited from Auckland, New Zealand, with an aim of even gender selection, with ages between 20—65 years. A person independent to the study coded participants names once healthy participants were accepted into the first trial of the first study, and they also randomly allocated them into one of the two arms of the study.
In the second trial, CD participants were drawn from the database of an earlier IBD study; therefore their existing codes were used for the CD participants, and they were randomly allocated by a person independent to the study into one of the two arms of the study. All participants and researchers were blinded to the treatment regime until the trial was completed.
Participants were randomly allocated to groups A or B. In each intervention period four weeks for the healthy population and six weeks for the CD population respectively , group A received the placebo first, while group B received the nutrient supplement first Figure 1.
After a washout period four weeks for the healthy and six weeks for the CD subjects respectively , group A received the supplement and group B received the placebo. For the interventions, participants were asked to take their two nutrient capsules or placebo capsules together daily, with their lunch or dinner meal.
A total of four fasting blood samples were collected from each individual during the study; one sample before the first intervention T1 , one sample after the first intervention T2 , one sample after the washout period T3 , and finally, one sample at the end of the last intervention T4; Figure 1. Both trials were conducted from the beginning of the autumn season to early winter in New Zealand from March to June.
The time periods for each intervention were based on the outcomes of previous intervention studies involving fatty acids, which showed the most effective length of time required for uptake, distribution, and interconversion of n -3 fatty acids. In healthy volunteers reaching steady levels can take up to four weeks [ 45 , 46 , 47 , 48 , 49 ].
All participants provided written consent. Inclusion and exclusion criteria were as follows. In order to confirm that the selected participants met the required conditions for the study, two questionnaires pre-screening and dietary were conducted online before the beginning of the trial Figure 2 and Figure 3.
Participants were excluded if they had: cancer in the last five years bar non-melanoma skin cancers ; intestinal disorders for the healthy participants e. Participants with CD also had to provide a list of current medicines and were screened by a gastroenterologist to ensure they were in remission at the start of the trial.
It also contained natural mixed tocopherols and vitamin E with ascorbyl palmitate 1 mg , and was enclosed in a soft gel composed of gelatin, glycerin soft gel, beeswax, and natural annatto. Two of these were taken daily. The heavy metal contamination guarantee was less than 10 ppm. The oil contained the following fatty acid distillates FAD C caproic 0. The one long chain n -3 PUFA fish oil placebo capsule used for the trial with CD participants contained purified fish oil mg.
Each capsule also contained orange oil 2. Each capsule was enclosed in a gelatin shell composed of gelatin mg , glycerol mg , and purified water 37 mg. This was sourced from the same GMP certificated facility as the nutrient capsule. Outcomes of interest were fatty acid and vitamin D serum levels, CRP plasma, and stool calprotectin Table 1.
Each outcome was measured from collected samples at each time point T1—T4. For each trial, a total of 19 fatty acids were measured. These n -3 PUFA were of particular interest as diets enriched in EPA and DHA in animal models have shown positive effects for chronic conditions, and studies have shown cancer and cardiovascular disease were lower in people whose diets had higher levels of these fatty acids [ 50 , 51 , 52 , 53 , 54 ].
Due to the short time for this trial four or six weeks in each intervention group , changes in fatty acids would be most notable in serum, rather than in red blood cells, thus serum measures of fatty acids were used instead of the red blood cell measures of fatty acids which is the preferred measure for the omega-3 index [ 16 ]. This measure is based on the changes in metabolites produced with the ingestion of the nutrient capsules or the placebo.
It is used to determine the extent to which the lipids ingested are utilized. Increasing the intake of lipids does not always equate to utilization and uptake. There are several methods to measure the biomarker for vitamin D [ 60 ]. The international vitamin D external quality assurance scheme reports 16 types of measures [ 52 ].
The plasma 1 mL samples for CRP analysis were collected in Eppendorf tubes and processed on the same day. Separate analyses were carried out for data from each of the two trials. Participant characteristics, anthropometric measurements, and outcome measurements were assessed for balance between groups A and B at baseline within each trial using a chi-square test for categorical variables, and the non-parametric Kruskall—Wallis test to compare continuous variables.
In order to describe the differences between the two trial populations, these baseline variables were also directly compared between healthy participants and CD participants. Generalized linear mixed models were used to estimate treatment differences in each outcome measure while accounting for within-subject correlations arising from the cross-over design.
