Mass spectrometric profiling of estrogen and estrogen metabolites in human stool and plasma partially elucidates the role of the gut microbiome in estrogen recycling

Estrogen and estrogen metabolites are commonly measured in human plasma and serum, but there exist almost no reports of estrogen measured in human stool. This methodological limitation in turn limits our understanding of the relationship between systemic and intestinal estrogen. We thus developed a highly sensitive liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for measuring free and conjugated forms of 15 estrogens and estrogen metabolites in human stool and plasma. We first investigated human stool and plasma estrogen in healthy control males; follicular and luteal phase premenopausal females; and postmenopausal females. Most estrogens were present in the plasma and stool of all groups, and plasma estrogen levels correlated with stool estrogen levels. In stool, estrogens were higher in premenopausal females, with estrogen levels rising across the menstrual cycle. We further combined these measures with shotgun metagenomic sequencing of the stool microbiomes. The level of estrogen deconjugation enzyme gene copy number (beta-glucuronidase + arylsulfatase) was higher in premenopausal females; while the gene copy numbers of beta-glucuronidase + arylsulfatase, but not beta-glucuronidase alone, correlated with reactivated stool estrogen in all groups. Moreover, deconjugation enzyme gene copy number correlated with plasma total estrogen in males and with individual plasma estrogen metabolites in all groups. These results support the hypothesis that gut microbial beta-glucuronidase and arylsulfatase control the reactivation of gut estrogen while modulating systemic levels through the uptake and recirculation of reactivated estrogen.