Ketone Body Assay Kit

Catalog No. ABIN1000317

Product Details

Target: Ketone Body

Application: Biochemical Assay (BCA)

Sample Type: Plasma, Serum, Urine

Specificity: 0.12 mM

Characteristics: Sensitive and accurate. Uses 10 µL sample. Linear detection range of 0.12 to 8 mM for each ketone body in 96-well plate assay.
Convenient. The procedure involves adding a single working reagent, and reading the optical density at room temperature.
High-throughput. Can be automated as a high-throughput 96-well plate assay for many samples per day.

Components: AcAc Buffer: 20 mL. BOH Buffer: 20 mL. AcAc Reagent: 1 mL. BOH Reagent: 1 mL. AcAc Standard: 200 µL. 80 mM BOH Standard: 200 µL. 80 mM HBDH Enzyme: 120 µL.

Material not included: Pipeting (multi-channel) devices. Clear flat-bottom 96-well plates (e.g. Corning Costar) and plate reader.

Application Details

Application Notes: Direct assays of ketone body in serum, plasma, urine and other biological samples.

Protocol: AcAc Assay:
2. Reaction. Prepare Working Reagent for H2O, Standard and Sample wells, by mixing 195 µL AcAc Buffer, 8 µL AcAc Reagent and 0.5 µL HBDH Enzyme for each well. The Blank Reagent is prepared by mixing, for each blank well, 195 µL AcAc Buffer and 8 µL AcAc Reagent (i.e., no enzyme). Add 195 µL Working Reagent to the H2O, Standard and Sample wells. Add 195 µL Blank Reagent to Sample Blank wells. Gently tap plate to mix.
3. Incubate 5 min at room temperature. Read OD340nm. Calculate the acetoacetic acid (AcAc) concentration from the OD values of the H2O, 8 mM Standard, Sample and Sample Blank wells.
BOH Assay
1. Standards. Prepare 8 mM standard by mixing 5 µL BOH standard with 45 µL distilled H2O. Transfer 5 µL H2O, 5 µL 8 mM BOH standard in separate wells of a clear, flat-bottom, 96-well plate. Samples. Transfer 5 µL sample into two wells, one Sample well and one sample Blank well.
2. Reaction. Prepare Working Reagent for H2O, Standard and Sample wells, by mixing 195 µL BOH Buffer, 8 µL BOH Reagent and 0.5 µL HBDH Enzyme for each well. The Blank Reagent is prepared by mixing, for each blank well, 195 µL BOH Buffer and 8 µL BOH Reagent (i.e., no enzyme). Add 195 µL Working Reagent to the H2O, Standard and Sample wells. Add 195 µL Blank Reagent to Sample Blank wells. Gently tap plate to mix.
3. Incubate 15 min at room temperature and read OD340nm. Calculate the 3-hydroxybutyric acid (BOH) concentration from the OD values of the sample, sample blank, Standard and H2O. Total ketone body (TKB) concentration is calculated as, [TKB] = [AcAc] + [BOH] Note: if the calculated [AcAc] or [BOH] is higher than 8 mM, dilute sample in H2O and repeat this assay. Multiply the results by the dilution factor.

Sample Preparation:

Serum and plasma samples should be non-hemolyzed and assayed immediately. If not assayed, samples can be stored at -80°C for up to 30 days. Reagent preparation: bring all reagents to room temperature prior to assay. During experiment, keep the HBDH enzyme on ice or in refrigerator (2-8°C).

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Syamsunarno, Iso, Yamaguchi, Hanaoka, Putri, Obokata, Sunaga, Koitabashi, Matsui, Maeda, Endo, Tsushima, Yokoyama, Kurabayashi: "Fatty acid binding protein 4 and 5 play a crucial role in thermogenesis under the conditions of fasting and cold stress." in: PLoS ONE, Vol. 9, Issue 3, pp. e90825, (2014) (PubMed).

Martin-Murphy, Kominsky, Orlicky, Donohue, Ju: "Increased susceptibility of natural killer T-cell-deficient mice to acetaminophen-induced liver injury." in: Hepatology (Baltimore, Md.), Vol. 57, Issue 4, pp. 1575-84, (2013) (PubMed).

Fujieda, Manno, Hayashi, Rhodes, Guo, Arita, Bamba, Fukusaki: "Inflammation and resolution are associated with upregulation of fatty acid β-oxidation in Zymosan-induced peritonitis." in: PLoS ONE, Vol. 8, Issue 6, pp. e66270, (2013) (PubMed).

Syamsunarno, Iso, Hanaoka, Yamaguchi, Obokata, Koitabashi, Goto, Hishiki, Nagahata, Matsui, Sano, Kobayashi, Kikuchi, Sasaki, Maeda, Murakami, Kitamura, Suematsu, YoshitoTsushima, Endo, Hotamisligil, Kurabayashi: "A critical role of fatty acid binding protein 4 and 5 (FABP4/5) in the systemic response to fasting." in: PLoS ONE, Vol. 8, Issue 11, pp. e79386, (2013) (PubMed).

Qi, Tu, Ouyang, Wang, Peng, Cai, Dai: "Comparison of the metabolic profiling of hepatitis B virus-infected cirrhosis and alcoholic cirrhosis patients by using (1) H NMR-based metabonomics." in: Hepatology research : the official journal of the Japan Society of Hepatology, Vol. 42, Issue 7, pp. 677-85, (2012) (PubMed).

Sharma, Nagrath, Yarmush: "Metabolic profiling based quantitative evaluation of hepatocellular metabolism in presence of adipocyte derived extracellular matrix." in: PLoS ONE, Vol. 6, Issue 5, pp. e20137, (2011) (PubMed).

Target Details

Target: Ketone Body

Background: Quantitative determination of ketone body (acetoacetate and 3-hydroxybutyrate) in serum, plasma, urine etc at 340 nm.
Procedure: 30 min.

Ketone Bodies (acetoacetic acid and 3-hydroxybutyric acid) are produced in the liver mainly from oxidation of fatty acids, and are normally present at low concentrations in urine and blood. Increased ketone concentrations in the blood may lead to metabolic acidosis, which has been associated with diabetes, childhood hypo-glycaemia, growth hormone deficiency, alcohol or salicylate intoxication and inborn errors of metabolism. Simple, direct and automation-ready procedures for measuring acetoacetic acid (AcAc) and 3-hydroxybutyric acid (BOH) are very desirable. This ketone body assay is based on 3-hydroxybutyrate dehydrogenase catalyzed reactions, in which the change in NADH absorbance, measured at 340nm, is directly related to the AcAc and BOH concentrations.