β hydroxybutyrate assay kit

Manufactured by Merck Group

Sourced in United States

The β-hydroxybutyrate assay kit is a laboratory tool used to quantify the concentration of β-hydroxybutyrate, a ketone body, in biological samples. It provides a reliable and efficient method for the measurement of this important metabolite.

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β-hydroxybutyrate (20 citations)

10 protocols using β hydroxybutyrate assay kit

Quantifying 3-Hydroxybutyrate Release from Wound Dressings

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The integrated wound dressing (about 3.83–4.0 g – 10 cm²) was incubated in buffered saline solution (10 ml, PBS) for 12 h at 37°C. After incubation, the solution was adjusted to pH 3 and extracted 3 times with ethyl acetate. The ethyl acetate fraction was dried under a vacuum, re-suspended in 1 ml of methanol and analysed in Ultra Performance Liquid Chromatography (UPLC; Waters Corp., Milford, USA).
For measuring the release of 3-hydroxybutyrate from dressing, a commercially available assay (β-hydroxybutyrate Assay Kit, Sigma-Aldrich) was used. In this assay, the concentration of β-hydroxybutyrate is determined by an enzymatic reaction wherein the product is determined colorimetrically (at 450 nm).

Skórkowska-Telichowska K., Mierziak-Darecka J., Wrobel-Kwiatkowska M., Gebarowski T., Szopa J, & Zuk M. (2021). Wound coverage by the linen dressing accelerates ulcer healing. Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii, 38(5), 827-841.

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Comprehensive Metabolic Assessment of Ketosis

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Capillary blood samples were analyzed for concentration of ketones and lactate (Lactate Pro 2, Akray, Japan). Venous blood samples were collected into 4 mL SST vacutainers with immediate analysis of a small aliquot for blood glucose concentrations (Cobas Integra 400 plus, Roche Diagnostics, Switzerland). This venous sample was then centrifuged at 1,500 g for 10 min at 4°C, and aliquots of serum were stored at –80°C for later analysis. Samples were analyzed for FFA concentrations using a non-esterified-fatty acid (NEFA) assay kit (Wako Pure Chemical Industries, Ltd, Osaka, Japan), βeta-hydroxybutyrate concentrations using a β-hydroxybutyrate assay kit (Sigma-Aldrich, Ltd, Australia) and acetoacetate (AcAc) concentrations using an acetoacetate assay kit (Abcam, Cambridge, UK), as per the manufacturer's instructions. Urine samples were analyzed for urine ketones (namely AcAc) using ketone reagent strips (Keto-Diastix, Bayer).

Leckey J.J., Ross M.L., Quod M., Hawley J.A, & Burke L.M. (2017). Ketone Diester Ingestion Impairs Time-Trial Performance in Professional Cyclists. Frontiers in Physiology, 8, 806.

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Metabolic Biomarker Analysis in Liver Samples

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Serum glucose and triglyceride levels were measured using the Accutrend Plus meter (Roche Diagnostics, Indianapolis, IN). Serum β-hydroxybutyrate and liver ALT levels were measured using the β-hydroxybutyrate Assay Kit and ALT Activity Assay Kit, respectively (Sigma-Aldrich, Saint Louis, MO). Lipids were extracted from the liver samples by the Bligh/Dyer method and analyzed by gas-liquid chromatography as previously described (Zadravec et al., 2010 (link)).

Rando G., Tan C.K., Khaled N., Montagner A., Leuenberger N., Bertrand-Michel J., Paramalingam E., Guillou H, & Wahli W. (2016). Glucocorticoid receptor-PPARα axis in fetal mouse liver prepares neonates for milk lipid catabolism. eLife, 5, e11853.

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Comprehensive Blood Biomarker Analysis

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The blood hemoglobin was measured spectrophotometrically with HemoCue Hb 201 Analyzer. Blood lactate was measured using enzymatic-amperometric detection (Lactate Scout+ -meter;SensLab/EKF Diagnostics). The serum fraction was collected from the terminal blood of the dams at sacrifice. Serum total cholesterol and triglyceride levels were determined by an enzymatic method (Roche Diagnostics). Fatty acids were determined with the fluorometric Free Fatty Acid Quantification Kit (abcam #ab65341) and ketoacids with β-Hydroxybutyrate Assay Kit (Sigma Aldrich #MAK041).

Määttä J., Sissala N., Dimova E.Y., Serpi R., Moore L.G, & Koivunen P. (2018). Hypoxia causes reductions in birth weight by altering maternal glucose and lipid metabolism. Scientific Reports, 8, 13583.

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Comprehensive Metabolic Profiling

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Blood Hb was measured spectrophotometrically with a HemoCue Hb 201 Analyzer. Total cholesterol, HDL cholesterol, and triglyceride levels were measured from the serum fraction by an enzymatic method (Roche Diagnostics). Fatty acids were determined with a fluorometric Free Fatty Acid Quantification Kit (ab65341, Abcam) and ketoacids with a β‐Hydroxybutyrate Assay kit (MAK041, Sigma Aldrich). Erythropoietin was determined with a Mouse Erythropoietin/EPO Quantikine ELISA kit (MEP00B, R&D Systems).

Sissala N., Myllymäki E., Mohr F., Halmetoja R., Kuvaja P., Dimova E.Y, & Koivunen P. (2022). Hypoxia ameliorates maternal diet‐induced insulin resistance during pregnancy while having a detrimental effect on the placenta. Physiological Reports, 10(9), e15302.

