Diazyme Laboratories
12889 Gregg Court
Poway, CA 92064, USA
Tel: 858-455-4768 / Fax: 858-455-3701
Email: [email protected]
Website: www.diazyme.com
Potassium Enzymatic Assay Kit
Configuration
The Diazyme Potassium reagent is provided in bulk and in the following kit configurations:
Instrument
Catalog No.
Kit size
R1: 4 x 45 mL
R1a: 4 x 45 mL
Universal
DZ113B-K
R2: 2 x 45 mL
R2a: 2 x 45 mL
Cal: 2 x 3 mL
Intended Use
For the professional in vitro quantitative determination of potassium
in human serum. Measurements obtained by this assay are used to
monitor electrolyte balance. For investigational use or export only.
Reagent Composition
R1 Diluent
Good’s Buffer, sucrose
4 x 45 mL
R1A. Lyophilized powder
0.2KU/L SPPase, 30KU/L PGM, 0.5KU/L G6PDH,
0.5mM ATP, 0.2mM NAD, 1mM glucose-1, 6bisphosphate and stabilizers
R2 Diluent
Good’s buffer
4 x 45 mL
2 x 45 mL
R2A. Lyophilized powder
1.0KU/L MAT, stabilizers
2 x 45 mL
Low potassium Calibrator
High potassium Calibrator
1 x 3 mL
1 x 3 mL
Clinical Significance
In healthy individual, an extracellular fluid level of potassium is
regulated to maintain at 3.5-5.5mM1. Small deviations from normal
level can have severe health consequences. Monitoring serum potassium concentration is important in both routine check and emergency rooms.
Assay Principle
Potassium is determined spectrophotometrically through a kinetic
coupling assay system using potassium dependent SAdenosylmethionine Synthase (MAT). NADH is generated after a
series of enzymatic reactions. The corresponding increase of optical
density at 340nm is proportional to the potassium concentration in
the serum.
MAT
Reagent Preparation
R 1 Reagent
Transfer the contents of one bottle of enzyme/substrate 1a to one
bottle of R1 diluent and dissolve by swirling gently ensuring that
the entire contents are completely dissolved (approximately 30
min). Avoid the formation of foam. The reconstituted R1 solution is
stable for 1 week at 2-8°C.
R 2 Reagent
Transfer the contents of one bottle of enzyme/substrate 2a to one
bottle of R2 diluent and dissolve by swirling gently ensuring that
the entire contents are completely dissolved (approximately 30
min). Avoid formation of foam. The reconstituted R2 solution is
stable for 1 week at 2-8°C.
L-methionine+ ATP+ H2O--------→ S-Adenosylmethionine + PPi +Pi
K+
Low and High Calibrators
Low and high K+ calibrators included are ready to use and are stable up to expiration date when stored under 2-8°C.
SPPAse/αPGM/G6PDH
NAD+ Pi+Sucrose ------------------------→NADH+6Phosphogluconate
Reagent Stability and Storage
Diazyme enzymatic potassium assay reagents including diluents
and lyophilized powder should be stored at 2-8°C. DO NOT
FREEZE. The reagents are stable when stored as instructed until the
expiration date on the label. Do not mix reagents of different lots.
Materials Required But Not Provided
An analyzer capable of dispensing two reagents and of measuring
absorbance at 340 nm with temperature control (37°C). Diazyme
Normal and Abnormal controls (Catalog No. DZ113B) are recommended.
Specimen Collection and Handling
The Diazyme Potassium Enzymatic Assay is formulated for use
with non-hemolysed serum. No special handling or pretreatment is
needed. Serum samples should be collected such that testing may be
performed as soon as possible after the specimen collection. The
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Effective: 2008-01-08
specimens may be refrigerated at 2-8°C for 7 days, or frozen at 20°C for a longer period of time. Note: Serum specimens and all
materials coming in contact with them should be handled and disposed as if capable of transmitting infection. Avoid contact with
skin by wearing gloves and proper laboratory attire.
mended to establish a range of normal values for the population in
their region.
Limitations
The assay is designed for use with human serum sample only. There
is a possibility that some substances that are not listed above may
interfere with the test.
