Hepatic tissue (100 mg) from each sample was homogenized with 200 μL of chloroform/isopropanol/MP40 (7/11/1). The mixtures were centrifuged and the supernatant dried at 50 °C overnight. The quantification for TC and free cholesterol (FC) was done with enzymatic assays kits (Wako Diagnostics, Mountain View, CA, USA) [28 (link)]. Esterified cholesterol (CE) was calculated manually by subtracting FC from TC. Liver TG were extracted (100 mg of tissue) with chloroform:methanol (2:1), dried under nitrogen at 60 °C and solubilized in 1% Triton X-100. The solubilized lipid isolates were analyzed by enzymatic methods (Wako Diagnostics, Mountain View, CA, USA), as previously described [29 (link)].
Enzymatic method
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Sourced in Japan
Enzymatic methods are analytical techniques that utilize enzymes to facilitate chemical reactions. These methods are designed to measure the concentration of specific compounds in a sample by monitoring the rate or extent of the enzyme-catalyzed reaction. Enzymatic methods are known for their high specificity and sensitivity, making them a valuable tool in various fields such as clinical diagnostics, food analysis, and environmental monitoring.
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6 protocols using enzymatic method
1
Hepatic Lipid Quantification Protocol
2
Metabolic Biomarker Assessment in Mice
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The blood glucose levels, serum insulin levels, triglyceride (TG) content in the liver, and glycogen content in the liver were determined using a Glutest Neo Super (Sanwa Chemical Co., Tokyo, Japan), an insulin kit (Morinaga, Tokyo, Japan), a leptin kit (Morinaga) and the Determiner-L TG II kit and the Determiner-GL-E kit (Wako Pure Chemical Industries, Osaka, Japan), respectively. The plasma alanine aminotransferase, free fatty acids, total cholesterol, LDL-cholesterol, and triglyceride levels were assayed using enzymatic methods (Wako Pure Chemical Industries). The plasma 3-hydroxybutyrate level was determined using the β Hydroxybutyrate Assay kit (Abcam, Cambridge, England). The blood glucose levels were checked just before and 4 hours after the administration of OSI-906 or the vehicle until day 7 or on days 8, 10, 14, 21, and 28. Blood samples were taken from the inferior vena cava immediately after sacrifice.
3
Comprehensive Metabolic Profiling of Glucose and Lipid Metabolism
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To avoid analytical variation, serum and plasma were stored at −80°C and analyzed as a single batch after completion of the study. Isotopic enrichment of glucose was measured at the Stable Isotope Core Laboratory of the Children's Nutrition Research Center, Baylor College of Medicine, Texas, USA. Penta‐acetate derivative of glucose was prepared, and the isotopic enrichment of [6,6‐2H2] glucose was measured by gas chromatography mass spectrometry, as previously described7 . Profiling of plasma amino acids and AC were carried out using liquid chromatography and tandem mass spectrometry at the Duke‐NUS metabolomics core facility to provide a comprehensive overview of substrate metabolism8 . Free fatty acid concentration was measured using enzymatic methods (Wako, Osaka, Japan) and pyruvate concentration using the gas chromatography mass spectrometry technique.
4
PTH Effects on WT and miR-143/145 Mice
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Intermittent PTH injections were conducted to 5-week-old female WT and miR-143/145Osx−/− mice (PTH: human PTH (1–34) [teriparatide; Asahi Kasei Pharma Corporation]). The dose of human PTH (1–34) was 40 µg/kg body weight per day for 5 days per week, delivered via subcutaneous injection for 4 weeks, as described previously36 . Micro-CT analysis, bone morphometric analysis, and measurement of the urine C-terminal telopeptide-1 (CTX-1) level were performed after PTH administration at 9 weeks of age (body weight 18–22 g). The right femurs were extracted for micro-CT analysis, and the left femurs were used for bone morphometric analyses with Villanueva bone staining embedded in methylmethacrylate as described above. For measurement of CTX-1, mice were fasted for 12 h before urine sampling by bladder puncture. CTX-1 and creatinine levels in the urine samples were measured by enzyme-linked immunosorbent assay (RatLaps TM; Immunodiagnostics Systems) and enzymatic method (Wako), respectively. The CTX-1 level was corrected for the creatinine concentration.
5
Serum urea and creatinine analysis
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Serum concentrations of urea nitrogen were determined by the urease‐indophenol method (Wako Pure Chemical, Osaka, Japan). Serum creatinine concentrations were measured by an enzymatic method (Wako Pure Chemical).
6
Serum Bile Acid Analysis in Propranolol Study
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The total serum bile acid pool was determined in fasting serum samples obtained immediately prior to each propranolol administration using a commercially available enzymatic spectrophotometric assay (TOTAL BILE ACIDS-HR, Enzymatic method, Wako, Osaka, Japan). For the purpose of subsequent calculations, the mean of the two baseline total SBA measurements was employed. As bile acid measurement took place prior to propranolol dosing, measurements were performed on samples taken at least 7 days apart from one another.
The serum concentrations of the individual bile acids were determined by gas chromatography mass spectroscopy as described previously [26] (link). Conventional liver function tests, other biochemical determinations and haematological investigations were performed using standard methods in the Departments of Clinical Chemistry and Haematology of the University Hospital of Basel.
The shunt index was estimated using the serum bile acid concentration according the equation published by Ohkuba and colleagues [21] (link):
The serum concentrations of the individual bile acids were determined by gas chromatography mass spectroscopy as described previously [26] (link). Conventional liver function tests, other biochemical determinations and haematological investigations were performed using standard methods in the Departments of Clinical Chemistry and Haematology of the University Hospital of Basel.
The shunt index was estimated using the serum bile acid concentration according the equation published by Ohkuba and colleagues [21] (link):
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