Table 2.

Proposed analyses of primary and secondary outcomes in the Alaska FIRST clinical trial

Measured outcomes	Samples analysed	Method of analysis	Description
Suppression of Ki-67 proliferative biomarkers Primary	Biopsies	IHC	Biopsies of the colon are taken by endoscopy before and at the end of the supplementation for measurement of Ki-67 epithelial proliferation.26 32
Mucosal inflammation Secondary	Biopsies	H&E Staining; IHC of CD3+ intraepithelial lymphocytes and CD68+ lamina propria macrophages	Biopsies of the colon are taken by endoscopy before and at the end of the supplementation for measurement.26 32
Change in human gene expression in response to diet and microbe metabolites Secondary	Biopsies	Affymetrix human transcriptome array and qPCR	Investigate the expression of host genes responsive to diet and microbial metabolites that might explain the mechanisms responsible for the changes in our biomarkers.38 Genes of specific interest are those implicated in butyrate transport39 40 and metabolite detoxification;41 mucosal redox homeostasis as a measure of stress42 and inflammation;43–45 cell cycle regulation;46 Wnt/β-catenin signalling;47 DNA damage repair.43 48
Colonic and oral microbiota composition and diversity Secondary	Biopsies, faeces and saliva	16S rRNA sequencing	Identify the effect of fibre on the composition of the colonic microbiome in faeces, saliva and mucosal biopsies collected pre and post supplementation.26 32
Targeted colonic functional microbial genes Secondary	Faeces	qPCR	Quantitation of specific functional genes responsible for butyrate synthesis and bile acids production by real time PCR microbiome in faeces, saliva and mucosal biopsies collected pre and post supplementation.26 32
Biofluid metabolome composition Secondary	Serum, faeces and urine	NMR and high-resolution LC–MS (semiquantitative/quantitative—relative and absolute)	Look for trends of saccharolytic and inflammatory nitrogenous metabolites in response to fibre intake in faeces collected at presupplement endoscopy, weeks 1–3, and post supplement endoscopy and urine samples collected at pre and post supplementation.26 32 49
Targeted colonic short chain fatty acids Secondary	Faeces and colonic evacuate	GC–FID (quantitative)	Look for changes in the colonic acetate, propionate and butyrate amounts in response to fibre intake in faeces collected at presupplement endoscopy, weeks 1–3, and post supplement endoscopy and evacuate samples collected at pre and post supplementation.26 32
Targeted colonic bile acids Secondary	Faeces and colonic evacuate	LC–MS (quantitative)	Look for changes in colonic primary bile acids—cholic and chenodeoxycholic acid and secondary bile acids—deoxycholic and lithocholic acid amounts in response to fibre intake in faeces collected at presupplement endoscopy, weeks 1–3, and post supplement endoscopy and colonic evacuate collected pre and post supplementation.50
Clinical blood labs Secondary	Blood	Clinical labs, GC–FID (RBC fatty acids)	Fasting clinical blood labs before and after supplementation—blood count, complete metabolic profile, lipoproteins, folate, vitamin B-12, vitamin A, vitamin D, triglycerides and RBC fatty acids (n-3:n-6 ratio).26 32

GC–FID, gas chromatography–flame ionisation detector; IHC, Immunohistochemistry; LC–MS, liquid chromatography–mass spectrometry; NMR, nuclear magnetic resonance; qPCR, quantitative PCR; RBC, red blood cells.