Insulin ria kit

Blood samples were drawn from the jugular vein at 0, 5, 10, 15, 30, and 60 min and immediately transferred into polypropylene tubes containing 1 mg/mL EDTA-2Na, aprotinin (final concentration, 500 kallikrein-inhibiting units (KIU)/mL), and DPP-4 inhibitor (10 μg/mL). The plasma glucose level was measured using the glucose oxidase method (SINNOWA-D360PLUS, Jiangsu, China) after centrifuging the blood samples at 3000 rpm at 4°C for 10 min. The separated plasma was then stored at 4°C to assay the various parameters. The plasma insulin concentrations were measured by a radioimmunoassay (RIA) using the double-antibody technique, with rat insulin as the standard (Insulin RIA Kit; LINCO Research, St. Charles, Missouri, USA). The serum GLP-1 concentrations were measured using the RIA kits (active GLP-1 (7–36) RIA Kit; LINCO Research, St. Charles, Missouri, USA). At first, the samples were extracted by 95% alcohol. The sensitivity of this assay is 3 poml/L when a 100 μL sample is used. The specificities of the assay to identify active GLP-1 (7–36) are 100% and the cross-reaction to GLP-1 (9–36) fragment was less than 1%.

Plasma glucose (Uni Kit III, 07367204; Roche) concentrations were analyzed with a COBAS-FARA semiautomatic analyzer (Roche). Insulin was analyzed by using a radioimmunoassay (Insulin RIA kit; LINCO Research Inc). Plasma (100 mL) for amino acid analyses was deproteinized on ice with 10 mg dry 5-sulphosalicylic acid, mixed, and the clear supernatant fluid was collected after centrifugation. Plasma amino acid concentrations were determined by using HPLC after precolumn derivatization with o-phthaldialdehyde [23 (link)]. For plasma enrichment measurements, plasma Phe and Tyr were derivatized to their t-butyldimethylsilyl derivatives and analyzed by using gas chromatography–mass spectrometry (GC-MS) (Agilent 6890N GC/5973N MSD; Agilent) by using selected ion monitoring of masses 336 and 341 for unlabeled and labeled (ring-²H₅) Phe, respectively; and masses 466, 468, and 470 for unlabeled and labeled (ring-²H₂ and ring-²H₄) Tyr, respectively [24 ]. Thereafter, ratios of labeled: unlabeled derivatives were analyzed by using gas chromatography–combustion isotope ratio mass spectrometry (FinniganMAT 252; ThermoFisher Scientific). Standard regression curves were applied in all isotopic enrichment analyses to assess the linearity of the mass spectrometer and to control for the loss of tracer.

Five-week-old male C57BLKS/J-db/db and C57BLKS/J-db/+ mice were purchased from Japan SLC Inc (Shizuoka Ken, Japan). Mice were housed in a temperature-controlled room (22 ± 2 °C), with a light/dark cycle of 12 h/12 h and fed ad libitum. At 8 weeks of age, mice were randomly assigned into either saline control group or SFC-treated group. Treated groups were injected intra-peritoneally every other day with 10 mg/kg of SFC for 10 weeks. Blood was collected from tail. Glucose levels were measured using Accu-check (Korea Roche Diagnostics, Seoul, Korea). Plasma insulin levels were determined using insulin RIA kit (Linco Research, Billerica, MA, USA).