Determination of berberine-upregulated endogenous short-chain fatty acids through derivatization by 2-bromoacetophenone.

Short-chain fatty acids (SCFAs) are a major group of endogenous metabolites generated by the gut microbiota and have been reported to play an important role in physical health, such as improving energy metabolism. Here, using 2-bromoacetophenone as the derivatization reagent (BP, 10 mg/mL, 40 °C for 20 min), a sensitive liquid chromatography-tandem mass spectrometric method was established for the quantitative determination of seven short-chain fatty acids in plasma and feces. The analyses were performed on a C18 column in positive multiple reaction monitoring mode. Specificity, linearity, accuracy, precision, recovery, and stability were observed within the quantitative limits of biological sample analysis. The established method has largely improved the sensitivity by 200- to 2000-fold than that in gas chromatography (GC). Especially for butyrate, the lower quantitative limit of 1 ng/mL, 1600-fold higher in sensitivity than that of GC (1.6 µg/mL), ensured the accurate determination of its low level in blood or feces (88 ± 29 ng/mL in blood, 176 ± 18 µg/g in feces). Then, the validated method was applied for therapeutic studies of berberine in hyperlipidemia hamsters in vivo and screening of 13 compounds (including five metabolites of berberine and eight typical isoquinoline alkaloids) in vitro. After berberine treatment (oral, 200 mg/kg, 2 weeks) to hyperlipidemia hamsters, the levels of butyrate were significantly upregulated in blood (77 ± 10 ng/mL vs. 117 ± 13 ng/mL, *P < 0.05) and feces (132 ± 11 µg/g vs. 547 ± 57 µg/g, ***P < 0.001), which further verified butyrate as an active endogenous metabolite in coordination with berberine to lower the blood lipids. Additionally, the berberine metabolites (M1, M2, M3), as well as two isoquinoline alkaloids (tetrandrine and dauricine), could also obviously induce the production of SCFAs (butyrate, etc.) in gut microbiota. In total, we have successfully established a new derivative LC-MS/MS method for the targeted quantitative determination of seven SCFAs in biological samples. Graphical abstract.

Modulación neuroendocrina de los efectos enterotróficos de los ácidos grasos de cadena corta colónicos

Los efectos enterotróficos de los ácidos grasos de cadena corta son cadena complejos y problabemente multifatoriales. La adición directa de energía a la mucosa intestinal es una contribución moderada, mientras que el incremento en el flujo sanguíneo puede aportar una contribución ligeramente mayor al enterotrofismo. Los mecanismos mediados localmente solos, como se describe, no pueden explicar por completo los efectos tróficos de los ácidos grasos de cadena corta, debido a que el trofismo intestinal se presenta tanto local como distalmente al sitio de infusión. Los ácidos grasos de cadena corta estimulan el crecimiento del intestino delgado, lo mismo que del intestino grueso cuando se infunden al colon de ratas. El incremento de las secreciones endocrinas pancreáticas, la estimulación de las hormonas gastrointestinales enterotróficas y el incremento en el sistema nervioso autónomo pueden ser mediadores adicionales, tanto de los efectos enterotróficos locales, como de los efectos enterotróficos sistémicos de los ácidos grasos de cadena corta. Definir los mensajeros hormonales específicos y los procesos celulares mediante los cuales son liberados, es una área muy activa de la investigación actualmente en marcha.