Name: ________________________________ Score:______________

Student no. ____________________________ Date: ______________

Acitivity 11
𝛄- GT Determination
(Kinetic Method)

Gamma-glutamyl transferase is one of the large group of enzymes known as peptides. Although
renal tissue has the highest level of γ-GT, the major source of the enzyme present in serum is hepatic
origin. Elevated levels of γ-GT are found in association with hepatobiliary and pancreatic disorders;
alcoholics and heavy drinkers, in myocardial disorders and in diabetics.

Unlike alkaline phosphatase activity, the serum γ-GT activity remains normal in disease affecting
bone and during normal bone growth. Therefore, a rise in serum GGT activity may be considered as a
sensitive and more specific indicator of liver disease than alkaline phosphatase activity.

The Teco γ-GT procedure has been optimized according to Szasz.

γ-GT
L - γ – glutamyl -3- carboxy -4- nitroanilide + glycylglycine
L - γ- glutamylglycylglycine + 5 – amino – 2 – nitrobenzoate

γ-GT catalyzes the transfer of a γ-Glutamyl group from L- γ-glutamyl-3-carboxy-4-nitroanilide.

The rate of liberation of 5-amino-2-nitrobenzoate is directly related to the γ-GT activity in the sample
and is quantitated by measuring the increase in absorbance at 405 nm.

REAGENTS
γ-GT Buffer (R1):
Tris, pH 8.25 100 mmol/L
Glycylglycine 100 mmol/L

γ-GT Substrate (R2):

l- γ-glutamyl-3-carboxy-4-nitroanilide 4.0 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 that 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 substrate liquid reagents are supplied ready-to-use. Prepare Working Reagent in the ration 5
parts Buffer (R1) to 1 part Substrate (R2), (i.e, 25mL Buffer 5 mL Substrate).

REAGENT STORAG AND STABILITY

Reagents are stable until the expiration date on their respective lables, when properly stoed at 2-8˚C
and protected from light. R1 should appear clear/colorless while R2 should appear clear/yellow. Discard
if either appears cloudy or contains particulate matter. The Working Reagent is stable for 4 weeks at 2-

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8˚C or 5 days at room temperature (15-25˚C). The Working Reagent should be discarded if the initial
absorbance, red against distilled water at 405 nm, is above 0.800.

MATERIAL REQUIRED BUT NOT PROVIDED

1. Spectrophotometer capable of absorbance reading at 405 nm and 1cm light path.

2. Constant temperature block/bath, 37˚C, or temperature controlled cuvette.
3. Accurate pipetting devices
4. Test tubes
5. Interval timer

SPECIMEN COLLECTION AND STORAGE

Serum or EDTA plasma, free of hemolysis, should be used. Complexing anticoagulants such as citrate,
oxalate, fluoride and must be avoided since the inhibit γ-GT activity. The loss of γ-GT activity is
minimized by storing the samples refrigerated for up to 7 days or frozen up to 2 months. Bilirubin levels
up to 40 mg/dL and triglyceride levels up to 2000 mg/dL show no interference in this test.

INTERFERING SUBSTANCES
γ-GT is an inducible enzyme. Consequently patients who are receiving antiepileptic drugs or
aminopyrine show elevated γ -GT activity. Chromic use of ethanol also increase serum γ -GT activity.
Certain drugs and other substances are also known to affect γ -GT values.

MANUAL PROCEDURE

1. Prepare γ-GT Working Reagent according to instructions.

2. Zero spectrophotometer at 405 nm with distilled water.
3. For each sample and control, add 1.0 mL Working Reagent to cuvette or test tube and warm to
37˚C for 3 minutes.
4. Add 100 µL (0.10 mL) serum to its respective tube and mix gently.
5. Read and record absorbance at 1 minute. Continue incubating at 37˚C and record absorbance
again at 2 and 3 minutes. Rate should be constant.
6. Determine the average absorbance per minute ( A/min), multiply by factor 1158 for results in
U/L.

NOTE: if cuvette is not temperature controlled, incubate samples at 37˚C between readings.

AUTOMATED PROCEDURE
Special adaptations for automated analyzers are available by contacting Teco’s Costumer Service
Department.

QUALITY CONTROL
It is recommended that controls be included in each set of assays commercially available control
material with established γ-GT values may be used for quality control. The assigned value of the control
material must be confirmed by this methodology. Failure obtain the proper range of values in the assay
of control material may indicate reagent deterioration, instrument malfunction or procedural errors.

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CALIBRATION
γ-GT activity is based on the “micromolar extinction coefficient” of 5 amino-2-nitrobenzoate at 405 nm
(see “Results” section). The instrument manufacturer/s calibration guidelines should be followed to
calibrate your analyzer.

RESULTS
Values are derived based on the “absorptivity micromoloar extinction coefficient” of 5-amino-2-
nitrobenzoate at 405 nm (0.0095). units per liter (U/L) γ-GT activity are the amount of enzyme that
produces one mmol/L of 5-amino-2-nitrobenzoate per minute.

∆A
U Min total volume
= x
L absorptivity sa mple volume

∆A
U Min
= x 1.10 /0.10
L 0.0095

U ∆A
= x 1158
L Min

LIMITATION
if the ∆A/Min. is greater than 0.259, dilute 1 part sample with 5 parts isotonic saline and re-assay.
Multiply results by 6.

EXPECTED VALUES
Normal Range: Males: 0 – 50 U/L (37°C)
Females: 0 - 30 U/L (37°C)

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 CHARACTRISTICS
Comparison: a group of 63 sera ranging in γ-GT activity from 15 – 714 U/L was assayed by the
described γ-GT reagent. Comparison of the results yielded a correlation coefficient of 1,000 and the
regression equation was y=0.94x + 1.9. (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 γ-GT (U/L) 40 74 206
Std. Deviation (U/L) 1.0 0.9 1.3
C.V. (%) 2.5 1.2 0.6

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 Total Precision
Serum 1 Serum 2 Serum 3
Mean γ-GT (U/L) 42 72 204
Std. Deviation (U/L) 0.6 0.6 0.7
C.V. (%) 1.5 0.9 0.4

Precision studies were performed according to NCCLS Tentative Guideline, EP5-T.

Linearity: Linear to 300 U/L at 37°C. Performed according to NCCLS Guideline EP6-P.

Sensitivity: based on an instrument resolution of A = 0.001, the method presented show a

sensitivity of 1.0 U/L.

Study Questions:

1. Provide methodology of analyses of GGT.

2. Provide specimen considerations in GGT determination.

3. Enumerate the clinical significance of GGT assay.

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