ALT (SGPT) REAGENT SET

(UV-KINETIC METHOD)

INTENDED USE REAGENT DETERIORATION

For the quantitative determination of alanine aminotransferase in The reagent should be discarded if:
human serum. 1. Turbidity has occurred; turbidity may be a sign of contamination.
2. Moisture has penetrated the vial and caking has occurred.
INTRODUCTION 3. The reagent fails to meet linearity claims or fails to recover
The enzyme alanine aminotransferase is widely reported in a variety of control values in the stated range.
tissue sources. The major source of ALT is of hepatic origin and has 4. The reconstituted reagent has a reagent blank absorbance less than
led to the application of ALT determinations in the study of hepatic 0.8 at 340 nm.
diseases. Elevated serum levels are found in hepatitis, cirrhosis, and
obstructive jaundice. Levels of ALT are only slightly elevated in SPECIMEN COLLECTION
patients following a myocardial infarction.l This assay is intended for use with serum. Reports indicate that ALT in
serum remains stable at 4 - 8°C for a minimum of seven (7) days.4
UV methods for ALT determination were first developed by Hemolyzed specimens should not be used as erythrocytes contain seven
Wroblewski and LaDue in 1956.2 The method was based on the times the ALT activity of serum.
oxidation of NADH by lactate dehydrogenase (LDH). In 1980, the
International Federation of Clinical Chemistry recommended a INTERFERING SUBSTANCES
reference procedure for the measurements of ALT based on the Pyridoxal phosphate can elevate ALT values by activating the
Wroblewski and LaDue procedure.3 The ALT reagent conforms to the apoenzyme form of the transaminase.5 Pyridoxal phosphate may be
formulation recommended by the IFCC. found in water contaminated with microbial growth.

PRINCIPLE High levels of serum pyruvate may also interfere with assay
The enzymatic reaction sequence employed in the assay of ALT is as performance. Young, et al., give a list of drugs and other substances
follows: that interfere with the determination of ALT activity.6

ALT MATERIALS REQUIRED BUT NOT PROVIDED

L-Alanine + -Ketoglutarate Pyruvate + L-Glutamate 1. Pipetting devices
2. Test tubes/rack
LDH 3. Timer
Pyruvate + NADH + H+ Lactate + NAD+ + H2O 4. Spectrophotometer with capability to read at 340 nm
5. Heating bath/block (37°C)
The pyruvate formed in the first reaction is reduced to lactate in the
presence of lactate dehydrogenase and NADH. The activity of ALT is GENERAL INSTRUCTIONS
determined by measuring the rate of oxidation of NADH at 340 nm. The reagent for ALT is intended for use either as an automated
Endogenous sample pyruvate is converted to lactate by LDH during the procedure on chemistry instruments or as a manual procedure on a
lag phase prior to measurement. suitable spectrophotometer.

