Impact of Aronia Berry Consumption on Inflammation, Metabolites, and the Gut Microbiome
Condition: Inflammation; Metabolic Disorder; Hypertriglyceridemia
MeSH terms: Hypertriglyceridemia; Metabolic Diseases; Inflammation
Study type: Interventional

Summary: 
      The goal of this project is to elucidate interactions between the gut microbiome,
      anti-inflammatory/anti-oxidant food metabolomic signatures, and human inflammation
      phenotypes. Inflammation plays both direct and indirect roles in the development of type 2
      diabetes (T2D), atherogenic cardiovascular diseases, and other causes of morbidity and
      mortality. Aronia melanocarpa (Aronia berries) are rich in bioactive polyphenolic compounds,
      which have been shown to lower inflammation and favorably impact metabolism. However, there
      is tremendous inter-individual variability in the bioavailability of polyphenolics and
      production of bioactive phenolic metabolites in the colon that depends, at least in part, on
      digestive metabolism by the gut microbiota. Little is known about the complex interactions
      among the gut microbiome, anti-inflammatory food metabolomic signatures, and human
      inflammation phenotypes. This study will utilize a systems-level approach to disentangle
      these complex interactions. The specific study objectives are as follows:

        1. to determine the impact of Aronia supplementation on inflammation, metabolic health, and
           gut microbiome composition

        2. to determine the static and dynamic metabolomic signature of Aronia based on an Aronia
           supplementation period and responses to a high-fat meal challenge
    

Detailed description: 
      To meet these objectives, a randomized, double-blind, placebo-controlled clinical trial of
      Aronia versus placebo treatment for 28-30 days in human adults will be conducted. Pre- and
      post-intervention assessments will be made for the following variables: makeup of the gut
      microbiome (microbial species and relative abundance), gut metabolome, postprandial response
      of TG, inflammatory cytokines, and serum metabolome to a high-fat meal challenge (established
      inflammation stimulus), fasting serum glucose, lipid, insulin, inflammation markers and
      metabolome, blood pressure, and anthropometric measures including weight, body composition,
      waist circumference, and quantity of visceral adipose tissue. Physical activity, sedentary
      behavior, and habitual diet will be measured so that these variables can be used to
      characterize participants and aid in analysis and interpretation of data.

      Procedures:

      Postprandial lipidemic and inflammation responses: High-fat meal challenges with 40 to 100 g
      of dietary fat are an established laboratory test to measure both postprandial
      triglyceridemic and inflammation responses. Investigators have used a 50 g dose of fat
      delivered in the form of butter on toast on > 50 individuals because this particular dose is
      effective at discriminating between low versus high TG and inflammation responders. In brief,
      participants will report to the laboratory after an overnight fast, and blood samples will be
      collected before, and 1, 2, 4, and 6 hours following ingestion of the high-fat meal. Samples
      will be analyzed in real time for TG (and full lipid panel plus glucose) using a clinical
      chemistry analyzer (Piccolo xpress), while serum samples will be aliquoted and stored at -80
      C until analysis for inflammatory cytokines, metabolomics, and insulin. Investigators will
      measure inflammatory cytokines (TNF-α, interleukin(IL)-1β, IL-6 IL-17, IL-23, and granulocyte
      macrophage colony stimulating factor (GM-CSF)) using high-sensitivity Luminex multiplexing
      technology (Bio-RadBio-Plex® 200 HTS) prepared by Millipore.

      Dietary intervention: Participants will be randomized to either experimental (Aronia) or
      placebo-matched control group. The experimental supplement will consist of a once daily dose
      of 100 mL of Aronia juice. The placebo-matched control supplement will have no polyphenol
      content and will consist of 100 mL of the following mixture: black cherry Koolaid, blue and
      red food coloring, sucrose and sorbitol. This placebo will match the sugar content of the
      chokeberry juice. The daily dose of 100 mL for both groups is consumed once daily for
      duration of 28-30 day supplementation period. All participants will be instructed to avoid
      consumption of foods with polyphenolic content for the duration of the supplementation
      period. A list of disallowed foods will be provided for participants to reference.

      Gut microbiome analysis: Bulk DNA will be extracted from fecal samples using the Powersoil®
      DNA Isolation Kit (Mo Bio Laboratories Inc.). DNA will be shipped overnight to the University
      of Michigan, Center for Microbial Systems, for Illumina MiSeq amplicon sequencing of the 16S
      V4 variable region. Raw sequencing reads will be processed and curated using the mothur
      (v.1.39.5) software package, following the mothur MiSeq standard operating procedure,
      potentially chimeric sequences will be identified and removed using the Uchime (v4.2.40)
      algorithm, and taxonomic classifications will be assigned using the Bayesian classifier of
      the Ribosomal Database Project, and operational taxonomic units (OTUs) will be assigned in
      mothur using the VSEARCH distance-based clustering algorithm at the 97% sequence similarity
      threshold.

      Metabolomic analysis: Samples will be analyzed by high resolution liquid chromatography mass
      spectrometry (LCMS). Hydrophilic interaction chromatography (HILIC) and reverse-phase (RP)
      columns will be used for deep coverage. Metabolite identification will use fragmentation
      pattern matching, authentic standards and database matching with METLIN and the Human
      Metabome Database (HDB). Novel features of significant interest will be characterized with
      liquid chromatography mass spectrometry solid phase extraction nuclear magnetic resonance
      (LCMS-SPE-NMR). Pathway analysis will use XCMS and mummichog.

      Dietary analysis: Long-term dietary habits may create adaptations that influence the response
      to the short-term supplementation of Aronia. This study will use the most recent version
      (2018) of the web-based Diet History Questionnaire (DHQ III), a food frequency questionnaire
      designed for adults 19 and older, developed by staff at the Risk Factor Monitoring and
      Methods Branch (RFMMB) of the NIH National Cancer Institute. The outputs of the DHQ III
      include carbohydrate constituents, carotenoids and tocopherols, dietary constituents from
      supplements, fats, fatty acids and cholesterol, macronutrients and energy, minerals, protein
      constituents, and vitamins are dietary constituents and food groups available in the DHQ III
      output files.

      Statistical analysis: Two-sample t-tests to compare the difference between pre- and
      post-intervention assessments. Investigators will identify changes in the gut microbiome
      using the methods utilized in our preliminary research to identify characteristics of the gut
      microbiome that differentiate low versus high TG responders, and then use regression analysis
      to determine the level of variability in changes to the Aronia and control treatments
      explained by changes in relative abundance of gut microbial species. Investigators will
      identify changes in the gut and serum metabolomes, and then determine the metabolic pathways
      associated with the metabolomic changes to identify potential mechanisms underlying health
      impacts of Aronia supplementation.
    

Intervention name(s): Aronia juice; Placebo
Intervention type(s): Dietary Supplement; Other
Intervention description(s): Once daily dose of 100 mL of aronia juice; Once daily dose of 100 mL of placebo juice containing no polyphenols and matched to experimental Aronia juice in color, taste, and macronutrient content
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: Double-blind, parallel, control trial with participant randomization to control versus intervention groups.
Masking: Double (Participant, Investigator)
Primary purpose: Prevention
Keywords: gut microbiome; metabolome; postprandial hypertriglyceridemia