Gut Online First, published on September 26, 2017 as 10.1136/gutjnl-2017-314968 Gut microbiota ORIGINAL ArticLE A randomised trial of the effect of omega-3 polyunsaturated fatty acid supplements on the human intestinal microbiota Henry Watson,1 Suparna Mitra,2 Fiona C Croden,3 Morag Taylor,4 Henry M Wood,4 Sarah L Perry,1 Jade A Spencer,5 Phil Quirke,4 Giles J Toogood,6 Clare L Lawton,3 Louise Dye,3 Paul M Loadman,5 Mark A Hull1 ► Additional material is ABSTRACT published online only. To view, Objective Omega-3 polyunsaturated fatty acids Significance of this study please visit the journal online (PUFAs) have anticolorectal cancer (CRC) activity. The (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2017- 314968). intestinal microbiota has been implicated in colorectal The naturally occurring omega-3 1 carcinogenesis. Dietary omega-3 PUFAs alter the mouse ► Institute of Biomedical and polyunsaturated fatty acids (PUFAs), Clinical Sciences, St James’s intestinal microbiome compatible with antineoplastic University Hospital, University of activity. Therefore, we investigated the effect of omega-3 eicosapentaenoic acid (EPA) and Leeds, Leeds, UK PUFA supplements on the faecal microbiome in middle- docosahexaenoic acid (DHA) have 2Institute of Biomedical and aged, healthy volunteers (n=22). anticolorectal cancer (CRC) activity. Clinical Sciences, Leeds General Design A randomised, open-label, cross-over trial of ► High-purity EPA and DHA can be provided in Infirmary, University of Leeds, soft-gel capsule form or as a ‘nutrition’ drink Leeds, UK 8 weeks’ treatment with 4 g mixed eicosapentaenoic 3Human Appetite Research acid/docosahexaenoic acid in two formulations (soft- providing greater than 2 g omega-3 PUFAs daily. Unit (Nutrition and Behaviour gel capsules and Smartfish drinks), separated by a 12- ► The intestinal microbiota are implicated in Research Group), School of week ’washout’ period. Faecal samples were collected colorectal carcinogenesis, as well as modulation Psychology, University of Leeds, of chemotherapy and immunotherapy of CRC. Leeds, UK at five time-points for microbiome analysis by 16S 4 ribosomal RNA PCR and Illumina MiSeq sequencing. Institute of Cancer and What are the new findings? Pathology, St James’s University Red blood cell (RBC) fatty acid analysis was performed Oral high-dose omega-3 PUFAs do not produce Hospital, University of Leeds, by liquid chromatography tandem mass spectrometry. ► marked changes in the intestinal microbiome Leeds, UK Results Both omega-3 PUFA formulations induced 5Institute of Cancer in healthy volunteers, even in individuals with similar changes in RBC fatty acid content, except Therapeutics, University of treatment-emergent diarrhoea. Bradford, Bradford, UK that drinks were associated with a larger, and more 6 Intake of 4 g daily mixed EPA/DHA for 8 weeks Department of Hepatobiliary prolonged, decrease in omega-6 PUFA arachidonic ► was associated with a reversible increase Surgery, St James’s University acid than the capsule intervention (p=0.02). There Hospital, Leeds, UK in Bifidobacterium, Oscillospira, Roseburia were no significant changes inα or β diversity, and Lachnospira species, but decreased or phyla composition, associated with omega-3 Correspondence to Coprococcus and Faecalibacterium. PUFA supplementation. However, a reversible Professor Mark A Hull, Institute Similar effects of omega-3 PUFAs on the faecal increased abundance of several genera, including ► of Biomedical & Clinical microbiome were observed for both capsule Sciences, St James’s University Bifidobacterium, Roseburia and Lactobacillus and drink formulations. Hospital, University of Leeds, was observed with one or both omega-3 PUFA Leeds, LS9 7TF, UK; M. A. Hull@ Capsule and drink formulations provide interventions. Microbiome changes did not ► leeds. ac. uk equivalent tissue omega-3 PUFA incorporation, as correlate with RBC omega-3 PUFA incorporation or measured by red blood cell levels, but only drinks HW and SM are joint first development of omega-3 PUFA-induced diarrhoea. were associated with prolonged suppression of authors. There were no treatment order effects. proinflammatory arachidonic acid levels. Conclusion Omega-3 PUFA supplementation induces Received 2 August 2017 Revised 22 August 2017 a reversible increase in several short-chain fatty acid- How might it impact on clinical practice in the Accepted 23 August 2017 producing bacteria, independently of the method of foreseeable future? administration. There is no simple relationship between ► An increase in short-chain fatty acid-producing the intestinal microbiome and systemic omega-3 PUFA bacteria may be relevant to the beneficial exposure. anti-CRC effects of EPA in both prevention and Trial registration