REAGENT COMPOSITION AUTOMATED PROCEDURE

When reconstituted as directed, the reagent for ALT contains the Refer to appropriate application manual available.
following:
(Concentrations refer to reconstituted reagent): -Ketoglutarate 13 MANUAL PROCEDURE
mM, L-Alanine 400 mM, NADH 0.2 mM, LDH 800 U/L, Buffer l00 1. Reconstitute reagent according to instructions.
mM, pH 7.8 ± 0.2, Non-reactive stabilizers and preservatives. 2. Zero spectrophotometer at 340 nm with distilled water.
3. Pipette l.0 ml of reagent into a test tube and allow to equilibrate to
WARNINGS AND PRECAUTIONS 37°C. If the spectrophotometer being used requires a final volume
1. For in vitro diagnostic use. greater than l.0 ml for accurate readings refer to ALTERNATE
CAUTION: In vitro diagnostic reagents may be hazardous. PROCEDURE.
Handle in accordance with good laboratory procedures which 4. Add 0.10 ml (100µl) of specimen to reagent and mix gently.
dictate avoiding ingestion, and eye or skin contact. 5. Maintain solution at 37°C. After 60 seconds, measure the
2. Specimens should be considered infectious and handled absorbance at 340 nm.
appropriately. 6. Take two additional absorbance readings at one (1) minute
3. Use distilled or deionized water where indicated. interval. Calculate the mean absorbance change per minute
(A/min).
STORAGE AND STABILITY 7. Multiply the A/min. by 1768 to calculate IU/L of ALT activity.
1. Store dry reagent at 2 - 8°C (refrigerated). 8. Sample with values above 500 IU/L should be diluted 1:1 with
2. The reconstituted reagent is stable for thirty days (30) if saline, re-assayed and the results multiplied by two (2).
immediately refrigerated and for twenty-four (24) hours at room
temperature. NOTE: If the spectrophotometer being used is equipped with a
temperature-controlled cuvette, the reaction mixture may be left in the
cuvette while the absorbance readings are taken.
PROCEDURE NOTES (MANUAL) QUALITY CONTROL
Turbid or highly icteric samples may give readings whose initial It is recommended that controls be included in each set of assays.
absorbance exceeds the capabilities of the spectrophotometer. More Commercially available control material with established ALT values
accurate results may be obtained by using 0.05 ml (50µl) sample and may be routinely used for quality control. The assigned value of the
multiplying the final answer by two (2). control material must be confirmed by the chosen application. Failure
to obtain the proper range of values in the assay of control material may
CALCULATIONS indicate reagent deterioration, instrument malfunction, or procedure
One International Unit (IU) is defined as the amount of enzyme that errors.
catalyzes the transformation of one micromole of substrate per minute
under specified conditions. TEMPERATURE CORRECTION 7
1. If the assay is performed at 37°C but is to be reported at 30°C,
ALT (IU/L) = A/min.  TV  1000 multiply the results by 0.7.
  SV  LP 2. If the assay is performed at 30°C but is to be reported at 37°C,
multiply the results by l.43.
= A/min.  1.10  1000 = A/min.  1768
6.22  0.1  1.0 EXPECTED VALUES8
Up to 26 IU/L (30°C) Up to 38 IU/L (37°C)
Where: A/min. = Average absorbance change per minute
TV = Total reaction volume (ml) It is strongly recommended that each laboratory establish its own
1000 = Conversion of IU/ml to IU/L normal range.
 = Millimolar absorptivity of NADH
SV = Sample volume (ml) PERFORMANCE CHARACTERISTICS
LP = Light path (cm) 1. Linearity: 500 IU/L
2. Comparison: Studies between the present method and a similar
Example: 1.480 = Initial absorbance method yield a correlation of 0.99 and a regression equation
1.350 = absorbance after one (1) minute Y = 0.98X + 1.32.
A/min. = 1.480-1.360 = 0.12 3. Precision studies:

Within Run
Then 0.12  1768 = 212.2 IU/L
Mean (IU/L) S.D. C.V. %
23.6 1.8 7.9
SI UNITS: To convert to SI Units (nkat/L) multiply IU/L by 16.67.
82.6 2.1 2.5
NOTE: If any of the test parameters are altered, a new factor must be
Run-to-Run
calculated using the formula above.
Mean (IU/L) S.D. C.V. %
23.4 1.9 8.0
ALTERNATE PROCEDURE
82.4 1.9 2.3
1. Reconstitute reagent according to instructions.
2. Pipette 2.8 ml of reagent into a test tube and allow to equilibrate REFERENCES
to 37C. 1. Henry, J.B.: Clinical Diagnosis and Management by Laboratory
3. Add 0.2 ml (200l) of specimen to reagent, and then mix gently. Methods, W.B. Saunders and Co., Philadelphia, PA. p 332-335
4. Maintain solution at 37C. After 1 minute, measure the (1974).
absorbance at 340 nm. (A1) 2. Wroblewski, F. and LaDue, J.S.: Proc. Soc. Exper. Biol. and
Med.91: 569 (1956).
5. After exactly three (3) minutes, read and record.
3. International Federation of Clinical Chemistry, J. Clin.
6. The difference in absorbance between readings (A1 - A2) Chem.Clin.Bio.18:5231(1980).
multiplied by the factor of 803. 4. Henry, R.J.: Clin. Chem. Principles and Techniques 2nd Ed.,
Harper and Row, New York.p.822 (1974).
ALTERNATE PROCEDURE CALCULATIONS 5. Rej, R., et al.: Clin. Chem. 19:92 (1973).
6. Young, D.S., et al.: Clin. Chem. 21:5 (1975).
ALT (IU/L) = (A1 – A2)  3.0  1000 = (A1 – A2)  803 7. Henry, R.J., et al.: Amer. J. Clin. Path. 34:381 (1960).
3  1  6.22  0.2 8. Tietz, N.W.: Fundamentals of Clinical Chemistry. W.B.
Saunders Co., Philadelphia, PA p. 682 (1976).
(A1 – A2) = absorbance change
Example: If A = 1.45, A = 1.35, A525: 07/2013
then (1.45-1.35)  803 = 0.10  803 = 80.3 IU/L

NOTE: If any of the above tests parameters have been altered, a new
factor must be calculated using the above formula.

LIMITATIONS
The reagent is linear to 500 IU/L. Sample that have ALT values greater
than 500 IU/L should be diluted 1:1 with saline, re-assayed and the
results multiplied by two (2).