Changes in fatty acid measures, calprotectin, and vitamin D levels were analyzed using a gamma distribution and a log link function to satisfy normality assumptions without the need for transformation of outcome variables [ 62 ]. All models included random effects for individuals, and fixed effects for period, treatment nutrient supplement vs.
The use of period-dependent baselines in analysis of cross-over data using random subject effects has been shown to result in biased estimation of treatment effects, and is therefore not recommended [ 63 ]. The average of the two baseline measurements for each participant was therefore included in the model as a covariate. All analyses were conducted using Stata version 16 [ 64 ]. Thirty participants enrolled in the trial as healthy subjects Table 2 , Figure 2 , of which 29 began and 27 participants completed the whole trial.
Twenty-seven participants with CD Table 2 and Table 3 enrolled for the trial, of which 25 began and 24 completed the whole trial Figure 3. The two trial participant groups did not differ significantly by any other factors Table 2. CRP: C-reactive protein. Table 3 summarizes the outcome measurements within each trial separately for each treatment group and at each study timepoint. Table 4 presents results from the generalized linear mixed model analysis of the outcome measures over the two study periods.
Results for analysis of treatment effects on outcome measures using generalized linear mixed models adjusted for average baseline values. Table 4 shows that there was a significant treatment effect for vitamin D, with increases for the nutrient supplement compared to the placebo in both trials; however the treatment effects were notably larger in the healthy participants There was a significant period effect for vitamin D, with lower values recorded in the second period of both trials, although a larger period effect was observed amongst the CD participants Table 3 , Figure 4.
There were no significant treatment effects for calprotectin in either trial, although models were imprecisely estimated with wide confidence intervals, particularly for the CD analysis Table 4. Results from the analysis of changes in CRP are not presented due to the high number of individuals with a CRP level below the reference range of 0. This skew was particularly evident in the trial with healthy participants, where In the CD trial, There was insufficient statistical power to treat CRP as a binary outcome using any clinically relevant cut off for CRP levels, due to the relatively low levels of CRP for the most recorded measurements in both study populations.
The significance of these results is discussed, as well as the possible effects of the MCT placebo in the trial with healthy participants. The challenges in interpreting the results of the two inflammation measures CRP and calprotectin are also highlighted.
The results of these two trials showed that the fatty acids of interest EPA, DHA, DPA, the omega-3 index, and the vitamin D serum levels significantly increased in those taking the nutrient supplement in both the healthy population and those with CD. The results of the first trial on healthy people compares favorably with the results of the RCT by Minihane at al.
This trial with each arm of an 8 week duration and a wash-out period of 12 weeks showed that for a fish oil supplement providing 0. However, a number of limiting factors were highlighted in these reviews: the small numbers in some trials, the cross-over design for trials testing for remission which was thought to be inappropriate considering the relapsing nature of IBD ; the combinations of n PUFA with prebiotics and antioxidants; and the use of placebos such as olive oil which have shown anti-inflammatory properties [ 66 ].
These fatty acids are now being used in specific dietary supplements in arthritis [ 1 , 14 ]. EPA is also being used in medical conditions such as hypertriglyceridemia. However, the latter was very recently discontinued as results were showing a lack of benefit to patients [ 70 ]. Therefore it is important that studies on omega-3 nutrient supplements continue in IBD populations so that the most optimal formulation and effective dose for a supplement containing EPA, DHA, and DPA, and also with additions like vitamin D can be found and for whom it would provide the most benefit.
In vitro models have shown that these fatty acids can have an effect on the tight junctions associated with the gut wall. EPA and DHA were shown to change the lipid environment in the membrane micro-domains of tight junctions, preventing occluding essential for tight junction stability and maintaining barrier function , Zonula occludens-1 ZO-1 redistribution, and the distortion of tight junction morphology [ 71 ]. CD is associated with defects in tight junctions, therefore these fatty acids may help improve the barrier function of people with this disease.
In studies with cancer induced cachexia, which is thought to be associated with intestinal permeability and endotoxemia, therapeutic interventions with EPA were associated with improved intestinal function and reduced inflammation [ 73 ]. People with IBD also show higher risk for bone loss than the general population [ 74 ]. EPA acid derived resolvin E1 RvE1 has been associated with prevention of bone loss and the induction of bone generation. In the treatment of the parietal bone in vivo from a uniform craniotomy, regeneration of the bone defect was also significantly enhanced both for wild type and chemR23tg tg mice [ 75 ].