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Blood Metabolite Quantification

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Collected by cardiocentesis or retro orbital puncture, PH mouse blood was centrifuged
(4°C, 3,000 rpm, 10 min) and serum was deproteinized using 30,000 MWCO (for nitrite assay)
or 10,000 MWCO (for β-hydroxybutyrate assay) filter membrane (Sartorius, Göttingen,
Germany). Serum nitrite concentration was measured with Griess Reaction System (Promega,
WI, USA) and β-hydroxybutyrate was measured with β-hydroxybutyrate assay kit
(Sigma-Aldrich, MO, USA).

Yu Y., Tamai M, & Tagawa Y.I. (2017). Nitric oxide is critical for avoiding hepatic lipid overloading via IL-6 induction during liver regeneration after partial hepatectomy in mice. Experimental Animals, 66(4), 293-302.

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Comprehensive Analytical Methods for Metabolic Assessment

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All chemicals used were of analytical grade. Triglycerides and glucose assay kits were purchased from BioSystems SA (Barcelona, Spain). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and retinol-binding protein 4 (RBP4) measurement kits were procured from BioVision Inc. (Milpitas, CA, USA). Recombinant human insulin, β-hydroxy butyrate assay kit, secondary antibodies, various standards such as retinol, β-carotene, β-apo-8′-carotenal, and fatty acids were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). C-peptide (EMD Millipore Corporation, Billerica, MA, USA), insulin (Crystal Chem Inc., Downers Grove, IL, USA), and fibroblast growth factor 21 (FGF21) (R&D Systems, Minneapolis, MN, USA) quantitation kits were used. Total RNA isolation kits were obtained from Qiagen GmbH (Hilden, Germany). For quantitative real-time polymerase chain reaction analysis (qRT-PCR), first-strand cDNA synthesis kits (New England Biolabs, Ipswich, MA, USA) and pre-validated universal probe for rats (Roche Diagnostics GmbH, Mannheim, Germany) were used. Experimental diets were obtained from OpenSource Diets (Research Diets Inc., New Brunswick, NJ, USA).

Mahesh M., Bharathi M., Reddy M.R., Kumar M.S., Putcha U.K., Vajreswari A, & Jeyakumar S.M. (2016). Carrot Juice Administration Decreases Liver Stearoyl-CoA Desaturase 1 and Improves Docosahexaenoic Acid Levels, but Not Steatosis in High Fructose Diet-Fed Weanling Wistar Rats. Preventive Nutrition and Food Science, 21(3), 171-180.

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Measurement of Iron Homeostasis Biomarkers

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Serum iron, serum ferritin (Tina-quant Ferritin kit; Roche Diagnostics, Milan, Italy), hemoglobin, and glucose were determined using an automated COBAS C501 counter (Roche, Milan, Italy) at the clinical-chemical laboratory of the University Hospital of Modena. Serum hepcidin was determined using an enzyme-linked immunosorbent assay kit (USCN Life Science, Hubei, China) according to the manufacturer's instructions, as previously reported.⁹ (link) Serum ketone bodies were analyzed using a β-Hydroxybutyrate Assay Kit (Sigma-Aldrich, Milan, Italy) following the manufacturer's instructions.
Liver and spleen tissue specimens were analyzed for non-heme iron content as previously reported.²¹ (link)

Vecchi C., Montosi G., Garuti C., Corradini E., Sabelli M., Canali S, & Pietrangelo A. (2014). Gluconeogenic Signals Regulate Iron Homeostasis via Hepcidin in Mice. Gastroenterology, 146(4), 1060-1069.e3.

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Quantification of Metabolic Biomarkers

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β-OHB contents in plasma and the liver were measured with a β-hydroxybutyrate assay kit (Sigma-Aldrich, MAK041) according to the manufacturer's instructions. Total cholesterol (TCHO), triglyceride (TG), and nonesterified free fatty acid (FFA) concentrations in tissues and plasma were quantified using commercial assay kits (Nanjing Jiancheng, China) according to the manufacturer's instructions. Plasma and liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured with the ALT Assay Kit and AST Assay Kit (Nanjing Jiancheng, China), respectively, according to the manufacturer's instructions. Cardiac ROS levels were measured with the OxiSelect in vitro ROS Assay Kit (Cell Biolabs) according to the manufacturer's instructions.

Guo Y., Liu X., Li T., Zhao J., Yang Y., Yao Y., Wang L., Yang B., Ren G., Tan Y, & Jiang S. (2022). Alternate-Day Ketogenic Diet Feeding Protects against Heart Failure through Preservation of Ketogenesis in the Liver. Oxidative Medicine and Cellular Longevity, 2022, 4253651.

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Quantifying Serum Lipid Biomarkers

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Serum triglyceride level was determined using enzymatic assay kit (Stanbio Laboratory, Boerne, TX, USA). Serum free fatty acids and β-hydroxybutyrate levels were determined using free fatty acid quantification kit and β-hydroxybutyrate assay kit (Sigma) following manufacturer’s instructions.

Bushman T., Lin T.Y, & Chen X. (2023). Depot-Dependent Impact of Time-Restricted Feeding on Adipose Tissue Metabolism in High Fat Diet-Induced Obese Male Mice. Nutrients, 15(1), 238.

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