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Amplite® Fluorimetric Beta-Hydroxybutyrate (Ketone Body) Assay Kit

β-Hydroxybutyrate (β-HB) dose response was measured with Amplite™ Fluorimetric β-Hydroxybutyrate Assay Kit on a solid black 96-well plate using a Gemini microplate reader. 
β-Hydroxybutyrate (β-HB) dose response was measured with Amplite™ Fluorimetric β-Hydroxybutyrate Assay Kit on a solid black 96-well plate using a Gemini microplate reader. 
β-Hydroxybutyrate (β-HB) dose response was measured with Amplite™ Fluorimetric β-Hydroxybutyrate Assay Kit on a solid black 96-well plate using a Gemini microplate reader. 
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Ketone bodies are produced by the liver and used peripherally as an energy source when blood glucose levels drop. The two main ketone bodies are Beta-hydroxybutyrate (Beta-HB) and acetoacetate, while acetone is the third abundant ketone body. Normally these two predominant ketone bodies are present in small amounts in the blood during fasting and prolonged exercise. In patients who have diabetes, alcohol or salicylate poisoning, hormone deficiency, childhood hypoglycemia and other acute disease states, large quantities of ketone bodies are found in the blood. The over-production and accumulation of ketone bodies in the blood (ketosis) can lead to pathological metabolic acidosis (ketoacidosis). In extreme cases, ketoacidosis can be fatal. Blood ketone testing methods that quantify Beta-HB, the predominant ketone body in the blood (approximately 75%) have been used for diagnosing and monitoring treatment of ketoacidosis. Amplite® Fluorimetric Beta-Hydroxybutyrate Assay Kit offers a sensitive fluorescent assay for measuring Beta-HB levels in biological samples. This assay is based on an enzyme coupled reaction of Beta-HB, in which the product NADH can be specifically monitored by a fluorescent NADH sensor. The fluorescence signal can be measured by a fluorescence microplate reader. With this Fluorimetric Beta-hydroxybutyrate Assay Kit, we were able to detect as low as 1.4 µM Beta-HB in a 100 µL reaction volume.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare β-HB working solution (50 µL)
  2. Add β-HB standards or test samples (50 µL)
  3. Incubate at room temperature for 10 - 30 min
  4. Monitor fluorescence increase at Ex/Em = 540/590 nm

Important notes
To achieve the best results, it’s strongly recommended to use the black plates. Thaw one vial of each kit component at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. NAD stock solution (100X):
Add 100 µL of H2O into the vial of NAD (Component C) to make 100X NAD stock solution.

2. β-HB standard solution (100 mM):
Add 1 mL of H2O or 1X PBS buffer into the vial of β-HB standard (Component D) to make 100 mM β-HB standard solution.

PREPARATION OF STANDARD SOLUTION

β-HB standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13831

Add 10 µL of β-HB standard solution (100 mM) into 990 µL 1x PBS buffer to generate 1000 µM β-HB standard solution (HB7). Take the 1000 µM β-HB standard solution and perform 1:3 serial dilutions in 1x PBS to get serial dilutions of β-HB standard (HB6 - HB1). Note: Diluted β-HB standard solution is unstable and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

1. Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Mix (Component A).

2. Add 50 µL NAD stock solution into the bottle of Component A+B, and mix well to make β-HB working solution (Component A+B+C). Note: This β-HB working solution is not stable, use it promptly and avoid direct exposure to light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of β-HB standards and test samples in a clear bottom 96-well microplate. HB = β-HB standard (HB1 - HB7, 1 to 1000 µM); BL = blank control; TS = test sample.

BLBLTSTS
HB1HB1......
HB2HB2......
HB3HB3  
HB4HB4  
HB5HB5  
HB6HB6  
HB7HB7  

Table 2. Reagent composition for each well.

