Los reactivos deben verse claro y sin color.

Desechelo si alguno aparece

turbio o si contiene particulas de materia. El reactivo a utilizar, esta
estable por 4 semanas si se mantiene de 2 a 8C o 5 dias a temperatura
ambiente ( de 15 a 30C). El reactivo para trabajar, debera ser
desechado si la absorbancia inicial, leida contra el agua destilada a 340
nm. , esta bajo de 0.800.

Stanbio AST/SGOT (Liqui-UV)

Procedimiento No. 2920
Para la determinacion Cinetica cuantitativa de la AST/
SGOT en suero, para procedimientos Manuales o
Automatizados.

Resumen y Principios
El aspartato de aminotransferasa (AST), es una de varias enzimas que
catalizan el intercambio de grupos amino y oxo entre alfa-amino cidos y
alfa-oxocidos. Est ampliamente distribuida en los tejidos corporales con
major cantidad de AST como el corazn e hgado.1 Los que est pero con
menor cantidad de AST son el msculo esqueltico, riones, pncreas,
vaso, pulmones y cerebro. El dao a estos tejidos de como resultado una
liberacin de la enzima AST a la circulacin general. En el infaarto del
miocardio, la AST sica, puede empezar a incrementarse dentro de las 6
- 8 horas despus del ataque, por dos dias y vuelve a la normalidad para
el cuarto quinto da despus del infarto. 2 En enfermedades como
hepatitis, cirrosis y metstasis en hgado, se han entrado tambin
incrementos en las concentraciones sricas de AST. 3 Karmen 4 fue el
primero en reportar un mtodo cintico para medir la actividad de AST en
suero. Subsecuentemente el mtodo ha sido actualizado y optimizado por
Bergmeyer et al.5 El mtodo de Stanbio para medir AST, es similar al
mtodo recomendado por la Federacin Internacional de Quimica Clnica.6
Las reacciones enzimaticas presentes en este procedimiento son:
AST
L-Aspartato + 2 Oxo-glutarato
L-Glutamato + Oxalacetato
MDH
Oxalacetato + NADH + H+
L-Malto + NAD+
La AST, cataliza la transferencia del grupo amino entre el L-Aspartato y el
2 Oxo-glutarato. El Oxalccetico formado en la primera reaccin, va a
reaccionar con el NADH en presencia de malato deshiddrogenasa (MDH)
para formar NAD. La actividad de AST se determina midiendo el valor
total de oxidacin del NADH a 340nm. La lactato deshidrogenasa se
incluye en el reactivo para convertir al piruvato endogeno de la muestra
a lactato, durante la fase lag anterior a la medicin.

Reactivos
AST Buffer (R1), Ref. No. 2921
Composicin:
Tris, pH 7.5
L-Aspartato
MDH (msculo de porcino)
LDH (msculo de conejo

80 mmol/L
240 mmol/L
600
U/L
600
U/L

Enzima AST (R2), Ref. No. 2922

Composicin:
2-Oxoglutarato
NADH (Sal Disodio)

12
mmol/L
0.18 mmol/L

Precauciones: Los reactivos son para uso de diagnostico in vitro.

Precauciones normales en el manejo de los reactivos en el laboratorio
deberan ser seguidas. Los reactivos contienen azide de sodio que puede
ser toxico si se ingiere. El azide de sodio tambien puede reaccionar con el
plomo y cobre de las caerias formando metales azide altamente explosivos.
Refierase a las hojas de Materiales de seguridad para estar al tanto de la
informacion de seguridad y riesgos.
Preparacion del Reactivo: Los reactivos liquidos de buffer y enzima,
son suministrados ya listos para su uso. Prepare el reactivo con el que
trabajara a razon de de 5 partes de buffer (R1) y 1 de enzima (R2) (ej:
25 mL de buffer y 5 mL de enzima)
Almacenamiento y Estabilidad del Reactivo: El reactivo se mantiene
estable hasta la fecha de expiracion que esta en la etiqueta y cuando se
almacena adecuadamente de 2 a 8C y protegido contra la luz.