Kajarabille et al. This receptor is located on the osteoclast and causes bone resorption, which directs osteoclast formation [ 76 ]. A study by Trebble et al. When the data for the omega-3 index, measured in serum in these trials were summarized, the results showed a significant increase when both groups took the nutrient supplement Table 3. This index, instigated by Harris and Von Schacky in was originally used as a risk factor for coronary heart disease [ 78 ].
Since its original conception, it has also been applied to cognitive impairment in the elderly, schizophrenia and depression [ 79 , 80 , 81 , 82 ], cardiovascular disease [ 83 , 84 , 85 , 86 , 87 ], and also on cancer. Outcomes of these and other clinical trials have led health authorities to recommend consumption of oily fish at least twice a week [ 88 , 89 , 90 ].
Others recommend daily supplementation for those people with coronary heart disease 1 g and those with hypertriglyceridemia 4 g [ 91 ]. Despite the number of clinical trials studies reviewing omega-3 fatty acids, not all come to the same conclusion.
In the review by Mori [ 94 ] it was suggested that a number of factors could have contributed to this. Examples of these were: using doses of omega-3 lower than — mg, the presence of confounding comorbidities, medication interactions, or people already having a high intake of omega-3 through their diet. The confounding factors which may have affected the more recent studies, have in this study, been accounted for in the study design.
Vitamin D serum levels significantly increased in the healthy population A larger dose of vitamin D IU may have increased vitamin D serum levels more. In contrast, in the NZ study CD participants had vitamin D levels well above this when they began their supplementation: Group A placebo then supplement , Vitamin D is one of the fat soluble vitamins, with the others being vitamins A, E, and K [ 96 ]. Although it can be sourced from food hence the description of vitamin its main source is sunlight [ 97 ].
However, many people do not have enough exposure to sunlight. This could be because they live mainly indoors, or at a latitude where sunlight sources of vitamin D are diminished i. With sun exposure in the appropriate UV range, the skin absorbs vitamin D and triggers its production endogenously [ 98 ]. The updated metanalysis suggested there needed to be more research to determine which individuals would derive the most benefit from the supplement.
Exploring the genetic profile of participants may identify these individuals. Some research suggests that sufficient levels of vitamin D may also protect against the development of IBD [ ], and a recent study by Janssen et al. Metabolites of vitamin D act on anti-inflammatory pathways and are involved in the maintaining the tight junctions between the epithelial cells of the intestine [ , ].
There is also emerging evidence that vitamin D supplementation could diminish the risk of influenza and COVID infections and deaths [ ]. Vitamin D supplementation could be particularly important for populations that are immune suppressed, such as those with IBD. A study by Arihiro et al. In the second time period T3—T4 there were significantly lower levels of vitamin D in both trials Figure 4.
This might reflect the time of the year this period of both trials was conducted, as both trials were conducted in the first half of the year from March to June , the equivalent to autumn and early winter in the southern hemisphere, when exposure to ultra-violet sources of vitamin D are lower. All the other participants The report on this survey noted that the deficiency started to rise in the month of March and peaked in the winter months of August, September, and October [ ].
In the healthy participant trials, there were decreases in vitamin D levels for both groups period 1 for group A, period 2 for group B when taking the control MCT capsule Figure 4 a. Four participants were below the recommended level before starting the control capsule, but after four weeks of having the MCT capsule, nine were still under the recommended level. Genetic variance may have been an influence, and this would need to be explored with further analysis.
There was also a decrease in sunlight time; by the end of the control period sunlight exposure was less. However, the intake of MCT may have contributed to the decrease in vitamin D levels. MCTs have a reduced chain length, which means that they are more rapidly absorbed and metabolized by the body.
MCTs are ketogenic and, for this reason, have been used as the basis of a ketogenic diet [ , ]. Long-term ketogenic diets increase the risk of bone fractures despite the use of calcium supplements [ ] and the formation of kidney stones, which suggests that bone metabolism is effected [ ]. An earlier study by Hahn et al. A more recent study using a rat model showed severe bone microstructure destruction with the ketogenic diet [ ].
Vitamin D 3 absorption was significantly higher with peanut oil than with MCTs in both fasting and non-fasting states [ ]. For this reason, and because MCT appeared to also affect the fatty acid levels, MCT was not used as a control supplement in the trial with CD participants and a refined fish oil was used instead [ 37 ].
