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Estimation of SGOT (AST) in Serum

1. Aim

To study the kinetic estimation of serum aspartate aminotransferase, traditionally called SGOT, and calculate the absorbance-change rate from a timed series. This is practical 11 in the PCI Medicinal Biochemistry list. Enzyme activity in U/L requires the assay kit's verified factor. [1, 2]

2. Requirements

Use an instructor-supplied serum control or deidentified specimen, an approved AST kit, calibrated micropipettes, cuvette, timer, temperature-controlled bath or analyzer, UV spectrophotometer, gloves, coat, eye protection, and biological-waste containers. The college manual calls its reagents I and II without identifying their full composition or conversion factor. Current kit identity, volumes, temperature, wavelength, blank, time points, linearity limits, and quality-control ranges must follow your college lab manual and instructor's method sheet from its instructions. [2]

3. Theory

AST transfers an amino group from aspartate to an acceptor, producing oxaloacetate. In a common coupled kinetic assay, oxaloacetate is converted to malate while NADH is consumed. NADH absorbs at about 340 nm, so a fall in absorbance over time can be related to AST activity. The manual identifies the 340 nm kinetic approach but does not give a complete kit-specific conversion factor. A falling trace alone cannot be reported as U/L. [2]

AST occurs in liver and other tissues, including muscle. A result therefore cannot diagnose a particular organ condition in isolation. Hemolysis, specimen handling, assay timing, instrument calibration, and kit-specific blanking may affect the measurement. The article teaches how to record a kinetic trace and check it for a reasonably linear interval. Patient interpretation belongs to a validated clinical laboratory and clinician. [2, 3]

4. Procedure

  1. Confirm specimen identity, condition, collection time, and biosafety controls. Use a control or deidentified material; no student blood collection is implied. [2]
  2. Read the exact AST kit instructions. Record reagent lot, expiry, temperature, wavelengths, sample and reagent volumes, blank, and U/L factor. Mark unavailable details Record here. [2]
  3. Set the instrument and temperature control using the approved method. Check that the reagent blank and quality controls pass before reading a test. [2]
  4. Under supervision, prepare the reaction using the verified kit sequence. Start timing at the method-defined event, since an uncertain start changes the slope. [2]
  5. Record absorbance at the scheduled times, preserving raw values and units. The college manual describes readings over a short 340 nm series. [2]
  6. Inspect the trace for an acceptable direction and linear interval. Calculate the mean change in absorbance per minute over the valid interval. Do not force a rate from an irregular trace. [2]
  7. Apply the kit's verified U/L factor only after controls and linearity pass. Record all deviations and submit the result for instructor review. [2, 3]

5. Calculation, graph, or images as appropriate

Time after method-defined start Absorbance at 340 nm Quality note
First scheduled reading Record here Timing Record here
Second scheduled reading Record here Direction Record here
Third scheduled reading Record here Linearity Record here
Reagent blank and control Record here Acceptance record from the assigned current monograph, approved method, or supervised case

Worked time calculation: The manual describes first, second, and third minute readings. The elapsed interval between the first and third readings is 3 minus 1 = 2 minutes. If their measured absorbances are A1 and A3, the average fall is (A1 minus A3) divided by 2 absorbance units per minute. The individual readings and the kit conversion factor must follow your college lab manual and instructor's method sheet, so no U/L value is invented. [2]

SGOT AST kinetic observation flow

6. Result

Report the valid absorbance trace and calculated rate if readings are supplied. Report AST activity in U/L only with a verified kit factor and passing quality controls. A missing factor or failed trace means the activity cannot be reported; record the missing information here. Clinical meaning requires qualified review; this page does not interpret the result. [2, 3]

7. References

  1. Pharmacy Council of India. Pharm.D. Regulations, 2008, Medicinal Biochemistry practical list.
  2. East Point College of Pharmacy. Medicinal Biochemistry Practical Manual, SGOT experiment.
  3. National Library of Medicine, MedlinePlus. AST Test.
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Theory notes: Medicinal Biochemistry