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EnzyChromTM Ketone Body Assay
Kit (EKBD-100)
Quantitative Colorimetric
Determination of Ketone Body at 340nm
DESCRIPTION
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. BioAssay Systems'
EnzyChromTM 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,
APPLICATIONS
Direct assays of ketone body
in serum, plasma, urine and other biological
samples.
KEY FEATURES
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.
Storage conditions. Store
all reagents at -20°C. Shelf life of at least 6
months (see expiry dates on labels).
Precautions: reagents are
for research use only. Normal precautions for
laboratory reagents should be exercised while
using the reagents. Please refer to Material
Safety Data Sheet for detailed information.
PROCEDURES
Samples: 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).
AcAc Assay
1.
Standards. Prepare 8 mM standard by mixing 5
μL AcAc standard with 45 μL distilled H2O.
Transfer 5 μL H2O, 5 μL 8 mM AcAc
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 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.
MATERIALS REQUIRED, BUT NOT
PROVIDED
Pipeting (multi-channel) devices. Clear
flat-bottom 96-well plates (e.g. Corning Costar)
and plate reader.
Standard Curves of
Acetoacetic Acid (AcAc) and 3-Hydroxybutyric
Acid (BOH)
LITERATURE
1. Nuwayhid, N.F., Johnson,
G.F. and Feld, R.D. (1988). Kinetic measurement
of the combined concentrations of acetoacetate
and b-3-hydroxybutyrate in serum. Clin. Chem.
34/9, 1790-1793.
2. Hansen, J.L. and Freier,
E.F. (1978). Direct assays of lactate, pyruvate,
b-3-hydroxybutyrate, and acetoacetate with a
centrifugal analyzer. Clin. Chem. 24/3. 475-479.
3. Siegel, L., Robin, N.I.
and McDonald, L.J. (1977). New approach to
determination of total ketone bodies in serum.
Clin. Chem. 23/1, 46-49. |
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