In the trial of people with CD, vitamin D serum levels significantly increased in both groups when the nutrition supplement was taken Table 3 , Figure 4. This suggests that the nutrient supplement was able to maintain the recommended levels of vitamin D even though sunshine hours were decreasing. Of note, the group of participants with CD began with higher average levels of vitamin D The half-life of vitamin D is 15 days [ 96 , ].
The age range in both trial groups was also similar. In the healthy group, the median ages were As people age, their ability to metabolize vitamin D from sunlight decreases. Aging has been reported to decrease the capacity of the skin to produce pre-vitamin D 3 by greater than two-fold [ , ].
Skin color also affects the ability of the skin to absorb vitamin D. These participants may have had more melanin pigmentation in their skin, which would decrease their capacity to absorb vitamin D from sunlight. There may also have been genetic variant differences with respect to the genes involved in vitamin D metabolism in the healthy group. This would have lowered the absorption of vitamin D and decreased their measured vitamin D levels [ ]. From the results of the trial with CD participants, it appears that in this sample of people with CD, while on the nutrient supplement, their vitamin D levels were well within the recommended range.
C-reactive protein CRP is produced in the liver and blood concentrations are used as an indicator of inflammation in the body. It is an acute phase reactant and rising levels are used regularly as an indicator of inflammatory conditions such as infections, atherosclerosis, heart disease, and rheumatoid arthritis [ , ]. The high-sensitivity hs CRP test, which is often used to check for risk of heart disease, was not used in these two trials.
No formal analyses for CRP were presented here due to issues with model convergence and low statistical power. However, from the summaries of CRP measurements across the time points in the two trials, it appeared that the nutrient supplement did not have any clear effects with respect to inflammation as measured by CRP. The majority of people in both trials had CRP levels that indicated they were in a non-inflammatory state.
Calprotectin is a key protein found in the intracellular fluid of inflammatory cells and can be measured in the feces as an indicator of the migration of neutrophils through the bowel wall to the fecal material [ ]. In this study, comparisons of the two participant groups at baseline showed that levels of calprotectin were higher on average in CD participants Table 2. In the CD group, fifty percent of the participants had calprotectin scores in the normal or clinically inactive range.
The other participants fluctuated in their scores through the trial. This reflects the cycle of inflammation that people with CD can experience. Only one participant consistently rose from the normal range at the start of the trial to the severe range by the end of the trial. It was observed that for this participant their quality of life QoL score for the numeric scale from 1—10 was seven at both the start and the finish of the trial.
However, for their IBD QoL score also out of 10 , the average for the ten questions decreased from six at the start, to 4. A strength of these trials is that they both used a random controlled cross-over design. This approach allows the comparison of treatments applied to the same participant, which is considered more accurate than a comparison between different participants. In addition, a cross-over trial requires fewer participants than a regular trial to achieve the same statistical power.
Another strength of the trials is that the uptake of fatty acids was measured specifically by the FAMEs analysis. Many trials do not measure the uptake of fatty acids in their interventions. A limitation of the trials is that the participant numbers were small in number 27 completed in the healthy participant trial and 24 completed in the CD participant trial.
This means the power to test for carry-over effects was limited, therefore the interpretation of the difference between treatment effects is dependent on a subjective assessment of the reality or not of equal carry-over effects. Another limitation is that the carry-over effects for vitamin D in the CD trial suggest that the washout period could have been longer.
Another limitation is using changes in fatty acids in serum rather than in red blood cells for the omega-3 index. Results from this study showed that there was a consistent treatment effect, with the nutrient supplement increasing EPA, DPA, DHA, the omega-3 index, and vitamin D serum levels compared to the placebo. Watch this item. This listing has ended. This amount is subject to change until you make payment. For additional information, see the Global Shipping Programme terms and conditions - opens in a new window or tab This amount includes applicable customs duties, taxes, brokerage and other fees.
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See all condition definitions opens in a new window or tab. Modified Item:. United Kingdom. Fish Oil.
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Omega-3 fatty acids are thought to help reduce the risk of heart disease. They have been used along with diet and exercise to help lower levels of a certain. SUPER TWIN EPA-DHA (OMEGA-3 FATTY ACIDS). Indications; Dosing; Drug Interactions; Contraindications/Warnings; Adverse Reactions; Precautions. Excellent fish oil supplement. High levels of both EPA and DHA, no PCBs or heavy metals, and no more fish burps than any other fish oil. Omega 3 to Omega 6.