GAMMA GLUTAMYL TRANSFERASE (GGT) PROCEDURE

For The Quantitative Determination of Serum GT

SUMMARY AND EXPLANATION

Gamma Glutamyl Transferase is the most sensitive enzymatic indicator of hepatobiliary disease available at present. GGT levels are elevated in most all cases of liver disease, however, it is of little value in attempting to discriminate between different kinds of liver disease (1). Elevated levels are also noted in moderate and heavy alcohol drinkers. The release of GGT into the serum reflects the toxic effects of alcohol on the microsomal structures of the liver cells (1). The test has been suggested for follow-up of chronic alcoholics undergoing treatment (2). High levels of GGT are present in the prostrate which may account for the fact that the activity of GGT in males is approximately 50% higher than seen in females. Prostatic malignancy at times may be a source of elevated GGT levels, however, in malignant disease in general, an increased GGT level must first arouse suspicion that the disease is metastatic to the liver (1).

Methods for determining GGT are based on the use of glutamyl derivatives of aromatic amines as the substrate (1). Orlowski and Meiser introduced g-Glutamyl-p-nitroanilide as a substrate in 1963 (3) with Kulhanek and Dimov adding glycylglycine in 1966 to increase the speed of the reaction (4). In 1969, Szasz introduced a modified method using L-g-glutamyl-p-nitroanilide as the substrate with glycylglycine serving as the g-glutamyl residue acceptor (5). The EAGLE GGT PROCEDURE is based on this modification and offers a simple kinetic determination.

PRINCIPLE

L-g-Glutamyl-p-nitroanilid + Glycylglycine g-GT p-nitroaniline +GutamylglycylglycinThe g-glutamyl group is transferred from L-g-glutamyl-p-nitroanilide to glycylglycine in the presence of GGT in the serum sample. The change in the absorptivity of p-nitroaniline at 405 nm under specified conditions is proportional to the GGT activity in the sample.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set (Cat. No. 2700) provides:

GGT REAGENT - (Cat. No. 2701)

REACTIVE INGREDIENTS:

After reconstitution, L-y-Glutamyl-p-nitroanilide - 4.2mM, Glycylglycine - 100 mM.  

RECONSTITUTION:

Reconstitute with specified amount of GGT Buffer as indicated on the vial label. Buffer should be at room temperature for best results. Mix gently to dissolve.

PRECAUTIONS:

Do not ingest. Toxicity has not been established.

STORAGE AND STABILITY:

Store at 2 - 8ºC. Stable until the expiration date if sealed tightly. After reconstitution, the reagent is stable for 21 days stored at 2 - 8ºC. Prolonged exposure to room temperature will shorten the usability of reagent.

DETERIORATION:

The reagent should be a dry powder. Caking would indicate that moisture has entered the vial and the reagent should not be used. After reconstitution, the reagent should not be used if it has an absorbance 0.85 at 405 nm when read D-H2O. Failure to achieve assayed values of freshly prepared control sera would also indicate deterioration.

GGT BUFFER - (Cat. No. 2702)

REACTIVE INGREDIENTS:

2-amino-2-Methyl-1, 3-Propanediol - 120 mM; Surfactant and 0.1% Sodium Azide added.

PRECAUTIONS:

Buffer contains Sodium Azide. Dispose with large amount of water to prevent azide reacting with plumbing. Avoid contact with skin, eyes and clothing. In case of contact, wash with large amount of water.

STORAGE AND STABILITY:

The buffer may be stored at room temperature (15 - 30ºC) or refrigerated (2 - 8ºC). If stored 2 - 8º C, allow to warm to room temperature before using to reconstitute the GGT REAGENT. Stable until expiration date if protected from contamination and evaporation.

DETERIORATION:

Failure to reconstitute the GGT REAGENT would indicate deterioration. After reconstitution of the GGT REAGENT, failure to achieve assayed values of freshly prepared control sera could indicate deterioration of this buffer.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 405 nm that is capable of maintaining temperature at 30ºC or 37ºC.

SPECIMEN COLLECTION

PRECAUTION:

1. Serum free from hemolysis is preferred. GGT activity is inhibited by citrate, oxalate

and flouride anticoagulants used in blood collection tubes.

2. EDTA plasma up to 1 mg/mL blood may be used (1).

3. Heparin produces turbidity in the reaction mixture.

SAMPLE STORAGE:

Serum GGT activity appears stable for at least a week stored 2 - 8º C and for two months frozen and protected from evaporation.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

1. Most anticoagulants (except EDTA) interfere with assay.

2. Anti-epileptic drugs such as phenytoin (Dilantin) and barbituates may show falsely elevated GGT levels (2).

3. Young, et al (6) and Tietz (7) have comprehensive reviews of drug effects of serum GGT levels.

PROCEDURE

MATERIALS PROVIDED:

GAMMA GLYTAMYL TRANSFERASE (GGT) REAGENT (Cat. No. 2701) and GGT BUFFER (Cat. No. 2702).

