Kh2po4

Intracellular ROS were measured using the DCFDA Cellular ROS Detection Assay Kit (Abcam) with the instructions provided by the manufacturer. Briefly, on day 9 of treatment of 3HBA on 3T3-L1 adipocytes (Fig. 4), or on day 8 of transfection with small interfering RNA (Fig. 7), culture medium was replaced with Krebs–Ringer Bicarbonate buffer (KRBB), composed of 25 mM NaHCO₃ (Nacalai), 119 mM NaCl (Nacalai), 4.74 mM KCl (Nacalai), 1.19 mM MgCl₂ (Nacalai), 1.19 mM KH₂PO₄ (Nacalai), 2.54 mM CaCl₂ (Nacalai), 10 mM 4-(2-hydroxyethyl)-1-iperazineethanesulfonic acid (Nacalai), and 0.05 mM Bovine Serum Albumin solution (Sigma), supplemented with 10 μM DCFDA. After staining with DCFDA for 1 h at 37 ℃ under 5% CO₂, 3T3-L1 adipocytes were wash 3 times with PBS and detected by fluorescence spectroscopy with excitation / emission at 485 nm / 535 nm.

Dopamine dihydrochloride (Sigma-Aldrich, St Louis, MO), SCH23390 (Tocris-Bioscience, Bristol, UK), (S)-(–)-sulpiride (Sigma-Aldrich), and SKF38393 hydrochloride (Sigma-Aldrich) were dissolved at 10 mM in water and stocked at 4°C. Immediately before use, they were diluted to the final concentration with aCSF containing (in mM): 127 NaCl (Nacalai Tesque, Kyoto, Japan), 1.6 KCl (Wako, Tokyo, Japan), 1.24 KH₂PO₄ (Nacalai Tesque), 1.3 MgSO₄ (Nacalai Tesque), 2.4 CaCl₂ (Wako), 26 NaHCO₃ (Wako), and 10 d-glucose (Wako).

On day 7 after the induction of differentiation, the medium of 3T3-L1 cells was replaced with Krebs-Ringer Bicarbonate buffer (KRBB), composed of 25 mM NaHCO₃ (Nacalai), 119 mM NaCl (Nacalai), 4.74 mM KCl (Nacalai), 1.19 mM MgCl₂ (Nacalai), 1.19 mM KH₂PO₄ (Nacalai), 2.54 mM CaCl₂ (Nacalai), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Nacalai), 0.05 mM Bovine Serum Albumin solution (Sigma), and 25 mM glucose (Otsuka), with various concentrations of 3-Hydroxybutyric acid (Sigma). The cells were collected after 24 hr, then grown at 37 °C under 5% CO₂ fully humidified air environment.

United States Pharmacopeia (USP) reference standard omeprazole was procured from USP Convention. Authentic omeprazole standard capsules (Losec) were provided by AstraZeneca. Lansoprazole (internal standard) was from Sigma Aldrich (India). NaH₂PO₄.2H₂O, Na₂HPO₄, Na₃PO₄, KH₂PO₄ and other chemicals of reagent grade were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Distilled water was used for the preparation of HPLC eluents.
The investigational samples consisted of 154 samples of hard gelatin capsules containing 20 mg of omeprazole in enteric-coated pellets and 2 tablet samples purchased from different drug stores in Cambodia in 2010 and Myanmar in 2014. In Cambodia, samples were collected from pharmacies, depots, wholesaler and outlets of Phnom Penh, Svay Rieng and Kandal provinces. In Myanmar, collected samples were from pharmacy, hospital, and wholesalers of Yangon region. These samples of different serial and batch number were imported to Cambodia and Myanmar from 53 different manufacturers in seven countries. Samples were stored below 25 °C after collecting and all the quality analysis of the samples was finished before the expiration date of the samples. After quality-testing as required by the indicated pharmacopoeia, we selected five samples for further investigation based on the gravity of their failure in the dissolution test.

Four mineralization buffers were prepared, solutions A, B, C, and D [25 , 26 (link)].
Mineralization buffer A was prepared with 2.12 mM calcium chloride dihydrate (CaCl₂·2H₂O: Fujifilm Wako Pure Chemical Co., Osaka, Japan), 1.27 mM K-PO₄, which is a mixture of potassium dihydrogenphosphate and dipotassium hydrogen phosphate (KH₂PO₄, K₂HPO₄: Kanto Chemical Co., Inc., Tokyo, Japan). Mineralization buffer B was prepared with 2.24 mM CaCl₂·2H₂O and 1.34 mM K-PO₄. Mineralization buffer C was prepared with 2.35 mM CaCl₂·2H₂O and 1.41 mM K-PO₄. The final pH of mineralization buffers A, B, and C was adjusted to 7.4 by 10 mM HEPES–KOH [prepared from 10 mM hydroxyethyl piperazine ethane sulfonic acid (HEPES: Nacalai Tesque, Inc., Kyoto, Japan) mixed with potassium hydroxide (KOH: Nacalai Tesque, Inc.), and 150 mM potassium chloride (KCl: Nacalai Tesque, Inc.)].
Mineralization buffer D was prepared with 2.58 mM CaCl2·2H2O, 1.55 mM KH₂PO₄, and 180 mM sodium chloride (NaCl); the pH was adjusted to 7.6 with 50 mM Tris–HCl.
With all mineralization buffers, pH adjustment was carried out using a pH meter (F-22: Horiba Ltd., Kyoto, Japan). All of the mineralization buffers used in the present study had a Ca/P molar ratio of 1.67; the degrees of solution saturations with respect to HAp were (A) 2.58 × 10⁷, (B) 3.85 × 10⁷, (C) 5.50 × 10⁷, and (D) 8.32 × 10⁷ [25 ].