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CC2-LAB

This document discusses gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) determination methods. It describes GGT as a membrane-bound enzyme involved in amino acid transport and glutathione metabolism. The Szasz-Rosalki kinetic method is recommended for GGT determination using L-gamma-glutamyl-p-nitroanilide as a substrate. ALP exists in alkaline and acid forms, with the alkaline form elevated in hepatobiliary and bone diseases. The Bowers-McComb kinetic method is recommended for ALP determination.

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14 views

CC2-LAB

This document discusses gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) determination methods. It describes GGT as a membrane-bound enzyme involved in amino acid transport and glutathione metabolism. The Szasz-Rosalki kinetic method is recommended for GGT determination using L-gamma-glutamyl-p-nitroanilide as a substrate. ALP exists in alkaline and acid forms, with the alkaline form elevated in hepatobiliary and bone diseases. The Bowers-McComb kinetic method is recommended for ALP determination.

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COC UNIVERSAL
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GAMMA-GLUTAMYL TRANSFERASE (GGT) DETERMINATION

Szasz and Rosalki - Kinetic Method


GAMMA-GLUTAMYL TRANSFERASE - first identified in the kidney tissue.

• One of a large group of enzymes, “PEPTIDASES”


• Originally termed as “TRANSPEPTIDASE”
➢ Changed to Transferase
PEPTIDASE – an enzyme that catalyzes the hydrolytic cleavage of peptides.
• Forms AMINO ACIDS or SMALLER PEPTIDES
• Membrane-bound enzyme whose active site faces the external side of the cell
• HEPATOBILIARY TRACT ENZYME
RENAL TISSUE or the KIDNEY = highest level of GGT

• but appears to originate primarily from the HEPATOBILIARY SYSTEM


• GGT is elevated in many forms of liver disease.
• Relatively high activity in the canalicular portion of
o HEPATOCYTE
o PANCREAS ACINAR
o PROSTATE
o BILE DUCT

MOST ELEVATED MODERATELY ELEVATED ELEVATED


(5-30 x normal) (2-5 x normal)
• intra- or post-hepatic • infectious hepatitis • Primary and secondary
biliary obstruction tumors
= less useful diagnostically than
transaminase determinations
with this condition.
FUNCTIONS:
1. Involved in the transfer of AA across the cell membrane.
2. Resorption of AA from glomerular filtrate and from the Intestinal Lumen
3. In GLUTATHIONE metabolism, transfer the GLUTAMYL MOIETY to various ACCEPTOR molecules.
➢ Glutathione was used for accessing gamma-GT activity in the early times.

PRINCIPLE:
Gamma-glutamyl transferase catalyzes the transfer of the gamma-glutamyl group (at pH 8.2) from the donor substrate
(L-gamma-glutamyl-p- nitroanilide) to the glycylglycine acceptor to yield p- nitroaniline (yellow compound).
INCREASE ABSORBANCE = INCREASE ACTIVITY (directly proportional)
REAGENT COMPOSITION: pH= 8.2 +/- 0.1
1. Gamma-GT Reagent
2. Gamma-glutamyl-p-nitroanilide = 4.79 mM
3. Glycylglycine = 100 mM
4. Tris = 180 mM
SAMPLE AND ASSAY CONSIDERATIONS:
Non-hemolyzed serum = preferred specimen (EDTA plasma also used)
Heparin may produce turbidity in the reaction mixture.
Citrate, oxalate, and fluoride = depress by 10-15 %
Hemoglobin (100-500 mg/dl) = minimal depression by 5-7%
Serum GGT = stable at least 8 hours (room temperature)
= 1 month (4 degrees Celsius)
= 1 year (-20 degrees Celsius)
Drugs: Barbiturate, phenytoin, other anticonvulsant drugs = FALSELY ELEVATED GGT levels
Bilirubin (20 mg/dl) = negligible interference <5% in Szasz-Rosalki method
LINEARITY: 600 IU/L SENSITIVITY: 0.02 mg/dl EXPECTED VALUES: Male= 0-45 IU/L
FEMALE= 0-30 IU/L

METHODS FOR GGT ACTIVITY DETERMINATION

Szasz-Rosalki Persijn and Van der Slik method Goldberg Method


*IFFC Recommended Method*
(*REFERENCE METHOD)
Kinetic Method (*used in lab act) Chromogenic Method
or Endpoint method
L-gamma-glutamyl-3-carboxy-4- Substrate = highly soluble Measures amount = beta
nitroanilide produces: • This method can easily be naphthylamide liberated
• More stable reagent adapted to any equipment
• More water soluble for automated assays
Substrate = L-gamma-glutamyl-p- Activities (measured optimum Substrate = L-gamma-glutamyl- beta
nitroanilide substrate concentration) under 37 naphthylamide
degrees Celsius
Product: 5-amino-2- nitrobenzoate -
Absorbance = 405 nm = slightly higher than with the Szasz
method under 30 degrees Celsius
INCREASE ABSORBANCE (substrate=L-glutamyl-p-nitroanilide)
= INCREASE ACTIVITY

Glycylglycine = introduced as an activator

• Szasz adapted this activator


▪ GGPNA to 30 degrees Celsius (kinetic photometric method)
• Rosalki adapted optimal concentrations of the activator
▪ GGPNA substrate to 37 degrees Celsius (colorimetric endpoint method)
• Glycylglycine = 5 times more effective as an acceptor that is either (GLYCINE or the TRIPEPTIDE)
• Rate of Peptidase transfer reaction = Faster than simple hydrolysis reaction
ALKALINE PHOSPHATASE (ALP) DETERMINATION
Bowers-McComb - Kinetic Method: AACC Recommendation

Phosphomonoesterases or Orthophosphoric Ester Monohydrolases


= The phosphatase of the blood
TWO TYPES: Differ in pH
1. Alkaline Phosphatase = 9.0
2. Acid Phosphatase
Serum alkaline phosphatase estimations

• diagnosis of hepatobiliary disease and bone disease with increased osteoblastic activity.

HIGHEST ELEVATION ELEVATION


(bone disorders)
• Paget’s disease •
• Osteomalacia
• Rickets
• Osteogenic Sarcoma

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