Impacts of Aronia on Inflammation and the Gut Microbiome
Condition: Metabolic Syndrome; Inflammation; Hypertriglyceridemia; Obesity
MeSH terms: Metabolic Syndrome; Hypertriglyceridemia; Inflammation
Study type: Interventional

Summary: 
      The overall goal of this project is to determine the inflammation lowering impact of
      anthocyanin-rich Aronia berries. Inflammation is an underlying mechanism driving the
      development of several diseases. While an elevation in immune signals in the systemic
      circulation is commonly attributed to adipose tissue, inflammation is not present in all
      obese individuals. Adipose tissue must become inflamed, and the inflammation trigger may come
      from other sources. Microorganisms (microbiome), host tissues, and immune cells residing in
      the gastrointestinal tract (GIT) are a key source of pro-inflammatory signals that may cause
      the host organism to become inflamed. Anthocyanins are bioactive compounds with established
      anti-inflammatory and microbiome altering properties. We hypothesize that the GIT microbiome
      is a key determinant of host inflammation than can be manipulated by anthocyanins-rich
      berries to lower inflammation. We assembled a cohort of individuals, characterized their GIT
      microbiome and performed anthropometric measurements, basal measures of metabolism and
      metabolic health, and triglyceridemic, metabolomic, and inflammation responses to a high-fat
      meal challenge.
    

Detailed description: 
      Anthropometrics. Measurements were collected from participants using the validated segmental
      multifrequency bioelectrical impedance analysis (SECA mBCA 515, Hamburg, Germany). Fat mass
      (%) and estimated visceral adipose (L) were used for analysis.

      High-Fat Meal Challenge. The high-fat meal contained salted butter (58.3 g, Tillamook) over 3
      pieces of whole wheat toast (127.5 g; Wheat Montana). Total energy content of the meal was
      714 kcal, with 43.1% from fat, with a macronutrient breakdown of 50 g fat, 54 g carbohydrate,
      and 12 g protein. Water was provided with the meal; caffeinated black tea was provided
      instead for participants who identified as habitual coffee consumers.

      Blood Sampling. Participants were instructed to avoid alcohol consumption and strenuous
      physical activity in the 24 hours before their visit and to complete an overnight fast (10 -
      12 hours) before blood collection. Participant blood samples were collected by a certified
      nurse or physician in the morning before ingestion of the meal and hourly for 4 hours after
      meal ingestion, totaling five time points. Whole blood in serum separating tubes was allowed
      to clot for 15 minutes before centrifugation at 1200 RPM for 15 minutes with resulting serum
      aliquoted and stored at -80ºC until analysis.

      Determination of blood markers. Blood markers of metabolic syndrome were determined from
      whole blood run on Picollo Xpress Chemistry Analyzer lipid panels (Abaxis, Union City, USA).
      Serum insulin (INS) was determined using an insulin ELISA kit (MP Biomedicals, Solon, OH)
      performed according to manufacturer instructions. Cytokine measurement was performed using
      high-sensitivity multiplexing technology (Bio-Rad Bio-Plex 200 HTS) following procedures by
      Millipore (EMD Millipore Corporation, Billerica, USA). Classic systemic pro-inflammatory
      cytokines were measured and include granulocyte macrophage colony stimulating factor
      (GM-CSF), interleukin (IL)-1B, IL-6, tumor necrosis factor (TNF)-α. InterleukinI-17 and
      IL-23, both of which serve a pro-inflammatory and regulatory role in the gut mucosa, were
      also measured. Serum samples at each time point during the high-fat meal challenge were run
      in duplicate.

      Stool Sample Collection. Collection kits were provided and participants were asked to follow
      included instructions for the self-collection of a stool sample in the 24 hours before their
      blood collection visit. After initial collection into a sterile disposable commode, a small
      portion of the sample was transferred into a sterile Eppendorf tube and transported to
      researchers. Samples were prepared and aliquoted in an anaerobic chamber then frozen at -80ºC
      until analysis.