Materiales Requeridos Pereo No Incluidos

Espectrofotometro capaz de leer absorbancias a 340 nm y 1 cm para
paso de luz Bao Maria (37C) o celdillas de temperatura controlada
Pipetas capaces de medir exacatamenteTubos de pruebaCronometro
7,8,9,11

Recoleccion y Preparacion de la Muestra

Un suero claro y no hemolizado libre de hemolisis, debera ser utilizado.

Siempre que sea posible, los espicmenes deben ser separados y analizados
el dia que se recolectan. El suero AST es relativamente estable por un
minimo de 7 dias, si la muestra es refrigerada ( de 2 a 8C). Guarde el suero
en tubos bien tapados.
Interferencia de Substancias: La hemlisis debe ser evitada porque la
concentracin de AST en las clulas rojas excede 10 veces la del suero.
Ciertas drogas y otras substancias tambien se conoce que afectan los
valores del AST. Los niveles de Bilirubina arriba de 40mg/dL y los de
trigliceridos hasta 2000 mg/dL, no muestran interferencia en esta
prueba.

Procedimiento Automatizado
Adaptaciones especiales para analizadores automatizados estan
disponibles solo contactando con el Departamento de Servicio al Cliente
de Stanbio.

Procedimiento Manual
1. Prepare el reactivo de AST con la que va a trabajar de acuerdo a las
intrucciones.
2. Calibre el cero del espectrofotometro a 340 nm con agua destilada.
3. Para cada muestra y control, anada 1.0 mL del reactivo dentro de la
cubeta o en un tubo de prueba e incube por 3 minutos a 37C.
4. Agregue 100L (0.100 mL) de suero a sus respectivos tubos y mezcle
suavemente.
5. Lea y anote el cambio en la absorbancia cada minuto. Continue
incubando a 37C y anote los cambios en la absorbancia nuevamente a 2 y
3 minutos. Los valores deberan ser constantes.
6. Determine el promedio de la absorbancia por minuto (A/Min),
multipliquelos por el factor 1746 para obtener resultados en U/L.
NOTA: Si la cubeta no tiene temperatura controlada, incube las muestras
a 37C entre cada lectura.
Control de calidad: El control de suero normal Ser-T-Fy I, Cat. No.
G427-86 y el control de suero abnormal Ser-T-Fy II, Cat. No. G428-86
son recomendables para para verificar precision y exactitud. Otros
controles comerciales disponibles con valores de AST ensayados por
este metodo son tambien convenientes. La actividad de la AST
determinada en esos materiales, por este procedimiento, debera caer
dentro de los rangos fijados para los controles. Dos niveles de controles
deben ser analizados cada dia que se prueben.
Calibracion: La actividad de la AST, se basa en un coeficiente de
extincion micromolar de NADH a 340 nm ( vea la seccion de
Resultados). La guia de calibracion del instrumento por el fabricante,
debe ser seguido para calibrar su analizador.

Resultados
Los valores se derivan en base al coeficiente de extincion de absorptividad
micromolar de NADH 340 nm (0.0063). La actividad en unidades por litro
(U/L) de AST/SGOT, es la cantidad de enzima que produce 1 nmol/L de
NADH por minuto.
U/L= /Min/Absorptividad

x Volumen total/Volumen de la muestra

U/L= /Min /0.0063 X 1.100 / 0.100

U/L= A / Min x 1746

Limitaciones
Si el A/Min. es mayor de .342, diluya una parte de la muestra con 9 partes
de salina isotonica y vuelva a probar. Multiplique los resultados por 10.

Valores Esperados10
El Rango normal (en adultos):
833 U/L (37C)
Este rango unicamente debe servir como guia. Se recomienda que cada
laboratorio establezca sus propios rangos de valores esperados, ya que
existen diferencias entre instrumentos, laboratorios y la poblacion local.

Caracteristicas8
Comparacion: Un grupo de 62 sueros dentro de un rango de actividad
AST de 12 a 463 U/L, fueron probados por el metodo AST descrito y por
un reactivo comercial similar disponible. Al comparar resultados se
produjo un coeficiente de correlacion de 0.993 y la ecuacion de regresion
fue y=0.988x + 0.43. ( Otros estudios comparativos fueron realizados de
acuerdo con las guias tentativas de la NCCLS, EP9-T.)
Precision: Dentro de una corrida, la precision fue establecida por 20
pruebas en 3 diferentes niveles de sueros de control comerciales. Valores
totales de precision se obtuvieron probando 3 controles comerciales por 5
dias seguidos.