Precautions
This reagent is for professional in vitro diagnostic use only. DO
NOT INGEST. Avoid contact with skin and eyes. Additional safety
information concerning storage and handling of this product is provided within the Material Safety Data Sheet for this product. Human source material was tested and found negative for HIV1, HIV2,
HBV, and HCV using FDA approved methods. To obtain a MSDS,
please contact our customer service department at 858-455-4768.
Assay Procedure
1.
2.
3.
4.
Reconstitute R1 and R2 reagents as described in Reagent
Preparation section and keep the reconstituted R1 and R2 reagents on ice bath.
In a cuvette, mix 180 µL of Reagent R1 and 10 µL of serum
sample and incubate at 37°C for 5 minutes and then add 90 µL
of Reagent 2.
Read absorbance (340nm) at 3 minutes after addition of Reagent 2 as A1. Incubate for 2 more minutes and read the absorbance as A2.
Calculate ΔA = A2 - A1
R1: 180 μL
Sample: 10 μL
340 nm
5
8
A1
Limit of Detection
The lower detection limit of this assay is 2 mM potassium.
Accuracy
The potassium assay was tested on the Olympus AU400 instrument
and results obtained were compared to a legally marketed ISE
method. A total of 65 serum samples ranging from 2-10 mM potassium were tested in both assays. The samples were mostly individual patient serum samples. To ensure the concentrations of potassium would be distributed across the reportable range, some potassium serum samples used for the study were spiked with potassium
to targeted concentrations. The above described accuracy study
showed that the Diazyme method had good correlation with existing
ISE method with a correlation coefficient of 0.95 with a slope of 1.0
and 0.01 intercept.
Precision
The potassium assay precision was tested on the Olympus AU400
instrument with two serum samples containing potassium that were
replicated 10 times. The %CVs obtained were 2.64% and 0.78% for
serum samples containing 4.5 mM and 6.5mM potassium, respectively.
R2: 90 µL
37°C
0
Performance Characteristics
10 min
A2
Linearity
Assay Procedure for Chemistry Analyzers
Parameter settings for automated chemistry analyzers are available
upon request.
Calibration
This assay should be calibrated using the enclosed low and high
Potassium standards. Potassium concentration in sample is
determined from linear calibration curve using the included low and
high potassium standards. Daily calibration is recommended.
Quality Control
Good laboratory practice recommends the use of control materials.
Users should follow the appropriate federal, state and local
guideline concerning the running of external quality control.
To ensure adequate quality control, normal and abnormal control
with known values should be run as unknown samples.
Diazyme potassium controls are available and sold separately. The
Catalog number is DZ113B-CON.
The assay has a linear range from 2-8 mM.
Interference
Interference testing was done to determine tolerance of the assay by
spiking the substances to be tested into pooled human sera. The
following cations and substances normally present in the serum
produced less than 10% deviation when tested at levels equal to the
concentrations listed: Na+ 200mM, NH4+ 0.5 mM, Ca2+ 5.0 mM,
Mg 2+ 2.0 mM, ascorbic acid 5.0 mM, bilirubin 45 mg/dL, Pi 1.5
mM, CuCl2 0.25 mM, ZnCl2 0.25mM, FeCl3 0.25mM, hemoglobin
200mg/dL, and alkaline phosphatase 1000U/L.
References
1.
2.
3.
4.
Results
Potassium results are printed out in mmol/L.
N. Tietz. Textbook of Clinical Chemistry, p.1841. W.B. Saunders Company, Philadelphia.(1986)
S. Kimura, S. Iyama, Y. Yamaguchi, S. Hayashi, R. Fushimimi
and N. Amino. Ann. Clin. Biochem (1997), 34:384-388
K. Inouye, I. Ueno, S. Yokoyama, and T. Sakaki J. Biochem.
131, 97-105(2002)
H. Bergmeyer, K. Gawehn, and M. Grassl in Methods of Enzymatic Analysis (Bergmeyer H. U. ed) 2nd Volume I, 505507, Academic Press, Inc. New York, NY (1974)
Reference Range
The normal range of potassium level in human serum is from 3.5 to
5.5 mM (13.7-19.9 mg/dL). However, each laboratory is recomDiazyme Laboratories
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