WellVolumeReagent
HB1-HB750 µLSerial Dilution (1 to 1000 µM)
BL50 µL1X PBS Buffer
TS50 µLTest Sample
  1. Prepare β-HB standards (HB), blank control (BL) and test samples (TS) into a solid black 96-well microplate according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of β-HB working solution to each well of β-HB standard, blank control, and test samples to make the total volume of 100 µL/well. For a 384-well plate, add 25 µL of β-HB working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, cut off at 570 nm).

Images


Citations


View all 3 citations: Citation Explorer
Reassessing the accuracy of enzyme-based assays for $\beta$-hydroxybutyrate production by the retinal pigment epithelium
Authors: Autterson, Gillian and Han, John Yeong Se and Philp, Nancy and Miller, Jason Matthew Lewis
Journal: Investigative Ophthalmology \& Visual Science (2023): 4466--4466
Highly differentiated human fetal RPE cultures are resistant to the accumulation and toxicity of lipofuscin-like material
Authors: Zhang, Qitao and Presswalla, Feriel and Calton, Melissa and Charniga, Carol and Stern, Jeffrey and Temple, Sally and Vollrath, Douglas and Zacks, David N and Ali, Robin R and Thompson, Debra A and others,
Journal: Investigative ophthalmology \& visual science (2019): 3468--3479
A platform for assessing outer segment fate in primary human fetal RPE cultures
Authors: Zhang, Qitao and Presswalla, Feriel and Feathers, Kecia and Cao, Xu and Hughes, Bret A and Zacks, David N and Thompson, Debra A and Miller, Jason ML
Journal: Experimental eye research (2018)

References


View all 54 references: Citation Explorer
Surface glycosylation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) membrane for selective adsorption of low-density lipoprotein
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Journal: J Biomater Sci Polym Ed (2014): 2094
Biocompatibility of electrospun poly(3-hydroxybutyrate) and its composites scaffoldsfor tissue engineering.
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Journal: Biomed Khim (2014): 553
Gamma-hydroxybutyrate (GHB) induces cognitive deficits and affects GABAB receptors and IGF-1 receptors in male rats
Authors: Johansson J, Gronbladh A, Hallberg M.
Journal: Behav Brain Res (2014): 164
Protective effects of exogenous beta-hydroxybutyrate on paraquat toxicity in rat kidney
Authors: Wei T, Tian W, Liu F, Xie G.
Journal: Biochem Biophys Res Commun (2014): 666
Cell attachment on poly(3-hydroxybutyrate)-poly(ethylene glycol) copolymer produced by Azotobacter chroococcum 7B
Authors: Bonartsev AP, Yakovlev SG, Zharkova, II, Boskhomdzhiev AP, Bagrov DV, Myshkina VL, Makhina TK, Kharitonova EP, Samsonova OV, Feofanov AV, Voinova VV, Zernov AL, Efremov YM, Bonartseva GA, Shaitan KV, Kirpichnikov MP.
Journal: BMC Biochem (2013): 12
d-beta-Hydroxybutyrate inhibited the apoptosis of PC12 cells induced by H2O2 via inhibiting oxidative stress
Authors: Cheng B, Lu H, Bai B, Chen J.
Journal: Neurochem Int (2013): 620
Biocompatibility studies and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/polycaprolactone blends
Authors: Lim J, Chong MS, Teo EY, Chen GQ, Chan JK, Teoh SH.
Journal: J Biomed Mater Res B Appl Biomater (2013): 752
Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)/collagen hybrid scaffolds for tissue engineering applications
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Journal: Tissue Eng Part C Methods (2013): 577
New synthesis and tritium labeling of a selective ligand for studying high-affinity gamma-hydroxybutyrate (GHB) binding sites
Authors: Vogensen SB, Marek A, Bay T, Wellendorph P, Kehler J, Bundgaard C, Frolund B, Pedersen MH, Clausen RP.
Journal: J Med Chem (2013): 8201
Lotus-leaf-like topography predominates over adsorbed ECM proteins in poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) surface/cell interactions
Authors: Zheng J, Li D, Yuan L, Liu X, Chen H.
Journal: ACS Appl Mater Interfaces (2013): 5882