Urinary SECO, ED, and ENL were assayed by isotope dilution gas chromatography-mass spectrometry in the SIM mode (HP 6890 GC, HP 5973 MSD; Agilent Technologies, Palo Alto, CA, USA) as described previously [15 (link)]. All lignan measures were normalized to urinary creatinine levels to adjust for urine concentration. Stool samples were thawed, homogenized, and genomic DNA was extracted as described previously [19 (link)]. DNA concentrations and purity were determined using the NanoDrop 8000 Spectrophotometer (ThermoFisher Scientific) and gel electrophoresis. Working stocks were diluted in AE buffer (QIAGEN, Germantown, MD, USA) from genomic DNA and samples were stored at −20 °C until shipped for sequencing. Pooled in-lab designed quality control samples were included in triplicate to assess variation in library preparation and sequencing batches [20 (link)]. The V1–V3 region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform to obtain 2 × 300 bp paired-end reads. Fecal bacterial DNA extraction did not meet quality control standards for 15 participants and 5 participants were excluded for higher baseline vs. post-intervention enterolignan excretion (post-preintervention difference ≤−10 nmol/mg creatinine). Therefore, the final sample sizes for the present analyses were 228 completing baseline assessments and 170 completing all 4 visits.
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