      Genomic DNA Extraction and Microbial Analysis. Extraction of bulk DNA from fecal samples was
      performed using Powersoil DNA Isolation Kit (Mo Bio Laboratories, Inc.) and bead beating. DNA
      was shipped overnight to the University of Michigan, Michigan Microbiome Project for Illumina
      MiSeq amplicon sequencing of the 16S rRNA V4 region. After DNA quantification, V4 amplicon
      libraries were generated with dual-index barcoded primers, then by library purification,
      pooling, and MiSeq paired-end sequencing. Raw sequencing reads were processed and curated
      using MOTHUR software (Version 1.35.1) following the MOTHUR standard operating procedure for
      the MiSeq platform39. In brief review, paired-end reads were assembled into contiguous
      sequences and screened for length and quality. The remaining contigs were aligned to the
      SILVA ribosomal RNA database (Release 132), a comprehensive collection of aligned rRNA
      sequences. Potentially chimeric sequences were identified and removed using the UCHIME
      algorithm in MOTHUR. Taxonomic classifications were assigned using the Bayesian classifier of
      the Ribosomal Database Project. Non-target reads were removed, and operational taxonomic
      units (OTUs) were assigned using VSEARCH distance-based clustering at the 97% similarity
      threshold. Alpha-, and β- diversity indices were generated using the vegan package in R40. An
      OTU-based data matrix was constructed for participants included in the ppTG phenotype.

      Metabolomic Analysis. Frozen serum samples were thawed and 20μL was placed in a clean tube.
      80μL of HPLC grade methanol was added to the sample after which it was vortexed briefly and
      placed in a -80 C freezer for 2 hours. After two hours, the sample was centrifuged at 20,000g
      for 10 minutes. The metabolite supernatant was collected and concentrated in a Speed Vac to
      dryness while the protein pellet was discarded. Samples were then stored at -80 C until ready
      for LCMS analysis at which time they were reconstituted with 40μL of methanol:water (50:50)
      and placed in a clean mass spectrometry vial. Analysis was completed on an Agilent 6538 Q-TOF
      MS coupled to an Agilent 1290 UHPLC using a 130A, 1.7μm, 2.1mm X 10mm Acquity BEH-HILIC HPLC
      column. Samples were ionized via electrospray ionization and runs were completed in positive
      mode. Mobile phase A was 15mmol/L ammonium formate and mobile phase B was ACN using a 10-40%
      A gradient over 6 minutes. Flow was kept at 400µL/minute and the column compartment
      temperature was set at 30 C. MSMS analysis was completed using the same LC conditions while
      targeting specific ions using retention time and m/z values from previous MS runs. After LCMS
      analysis completion, raw data files were converted to .xml files using MSConvert. Data was
      then mined with mzMine using an intensity minimum value of 1,000 based on a visual inspection
      of the total ion chromatogram to remove noise. Blank samples were also ran and the resulting
      features were removed from the biological data if present at a ratio under 5:1 in the sample
      compared to the blank. Mined data was then input into MetaboAnalyst for statistical analysis.
      Tandem MS data was analyzed with Sirius software to identify features.
    

Intervention name(s): Aronia berry juice; Sham comparator
Intervention type(s): Dietary Supplement; Other
Intervention description(s): A juice blend of three different cultivars was used: Viking, MacKenzie and Autumn Magic. Raw juice was heat pasteurized before provided to participants. Participants consumed 100 mL of juice per day.; The placebo was flavor, color, and carbohydrate-matched to aronia juice. The placebo consisted of 28.8 g black cherry Koolaid mix (no sugar added), 128.5 g sorbitol, 74.5 g glucose, 77.9 g fructose, 4 oz lemon juice, 16 drops of blue food coloring, and enough water to create 1 L of solution. Participants consumed 100 mL of juice per day.
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Single (Participant)
Primary purpose: Prevention
Keywords: gut microbiome; metabolome; postprandial hypertriglyceridemia; low-grade inflammation; visceral adipose tissue