Media AST (U/L)

Desv. Std (U/L)
C.V. (%)

Durante de una corrida

Suero 1
Suero 2
Suero 3
25
51
116
0.8
1.6
0.9
3.3
3.1
0.8

Media AST (U/L)

Desv. Std (U/L)
C.V. (%)

Precisin Total
Suero 1
Suero 2
26
49
1.1
0.7
4.4
1.4

Suero 3
115
0.8
0.7

Estudios de precision fueron realizados de acuerdo con las guias

tentativas de la NCCLS, EP5-T.
Linealidad: Es lineal a 600 U/L y a 37C. Llevado a cabo de acuerdo a
las guias EP6-P de la NCCSL.
Sensitividad: Esta basada en la resolucion de un instrumento de
A=0.001, el metodo presentado muestra una sensitividad de 1.75 U/L.

Referencias
1. Wilkinson JH. Principles and Practice for Diagnosis Enzymology. Year Book
Medical Publishers, 1976
2. Kachmar JR: Enzymes. In Fundamentals of Clinical Chemistry, NW Tietz, Editor,
Saunders, Philadelphia, 1976, p 674.
3. Sacks HJ, Lanchantin GF; An elevation of serum transaminase in the jaundice state.
Am J Clin Pathol 33:97, 1960
4. Karmen A: A note on the spectrophotometric assay of glutamic-oxolacetic transaminase in human blood. J Clin Invest 34:131, 1955
5. Bergmeyer HU, Scheibe P, Wahlefeld AW: Optimization of methods for aspartate
aminotransferase and alanine aminotransferase. Clin Chem 24: 58, 1978.
6. Expert Panel of Enzymes of the International Federation of Clinical Chemistry: Part
3. Revised IFCC method for aspartate aminotransferase. Clin Chem 24:720, 1978.
7. Demetriou JA et al. In Clinical Chemistry - Principles and Technics 2nd ed. RJ Henry
et al. Eds. Harper & Row, Hagerstown MD, 1974, p 873.
8. International Federation of Clinical Chemistry. Provisional Recommendations on
IFCC Methods for the Measurement of Catalytic Concentrations of Enzymes. Clin
Chem 23: 887, 1977.
9. Young D.S. Effects of drugs on clinical laboratory tests. AACC Press, Washington
D.C., 1990
10.Henry JB. Clinical Diagnosis and Management by Laboratory Methods, 17th ed. WB
Saunders Co., 1984, p 1437.
11.Stanbio Laboratory Data

Para asistencia tecnica llame: 800-531-5535 (830) 249-0772

Fax (830) 249-0851 e-mail: [email protected]
http://www.stanbio.com
Stanbio Laboratory 1261 North Main Street Boerne, Texas 78006
DN: RBR.2920CE.02 Last Revision: 07/04 Procedure No. 2920CE

ing Reagent is stable for 4 weeks at 2-8C or 5 days at room temperature

(15-30C). The Working Reagent should be discarded if the initial absorbance, read against distilled water at 340 nm, is below 0.800.

Material Required But Not Provided

Spectrophotometer capable of absorbance reading at 340 nm and 1 cm
lightpath. Constant temperature block or bath, 37C, or temperature
controlled cuvette well. Accurate pipetting devices, Test tubes and
Interval timer

Stanbio AST/GOT (Liqui-UV)

Procedure No. 2920

Specimen Collection and Storage

For the Kinetic Quantitative Determination of AST/GOT

in Serum for Manual and/or Automated Procedures
Summary and Principle
Aspartate aminotransferase (AST) is one of several enzymes that catalyze
the exchange of amino and oxo groups between alpha-amino acids and
alpha-oxo acids. It is widely distributed throughout body tissues with
significant amounts in the heart and liver.1 Lesser amounts are found in
skeletal muscle, kidneys, pancreas, spleen, lungs and brain. Injury to these
tissues results in release of the AST enzyme to the general circulation. In
myocardial infarction, serum AST may begin to rise within 6-8 hours after
onset, peak within two days and return to normal by the fourth or fifth day
of post infarction.2 An increase in serum AST is also found with hepatitis,
liver necrosis, cirrhosis and liver metastasis.3
Karmen4 first reported a kinetic method for measuring AST activity in
serum. Subsequently, the method has been modified and optimized by
Bergmeyer et al.5 The Stanbio assay procedure for AST measurement is
similar to the method recommended by the International Federation of
Clinical Chemistry.6 The enzymatic reactions involved in the assay procedure are as follows:
AST
L-Aspartate + 2- Oxoglutarate
L-Glutamate + Oxalacetate
Oxalacetate + NADH + H+