number ISRCTN18662143. adjuvant treatment settings. ► Clinical evaluation of the anticancer properties of omega-3 PUFAs needs to consider INTRODUCTION the intestinal microbiota and its role in T Watson H,o cite: Mitra S, The two main omega-3 polyunsaturated fatty acids carcinogenesis and immune regulation. Croden FC, et al. Gut Published Online First: [please (PUFAs), eicosapentaenoic acid (EPA; C20:5 ω3) include Day Month Year]. and docosahexaenoic acid (DHA; C22:6 ω3) are Multiple health benefits have been claimed for these doi:10.1136/ widely used as nutritional supplements, as fish long-chain omega-3 PUFAs, including secondary gutjnl-2017-314968 oil or in more concentrated ‘nutraceutical’ form.1 prevention of ischaemic heart disease,2 treatment of Watson H, et al. Gut 2017;0:1–10. doi:10.1136/gutjnl-2017-314968 1 Copyright Article author (or their employer) 2017. Produced by BMJ Publishing Group Ltd (& BSG) under licence. Gut microbiota rheumatoid arthritis3 and anticancer activity,4 some of which are of the peach-flavoured drink and also swallowed two study supported by evidence from randomised trials.5 6 capsules with water, after providing written informed consent to The mechanism(s) underlying the colorectal cancer (CRC) confirm likely compliance and minimise dropout. chemopreventative activity of EPA reported by West et al is Visit 1 occurred within 2 weeks of the screening visit, at unclear.5 It has been proposed that the intestinal microbiota which participants were randomised to take either two 200 mL may play a role in colorectal carcinogenesis based on the asso- Smartfish Remune drinks (see online supplementary methods ciation of CRC with a specific intestinal microbiome profile, or for content) per day (providing approximately 2000 mg EPA so-called dysbiosis, characterised by low phylogenetic diversity, and 2000 mg DHA, as the triglyceride) at any suitable time altered Firmicutes/Bacteriodetes ratio, under-representation of of day or four soft-gel capsules (each containing 250 mg EPA short-chain fatty acid (SCFA)-producing genera such as Rose- and 250 mg DHA as the ethyl ester) twice daily with meals buria and Eubacterium as well as presence of putative pathobi- (providing 2000 mg EPA and 2000 mg DHA per day), both for onts such as Fusobacterium nucleatum.7 8 One possibility is that 8 weeks (intervention A; figure 1). After a 12-week ‘washout’ modulation of the intestinal microbiota may contribute to the period, participants took the second intervention for 8 weeks cancer-preventative properties of omega-3 PUFAs. (Intervention B; figure 1). We also included a final study visit Data from mouse models suggest that dietary omega-3 PUFA after a second 12-week ‘washout’ period (V5; figure 1). Rando- intake or high tissue levels of omega-3 PUFAs are associated with misation was performed by Leeds Teaching Hospitals Pharmacy differences in intestinal microbiota, including increased quanti- using random permuted block allocation in concealed envelopes. ties of certain genera, including Bifidobacterium and Lactoba- Neither participants nor researchers were blinded to the inter- cillus.9 10 There has been only one case report of the effect of ventions and hence allocation order. an omega-3 PUFA-rich diet on human intestinal microbiota.11 At each visit, adverse event (AE) monitoring was undertaken In this case, there was a notable increase in several SCFA (butyr- by a brief interview based on questioning for recognised AEs of ate)-producing genera including Blautia, Bacterioides, Roseburia omega-3 PUFA supplements, including loss of appetite, eructa- and Coprococcus.11 tion (‘fishy’ burping), nausea, vomiting, dyspepsia, abdominal Therefore, a plausible hypothesis is that omega-3 PUFA intake pain and diarrhoea, as well as bleeding events. Review of AEs was alters the composition of human intestinal microbiota, thereby performed by an independent data monitoring committee every attenuating the intestinal dysbiosis associated with colorectal 3 months. Tolerability of both drinks and capsules was assessed carcinogenesis. with a palatability questionnaire and capsule acceptability ques- Nutritional supplementation with omega-3 PUFAs can occur tionnaire at the end of each 8-week intervention period (visit 2 in several ways, either as unrefined fish oil, in ‘nutraceutical’ or 4). Participants’ height and weight were measured at the start form, usually as the triglyceride or ethyl ester conjugate in of the study. soft-gel capsules, taken with food or more recently as an emul- Blood and urine were collected at each visit and a faecal sample sion in drink form.1 (obtained with a Fe-Col faecal collection device) was returned To address the above hypothesis and, at the same time, by hand or by Royal Mail