MATERIALS NEEDED BUT NOT PROVIDED:

1. 0.05 mL micropipettor

2. 1.0 mL pipet or dispenser

3. Timer, test tubes and rack

4. Incubator capable of maintaining 30ºC or 37ºC

5. Instrument capable of reading at 405 nm

REACTION CONDITIONS:

Wavelength 405 nm

Reaction Tpye                 Kinetic

Prewarm Substrate          5 minutes

Preincubation Time          3 0 seconds

Incubation Time          1  minut

Sample Volume                0.05 mL

Reagent Volume              1.0 mL

Total Volume                     1.050 mL

Calculation Factor             2121

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

1. Pipet 1.0 mL of reconstituted reagent in tubes labeled Control, Sample 1, etc. Prewarm for five minutes at 37ºC.

2. Adjust instrument to zero absorbance at 405 nm using D-H2O.

3. Add 0.05 Specimen into appropriate tube, mix and incubate at 37ºC for 30 seconds.

4. After the 30 seconds preincubation, record the absorbance (A1). Return the tube to the 37ºC incubator.

5. After exactly one minute, read and record the abosrbance (A2). It is recommended that a second one minute reading be taken for better accuracy. Average the two readings to obtain change per minute.

NOTE: If instrument is equipped with a temperature controlled cuvette, the reaction mixture may be left in the cuvette while the absorbance readings are taken.

STABILITY OF FINAL REACTION:

The reaction is ongoing, the timing of the readings must be accurate.

One International Unit (IU/L) is defined as the amount of enzyme that catalyzes the transformation of one micromole of substrate per minute.

IU/L    =   (A1 - A2) x 1.050 x 1000 or (A2 - A1) x 2121

                                1 x 9,900 x 0.05

WHERE:

(A1 - A2) = Change in absorbance

1.050 = Total reaction volume

1000 = Conversion of IU/mL to IU/L

1 = Light pathincm

9,990 = Molar absorptivity of p-nitroaniline

0.05 = Sample volume

EXAMPLE:

The initial reading (A1) abs. = 0.350, the second reading (A2) abs. = 0.400, the (A1 - A2)

= 0.150, then 0.150 X 2121 (factor) = 318 IU/L.Conversion to SI Units (nkat/L): IU/L x 16.67=SI

CALIBRATION:

The reaction is standardized by means of the molar absorptivity of p-Nitroaniline taken as 9.90 x 103 at 405 nm under the test conditions outlined. Results are based on the change in absorbance per minute. All parameters must be known and controlled.

QUALITY CONTROL:

The realiability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner as the Unknowns. EAGLE DIAGNOSTICS makes available CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera should be thoroughly investigated before patient values are reported.

LIMITATIONS

Specimens with values greater than 600 IU/L should be diluted 1:1 with 0.9% saline and reassayed multiplying the result by two. This procedure is designed to measure g-GT in serum regardless of its source.

EXPECTED VALUES:

Male:             9  -  54 IU/L       at     37ºC

                        18 - 37IU/L        at     37ºC

Female:            8  -   35 IU/L      at 37ºC

6 -      24 IU/L     at    30ºC

This range represents the 95% confidence interval from a clinically normal population. Each laboratory should establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear to 600 IU/L.

PRECISION:

Within Run - Normal and abnormal control sera were assayed 20 times each for within run precision.

MEAN / ST. DEV. / %CV

Normal 23 / 0.7 / 3.0

Abnormal 108 / 1.5 / 1.4

Run to Run - Normal and abnormal control sera were assayed for 10 working days to estalbish run to run precision.

Normal 23 / 0.8 / 3.5

Abnormal 108 / 2.0 / 1.8

SPECIFICITY:

A comparison of this procedure with another widely used commercial method showed a 99% correlation with a regression equation of y = 1.04x - 2.7.

SENSITIVITY:

This procedure has a sensitivity of 2.1 IU/L per 0.001 absorbance unit.

REFERENCES

1. Tiez, N.W., Fundamentals of Clinical Chemistry, W.B. Saunders Co., Philadelphia, 1986, p. 723.

2. Tiez, N.W., Clinical Guide to Laboratory Tests, 2nd ed., W.B. Saunders Co., Philadelphia, 1990, p. 262.

2. Henry, R.J., et al., Am. Journ. Clin. Path., Vol. 34, p. 381 (1960).

3. Orlowski, M., Meister, A., Biochem Biophys. Acta 73, p. 679 (1963).

4. Kulhanek, V., Dimov, D.M., Clin. Chem. Acta 14, p.619 (1966).

5. Szasz, G., Clin. Chem. 15, p. 124 (1969).

6. Young, D.S., Pestaner, L.C. and Gibberman, V., Clin. Chem., Vol. 21, p. 272D (1975).

7. Tiez, N.W., Clinical Guide to Laboratory Tests, 2nd ed., W.B. Saunders Co., Philadelphia, 1990, p. 605