MDH

L-Malate + NAD+

AST catalyzes the transfer of the amino group aspartate to 2-oxoglutarate

to yield oxalacetate and glutamate. The oxalacetate formed in the first
reaction is then reacted with NADH in the presence of malate dehydrogenase (MDH) to form NAD. AST activity is determined by measuring the
rate of oxidation of NADH at 340 nm. Lactate dehydrogenase is included
in the reagent to convert endogenous pyruvate in the sample to lactate
during the lag phase prior to measurement.

Reagent
AST Buffer (R1), Ref. No. 2921
Composition:
L-Aspartate
MDH (porcine muscle)
LDH (rabbit muscle)
Tris Buffer, pH 7.5

240
600
600
80

mmol/L
U/L
U/L
mmol/L

AST Enzyme (R2), Ref. No. 2922

Composition: 2 - Oxoglutarate
NADH (Disodium salt)

12
0.18

mmol/L
mmol/L

Precautions: The reagents are for "In Vitro Diagnostic Use". Normal
precautions exercised in handling laboratory reagents should be followed.
The reagents contain sodium azide which may be toxic if ingested. Sodium
azide may also react with lead and copper plumbing to form highly
explosive metal azides. Refer to Material Safety Data Sheet for any updated
risk, hazard or safety information.
Reagent Preparation: Buffer and Enzyme liquid reagents are supplied
ready-to-use. Prepare Working Reagent in the ratio of 5 parts Buffer (R1)
to 1 part Enzyme (R2) (i.e., 25 mL Buffer and 5 mL Enzyme).
Reagent Storage and Stability: Reagents are stable until the expiration
date on their respective labels, when properly stored at 2-8C and
protected from light. Reagents should appear clear and colorless. Discard if either appears cloudy or contains particulate matter. The Work-

Non-hemolyzed serum is the specimen of choice, yet EDTA treated

plasma or heparinized plasma can be used.7 Whenever possible specimens
should be separated and analyzed on the day of collection. Store serum in
stoppered tubes. The enzyme in serum is reportedly stable for a minimum
of 7 days at 2-8C.8
Interfering Substances: Hemolysis must be avoided as the concentration
of AST in red cells is roughly 10 times that of serum.7 Bilirubin levels up to
40 mg/dL and triglyceride levels up to 2000 mg/dL show no interference
in this test.11 Certain drugs and other substances are also known to affect
AST values.9

Manual Procedure
1. Prepare AST Working Reagent according to instructions.
2. Zero spectrophotometer at 340 nm with distilled water.
3. For each sample and control, add 1.0 mL Working Reagent to cuvette
or test tube and warm to 37C for 3 minutes.
4. Add 100 uL (0.10 mL) serum to its respective tube and mix gently.
5. Read and record absorbance at 1 minute. Continue incubating at 37C
and record absorbance again at 2 and 3 minutes. Rate should be
constant.
6. Determine the average absorbance per minute (DA/min), multiply by
factor -1746 for results in U/L.
NOTE: If cuvette is not temperature controlled, incubate samples at 37C
between readings.
Quality Control: Stanbio Ser-T-Fy I, Normal Control Serum, Cat. No.
G427-86 and Stanbio Ser-T-Fy II, Abnormal Control Serum, Cat. No. G42886 are recommended for each run. Other commercially available controls
with AST values assayed by this method are also suitable. AST activity
determined in these materials, by this procedure should fall within the
ranges stated for the controls. Two levels of controls should be analyzed
with each run.
Calibration: AST activity is based on the " micromolar extinction coefficient" of NADH at 340 nm (see "Results" section). The instrument
manufacturer's calibration guidelines should be followed to calibrate your
analyzer. Assaying the AST contents of a control serum with known AST
values can be used to assure instrument calibration has been performed
correctly.

