ALT AST ALP

Major source: liver (hepatic origin)
Used to study hepatic diseases: hepatitis, cirrhosis, obstructive jaundice
Slightly elevated after myocardial infarction
UV methods for ALT determination first developed by Wroblewski and LaDue (1956), modified by IFCC

Clinical Significance of Alanine Aminotransferase (ALT)

IFCC without pyridoxal phosphate (P-5’-P); kinetic UV method

ALT Measurement Method

ALT transfers amino group from alanine to 2-oxoglutarate → forms glutamate and pyruvate
Pyruvate is reduced to lactate by LDH, oxidizing NADH to NAD+
Rate of decrease in absorbance at 340 nm is proportional to ALT activity

ALT Measurement Principle

ALT Buffer (R1): Tris buffer (pH 7.5), L-alanine, LDH, Sodium azide
Enzyme (R2): α-ketoglutarate, NADH
Working reagent: 5 parts Buffer (R1) + 1 part Enzyme (R2)
Stable for 5 days (room temp) or 4 weeks (2-8°C)

ALT Reagents and Preparation

Serum, free from hemolysis
Stability:
- 3 days at room temperature
- 7 days at 2-8°C
- Better stability at -70°C

ALT Sample Requirements and Stability

Activity (U/L) = ΔA/min × 1746

ALT Calculation

3-35 U/L

ALT reference values

Hemolysis must be avoided (ALT in RBCs ~5x serum)
Gross icteric or turbid specimens may require sample blanks
Bilirubin up to 40 mg/dL and triglycerides up to 2000 mg/dL: no interference
Certain drugs can affect results

ALT Limitations and Interferences

Catalyzes amino/oxo group transfer between amino acids and oxo acids
Significant in heart and liver; lesser amounts in skeletal muscle, kidney, pancreas, spleen, lungs, brain
Elevated in myocardial infarction, hepatitis, liver necrosis, cirrhosis, liver metastasis

Clinical Significance of Aspartate Aminotransferase (AST)

Modified Karmen method (Bergmeyer), kinetic UV method

AST Measurement Method

AST catalyzes transfer from aspartate to 2-oxoglutarate → oxaloacetate + glutamate.
Oxaloacetate reacts with NADH (via MDH) → malate + NAD+.
NADH oxidation rate at 340 nm reflects AST activity.

AST Principle

AST Buffer (R1): Tris buffer (pH 7.5), L-alanine, LDH.
AST Enzyme (R2): NADH, 2-ketoglutarate.
Working reagent: 5 parts (R1) + 1 part (R2).
Working stability: 4 weeks (2-8°C) or 5 days (room temp).

AST Reagents and Preparation

Sample: Serum (no hemolysis); lithium heparinized or EDTA plasma acceptable.
Storage:
- 24 hrs at room temp,
- 7 days at 2-8°C,
- 3 months at -20°C.

AST Sample Requirements, Storage

Activity (U/L) = ΔA/min × 1746

AST formula

Reference Values (Serum/Plasma, 37°C): 8–33 U/L.
Higher in infants.
Hemolysis must be avoided (RBC AST ~10x serum).
Bilirubin (≤40 mg/dL) and triglycerides (≤2000 mg/dL) do not interfere.

AST Reference Values and Interferences

Maximum activity at alkaline pH.
Found in: liver, osteoblasts, intestine, kidneys, placenta.
Elevated in: bone diseases (Paget’s, osteogenic cancer), extra/intra-hepatic obstruction, hepatitis, cirrhosis, bone growth (children/teenagers), third trimester pregnancy.

Clinical Significance of Alkaline Phosphatase (ALP)

Based on DGKC and SCE methods.

ALP Measurement Method

Principle: ALP catalyzes p-nitrophenylphosphate + H₂O → inorganic phosphate + p-nitrophenol (yellow).
Measured at 405 nm.

ALP Principle

R1: Diethanolamine buffer (pH 10.2), Magnesium chloride.
R2: p-Nitrophenylphosphate.
Sample: Serum free from hemolysis.
Fresh specimens preferred (analyze within 4 hrs).

ALP Reagents and Sample Requirements

Storage:
- 1 week at room temp or 2-8°C,
- 2 months at -20°C.
Reference Values (37°C):
- Men: ≤270 U/L
- Women: ≤240 U/L
- Higher for children/teens during growth.

ALP Storage and Reference Values

(ΔA_sample/ΔA_standard) × standard concentration

ALP Calculation

No major interference from glucose (≤500 mg/dL), ascorbic acid (≤40 mg/dL), turbidity (≤600 mg/dL).
Hemoglobin (≥400 mg/dL) may cause negative bias in normal serum.
Rare cases: monoclonal gammopathies (e.g., Waldenstrom's macroglobulinemia) cause unreliable results.

ALP Limitations and Interferences