Resistant starch type-4 intake alters circulating bile acids in human subjects

Abstract

Background: Resistant starch (RS) type 4 (RS4) is a type of RS, a class of non-digestible prebiotic dietary fibers with a range of demonstrated metabolic health benefits to the host. On the other hand, bile acids (BA) have recently emerged as an important class of metabolic function mediators that involve host-microbiota interactions. RS consumption alters fecal and cecal BA in humans and rodents, respectively. The effect of RS intake on circulating BA concentrations remains unexplored in humans.

Methods and results: Using available plasma and stool samples from our previously reported double-blind, controlled, 2-arm crossover nutrition intervention trial (Clinicaltrials.gov: NCT01887964), a liquid-chromatography/mass-spectrometry-based targeted multiple reaction monitoring, and absolute quantifications, we assessed BA changes after 12 weeks of an average 12 g/day RS4-intake. Stool BA concentrations were lower post RS4 compared to the control, the two groups consuming similar macronutrients (n = 14/group). Partial least squares-discriminant analysis revealed distinct BA signatures in stool and plasma post interventions. The increased circulating BA concentrations were further investigated using linear mixed-effect modeling that controlled for potential confounders. A higher plasma abundance of several BA species post RS4 was observed (fold increase compared to control in parenthesis): taurocholic acid (1.92), taurodeoxycholic acid (1.60), glycochenodeoxycholic acid (1.58), glycodeoxycholic acid (1.79), and deoxycholic acid (1.77) (all, p < 0.05). Distinct microbiome ortholog-signatures were observed between RS4 and control groups (95% CI), derived using the Piphillin function-prediction algorithm and principal component analysis (PCA) of pre-existing 16S rRNA gene sequences. Association of Bifidobacterium adolescentis with secondary BA such as, deoxycholic acid (rho = 0.55, p = 0.05), glycodeoxycholic acid (rho = 0.65, p = 0.02), and taurodeoxycholic acid (rho = 0.56, p = 0.04) were observed in the RS4-group, but not in the control group (all, p > 0.05).

Conclusion: Our observations indicate a previously unknown in humans- RS4-associated systemic alteration of microbiota-derived secondary BA. Follow-up investigations of BA biosynthesis in the context of RS4 may provide molecular targets to understand and manipulate microbiome-host interactions.

Keywords: circulating bile acid; dietary fiber; metabolic syndrome; metabolomics; microbiota; resistant starch type 4.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Dhakal and Dey.

Figures

FIGURE 1

Participant flow in the study.

FIGURE 2

Estimated nutrient intakes at baseline and post diets. Bar graphs with mean ± SD showing similar macronutrient intakes at baseline and after interventions (all, p > 0.05). The difference in fiber intake was due to RS4 supplementation in the test group; n = 14/group; RS4, resistant starch type 4; CHO, Carbohydrates.

FIGURE 3

Effects of RS4 intervention on plasma and stool bile acids. Multivariate partial least squares-discriminant analysis (PLS-DA) score plots of control (brown) vs. RS4 (blue) in plasma (A) and stool (B) bile acids with their corresponding variable importance in projection (VIP) scores plots; Each dot represents the unique signature of 15 primary and secondary bile acid species for an individual; species with VIP > 1 are shown (C) bar graph showing fold change in bile acid group abundance; p calculated using linear mixed-effect model controlling for body weight, intervention sequence, and participant ID; n = 14/group; RS4, resistant starch type 4; LV, latent variable; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; G, T, and U represent glyco-, tauro-, and urso-, respectively.

FIGURE 4

RS4 altered secondary bile acids. Box plots comparing concentrations of individual primary, secondary, and conjugated bile acid species: at baseline and after RS4 intake (A) after control vs. RS4 interventions (B); for (A,B)p-values were calculated using linear mixed-effect model controlling for body weight, intervention sequence, and participant ID; lower and upper hinges of the boxplot denote 25th and 75th percentile, line denotes median, and whiskers are drawn to minimum and maximum values but not further than 1.5x interquartile range. Outliers are displayed as black dots; (C) heatmap showing individual study participants on x-axis and bile acid species on y-axis to present differential abundance of circulating bile acids between individuals and groups; the colored cells denote abundance (red- high abundance, blue- low abundance); variables were autoscaled; For all panels: n = 14/group; RS4, resistant starch type 4; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; G, T and U represent glyco-, tauro-, and urso-, respectively.

FIGURE 5

Impact of RS4 on gut microbiota functional capability. (A) Principal component analysis (PCA) of KEGG orthologs based on 16s rRNA gene sequences showing higher variability in control as illustrated by wider 95% CI (elliptical boundaries) vs. RS4 intervention that clustered the predicted orthologs toward zero on both axes within a narrow spatial area; the x and y axes explain 31 and 16.5% variability, respectively, in the control vs. RS4 the distinction between diets was less evident with baseline vs. RS4; each dot represents a unique signature of an individual participant’s gut microbiota for the 8,621 KEGG orthologs; dataset was log transformed. (B) Heatmap showing individual study participants on x-axis and KEGG orthologs on y-axis to present differential abundance of orthologs between individuals and groups; the colored cells denote abundance (red- high abundance, blue- low abundance); variables were autoscaled; and n = 14/group; RS4, resistant starch type 4; KEGG, Kyoto encyclopedia of genes and genomes.