Results
Values are derived based on the "absorptivity micromolar extinction
coefficient" of NADH at 340 nm (0.0063). Units per liter (U/L) of AST/GOT
activity is that amount of enzyme which oxidizes one mol/L of NADH
per minute.
U/L = (A/Min Absorptivity) x (Total Volume/Sample Volume)
.
U/L = (A/Min/Min / 0.0063) x (1.10/0.10)
.

U/L = A/Min x 1746

Limitations
If the A/min. is greater than 0.342, dilute 1 part sample with 9 parts
isotonic saline and re-assay. Multiply the result by 10. AST values for
neonatal patients have not been established with this procedure.
Grossly icteric or turbid specimen may require the use of a sample
blank.

Expected Values10
Normal Range:
8 - 33 U/L (37C)
This range should serve only as a guideline. It is recommended that each
laboratory establish its own range of expected values, since differences
exist between instruments, laboratories, and local populations.

Performance Characteristics
Comparison: A group of 62 sera ranging in AST activity from 12 - 463
U/L was assayed by the described AST method and by a similar
commercially available AST reagent. Comparison of the results yielded
a correlation coefficient of 0.993 and the regression equation was y =
0.988x + 0.43. (Comparison studies were performed according to
NCCLS Tentative Guideline, EP9-T.)
Precision: Within-run precision was established by 20 assays on three
different levels of commercial serum controls. Total Precision values
were obtained by assaying the 3 commercial controls for 5 consecutive
days.
Within-Run
Serum 1 Serum 2 Serum 3
Mean AST (U/L)
25
51
116
Std. Deviation (U/L)
0.8
1.6
0.9
C.V. (%)
3.3
3.1
0.8
Total Precision
Serum 1 Serum 2 Serum 3
Mean AST (U/L)
26
49
115
Std. Deviation (U/L)
1.1
0.7
0.8
C.V. (%)
4.4
1.4
0.7
Precision studies were performed according to NCCLS Tentative
Guideline, EP5-T.
Linearity: Linear to 600 U/L at 37C.8 Performed according to NCCLS
Guideline EP6-P.
Sensitivity: Based on an instrument resolution of A = 0.001, the method
presented shows a sensitivity of 1.75 U/L.

References
1. Wilkinson JH. Principles and Practice for Diagnosis Enzymology. Year
Book Medical Publishers, 1976
2. Kachmar JR: Enzymes. In Fundamentals of Clinical Chemistry, NW
Tietz, Editor, Saunders, Philadelphia, 1976, p 674.
3. Sacks HJ, Lanchantin GF; An elevation of serum transaminase in the
jaundice state. Am J Clin Pathol 33:97, 1960
4. Karmen A: A note on the spectrophotometric assay of glutamic-oxolacetic
transaminase in human blood. J Clin Invest 34:131, 1955
5. Bergmeyer HU, Scheibe P, Wahlefeld AW: Optimization of methods for
aspartate aminotransferase and alanine aminotransferase. Clin Chem
24: 58, 1978.
6. Expert Panel of Enzymes of the International Federation of Clinical
Chemistry: Part 3. Revised IFCC method for aspartate aminotransferase. Clin Chem 24:720, 1978.
7. Demetriou JA et al. In Clinical Chemistry - Principles and Technics 2nd
ed. RJ Henry et al. Eds. Harper & Row, Hagerstown MD, 1974, p 873.
8. International Federation of Clinical Chemistry. Provisional Recommendations on IFCC Methods for the Measurement of Catalytic Concentrations of Enzymes. Clin Chem 23: 887, 1977.
9. Young D.S. Effects of drugs on clinical laboratory tests. AACC Press,
Washington D.C., 1990
10.Henry JB. Clinical Diagnosis and Management by Laboratory Methods,
17th ed. WB Saunders Co., 1984, p 1437.
11.Stanbio Laboratory Data
Manufactured By:
Stanbio Laboratory 1261 North Main Street Boerne, Texas 78006 USA
Ph: (830) 249-0772 Fax (830) 249-0851 e-mail: [email protected]
http://www.stanbio.com
DN: RBR.2920CE.02 Last Revision: 07/04 Procedure No. 2920CE