Acetate colorimetric assay kit

Acetic acid was quantified using the Acetate Colorimetric Assay Kit (Sigma‐Aldrich, St. Louis, MO, USA). Briefly, a 30–50 mg fecal sample was mixed with 500 μL Milli‐Q water containing internal standards (H3304‐1002, HMT). The mixture was centrifuged at 2300× g for 5 min at 4°C, after which 350 μL supernatant was centrifugally filtered using a 5‐kDa cutoff filter (Ultrafree‐MC PLHCC, HMT) at 9100× g for 120 min at 4°C to remove macromolecules. Subsequently, 15 μL filtrate was mixed with the assay buffer supplied with the kit. The absorbance of the sample was measured at 450 nm using an Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland). Five‐point calibration curves were prepared using standard compounds to estimate acetate concentrations in the fecal samples.

Blood samples were centrifuged at 3000 g for 10 minutes at room temperature to separate serum. ALT, AST and ammonia concentrations were evaluated with a dry chemistry analyser (FUJI DRICHEM 7000 V, FUJIFILM, Tokyo, Japan). Fecal samples (100 mg) were suspended in 1 ml PBS, and supernatants were obtained by centrifuging at 13000 g for 10 minutes. The cerebrum of mice was fully ground in 500ul PBS, and the supernatants were spun at 13000 g for 10 minutes at room temperature to obtain the supernatants. The fecal and brain ammonia levels were also detected by the dry chemistry analyser. The acetate levels were determined with Acetate Colorimetric Assay Kit (Sigma-Aldrich, US). Proline levels were detected using the Proline (PRO) Content Assay Kit (Solarbio, China).

MZ0003 deacetylase activity was detected using the Acetate Colorimetric Assay Kit (Sigma-Aldrich, St Louis, MO, USA). The substrates used, purchased from Sigma-Aldrich, were: 4-methylumbelliferyl acetate and 7-aminocephalosporanic acid (dissolved in DMSO); α-naphtyl acetate, β-D-glucose pentaacetate and β-D-galactose pentaacetate (dissolved in methanol); 5-bromo-4-chloro-3-indoyl acetate (dissolved in ethanol); p-NP acetate and cellulose acetate (dissolved in acetonitrile). The reaction mixtures, each containing 10 μg of protein and substrate, were pre-incubated in 5 mM Na-phosphate buffer pH 7.3 for 30 min at 35°C. Thereafter the assay was developed according to the manufacturer’s instructions and the absorbance was read at 450 nm. Values for each substrate were taken as the mean of three independent measurements after subtraction of a blank sample, without MZ0003, to correct for background degradation. The amount of acetate liberated was calculated in nM μl^-1 from a standard curve according to the manufacturer’s instructions.

The consumption of acetate was measured from 20 mL batch cultures (three biological replicates for each strain) grown in 100 mL Erlenmeyer bottles. The cultures were diluted to the OD₇₅₀ of 0.05, induced with 0.5 mM IPTG, and grown in BG-11 supplemented with antibiotics at ambient air under continuous low light (20 μmol photons m⁻² s⁻¹). Samples were collected on days 0, 2, 4, 6, 8, 10 and 12 followed by sample collection (0.5 mL each) and storage at −80 °C until analysis. Acetate quantitation was carried out from the growth media with Acetate Colorimetric Assay kit (Sigma-Aldrich) according to manufacturer’s instructions.

The levels of the metabolites acetate, glucose, lactate, and succinate were measured in the time-course samples (0, 2, 6, 10, 24, 48, 72 h) of vcDMEM and ccDMEM, as well as the endpoint sample VF (72 h) using the Acetate Colorimetric Assay Kit (MAK086, Sigma, USA), the Glucose-Glo TM Assay kit (J6021, Promega, USA), the Lactate-Glo TM Assay kit (J5021, Promega, USA), and the Succinic Acid Assay kit (K-SUCC, Megazyme, Germany) according to the manufacturers’ protocols. Measurements were made on an EnSpire 2300 plate reader (PerkinElmer Life Sciences, Schwerzenbach, Switzerland). Acetate, glucose, lactate, and succinate concentrations were calculated by linear regression based on the standard (0.00 to 10.00 nmol for acetate, 4.39 to 50.00 mM for glucose, 0.20 to 50.00 mM for lactate, and 0.13 µg to 4.00 µg total succinate) after subtraction of the respective enzyme blanks. Biological triplicates were analyzed for each sample and median and range (min, max) were calculated to represent the measured values. Linear reduction/accumulation rates were calculated using linear regression analysis in R and expressed as concentration changes in mM per hour.

For plasma GLP-1 quantification, blood samples were collected in test tubes containing a dipeptidyl peptidase IV inhibitor (Millipore). Colonic GLP-1 was extracted according to the method described by Cani et al.33 (link). The homogenate was centrifuged at 2000 × g for 20 min at 4 °C, and the supernatant fraction was decanted and diluted 1000-fold in assay buffer. The active GLP-1 concentration was determined using an enzyme-linked immunosorbent assay (ELISA) method (Active GLP-1 ELISA Kit, Shibayagi). Plasma and caecal acetate levels were enzymatically measured in duplicate using a commercial kit (Acetate Colorimetric Assay Kit, Sigma-Aldrich). The analysis of other caecal SCFAs was performed as follows: Caecal samples were suspended in 5% (w/v) metaphosphate-PBS (49 mL/g sample). Suspensions were subsequently filtered using a 3-kDa MWCO spin column (Pall Corporation) and analysed on a gas chromatography-flame ionisation detector system (Shimadzu GC2014, Shimadzu, Kyoto, Japan) equipped with a glass column packed with 60/80 SHINCARBON A coated with Themon-3000 (Shinwa-kakou). Plasma triglyceride (Wako) and insulin (Ultrasensitive Mouse Insulin ELISA, Mercodia) levels were determined using the corresponding commercial kits.

Acetate concentration was measured from the supernatant of cells after 24 h of stimulation, using the Acetate Colorimetric Assay Kit accordingly to the protocol’s manufactures Sigma (Saint Louis, MO).