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

Primary estrogens and estrogen metabolites are commonly measured in human plasma and serum, but there exist almost no recent reports for human stool. This knowledge gap limits our understanding of the relationships between systemic and gut estrogens. We developed a highly sensitive liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method to determine, in human plasma and stool, the free and conjugated levels of estrone, estradiol, and estriol together with their additional hydroxyestrogen and methoxyestrogen metabolites. We investigated human stool and plasma estrogens in healthy control men; in follicular and luteal phase premenopausal women; and in postmenopausal women. Most estrogens were present in plasma and stool of all groups, while the plasma and stool levels of hydroxyestrogen and methoxyestrogen metabolites but not estrone were correlated. In stool, estrogens were higher in premenopausal women, with estrogens increasing across the menstrual cycle. We combined these LC-MS/MS measures with shotgun metagenomic sequencing of the stool microbiomes. Estrogen deconjugation enzyme gene copy numbers (β-glucuronidase and arylsulfatase) were higher in premenopausal women; while the gene copy number of β-glucuronidase + arylsulfatase, but not β-glucuronidase alone, correlated with deconjugated stool estrogens in all groups. Moreover, β-glucuronidase + arylsulfatase gene copy numbers correlated with combined plasma estrogens in men and with individual plasma estrogen metabolites in men and premenopausal women. These results support the hypothesis that gut microbial β-glucuronidase and arylsulfatase control the deconjugation of gut estrogens while modulating systemic levels through the uptake and recirculation of these deconjugated estrogens. The intestine may thus constitute an important additional compartment in estrogen physiology.