Hionic fluor

Breath samples for hydrogen analysis were collected in Exetainers^© (Labco Ltd., Ceredigion, UK) and analyzed using a hydrogen monitor (M.E.C., Brussels, Belgium). Hydrogen excretion was expressed in parts per million (ppm). A significant increase in H₂ in breath was defined as an increase of 2.5 times the standard deviation of all previous points above the running average of all previous points [22 (link)]. Breath samples for analysis of ¹⁴CO₂ were collected by blowing through a pipette into a vial containing 2 mmol hyamine hydroxide until discoloration of the thymolphtaleine indicator, corresponding to the capture of 2 mmol CO₂. The amount of ¹⁴CO₂ was measured using β-scintillation counting (Packard Tricarb Liquid Scintillation Spectrometer, model 3375; Packard Instruments, Downers Grove, IL, USA) after addition of 10 mL of Hionic fluor (Perkin Elmer, Boston, MA, USA) and expressed as disintegrations per minute (dpm). The time of arrival of the meal in the colon was defined as the time at which a significant increase in ¹⁴CO₂ was observed in the breath and was determined in a similar way as for H₂.

The root Mg²⁺ uptake rate was determined radiometrically, as previously described by Tanoi et al. (2013) (link). The radioisotope ²⁸Mg was produced with ²⁷Al(α, 3p)²⁸Mg reaction in a cyclotron and was purified following the procedure of Iwata et al. (1992) (link). Four plants from each treatment were fed with 1/30-strength MGRL solution containing ²⁸Mg (6 MBq L^–1; Mg^{2 +}, 50 μM) for 1 h at 22°C under light conditions (100 μmol m^–2 s^–1). Roots were subsequently rinsed for 10 min with an ice-cold MGRL solution to wash out ²⁸Mg from the apoplast. After harvest, the whole plant tissue was solubilised with 0.5 mL of Soluene^® -350 (PerkinElmer, Waltham, MA, United States) and mixed with 3 mL of liquid scintillation cocktail (Hionic-Fluor, PerkinElmer, Waltham, MA, United States). Magnesium taken up by each plant was quantified using a liquid scintillation counter (LSC-6100, Aloka, Tokyo, Japan), as previously described by Sugita et al. (2013) (link). Since the ²⁸Mg half-life is ca. 21 h and its radioactivity decreased while all the samples were analysed, the activity at the same time point was calculated for all the samples.

Carboxyl-¹⁴C Leu–His (¹⁴C-LH) was prepared by Sekisui Medical Ltd. Its radiochemical purity was 97.1%, measured using HPLC, and its specific radioactivity was 7.90 MBq/mg. The test solution of 0.2 mg/mL of ¹⁴C-LH dissolved in distilled water was prepared immediately before administration. SD rats (n = 3) were starved for 16 h and orally administered the radioactive dipeptide (2 mg/kg, 10 mL/kg body weight). Plasma samples were collected 2, 5, 10, and 30 min and 1, 2, 4, 6, 8, 10, 24, 48, and 72 h after administration. Radioactivity was quantified using a liquid scintillation counter (HIONIC-FLUOR, PerkinElmer, Waltham, MA, USA). We prepared organ homogenates two hours after administration, and radioactivity was quantified according to a published method [10 (link)].

The whole brains were thawed and homogenized in 2.5 mL 50 mM Tris HCl buffer (pH 7.4) with 0.5% Triton, using a glass Teflon homogenizer (15 strokes, 900 rpm; Eurostar Power-B, IKA-Werke). Duplicates of 100 and 250 µl were transferred to glass scintillation vials (Perkin Elmer, 20 mL) and 15 mL Hionic-Fluor (Perkin Elmer) was added. The samples were shaken for 60 s and stored in darkness for 30 min before radioactivity was counted in a liquid scintillation analyzer (Tri-Carb 2810TR; PerkinElmer). Brain radioactivity was expressed as the percentage of the total injected radioactivity left in the brain.

Subjects deposited breath samples for the analysis of ¹³CO₂ by blowing through a straw, into a 12-mL glass tube (Exetainer^®, Labco Ltd., Ceredigion, UK), whereas samples for the measurement of ¹⁴CO₂ were collected by blowing through a pipet, into a plastic scintillation vial (Sarstedt, Nümbrecht, Germany) containing 4 mL of a 0.5 M hyamine hydroxide solution (Perkin Elmer, Boston MA, USA). Thymolphtaleine acted as a color indicator, which became discolored when 2 mmol CO₂ was exhaled.
The abundance of ¹³CO₂ was measured using isotope ratio mass spectrometry (ABCA, Sercon, Crewe, UK) and the results were expressed as delta over baseline (DOB). ¹⁴CO₂ was measured using β-scintillation counting (Packard Tricarb Liquid Scintillation Spectrometer, model 3375, Packard Instruments, Downers Grove, IL, USA), after addition of 10 mL hionic fluor (Perkin Elmer, Boston MA, USA), and was expressed as disintegrations per minute (DPM) [25 (link)]. The arrival time of the breakfast in the colon (OCTT) and the start of the fermentation, were defined as the time at which a significant increase in ¹⁴CO₂ and ¹³CO₂, respectively, from the background was observed in the breath. This increase was defined as 2.5 times the standard deviation of all previous points, above the running average of all previous points [26 (link)].

The test drugs were quantified using a high-performance liquid chromatography (HPLC)-tandem mass spectrometry system composed of a Nexera-XR (Shimazu, Kyoto, Japan) HPLC system connected to a Qtrap4500 (AB Sciex, Foster City, CA, USA) mass spectrometer with an electrospray ionization interface. The conditions are described in detail in the Supplementary Material. ³H and ¹⁴C-labeled samples were prepared as scintillation mixtures with Hionic-Fluor (Perkin Elmer) and the radioactivity was counted in a liquid scintillation counter (Tri-Carb 3110TR, Perkin Elmer).