Introduction
Alkaline Phosphatase (ALP) is an important enzyme present in many tissues of the human body. It belongs to the group of enzymes that hydrolyze phosphate esters at an alkaline pH. Measurement of ALP activity in serum or plasma is widely used in clinical laboratories to assess liver function, bone metabolism, and certain metabolic disorders.
The ALP test is commonly included in routine biochemical investigations such as liver function tests (LFTs). Abnormal levels of ALP help clinicians identify diseases related to the liver, bones, and other tissues.
Important points about ALP include:
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ALP is present in liver, bone, placenta, kidney, intestine, and mammary glands.
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In adults, most circulating ALP originates from the liver.
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In children and adolescents, bone ALP is higher due to active bone growth.
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Pregnancy increases ALP levels due to placental enzyme production.
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The enzyme works optimally in an alkaline environment.
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ALP estimation is useful for diagnosing liver and bone diseases.
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The test is performed using spectrophotometric methods.
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It is an important parameter in clinical biochemistry laboratories.
Principle
The ALP test is based on an enzymatic reaction where p-nitrophenyl phosphate (pNPP) acts as the substrate.
Reaction Principle
Alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate into p-nitrophenol and phosphate in an alkaline medium.
Reaction:
p-Nitrophenyl phosphate + H₂O → p-Nitrophenol + Phosphate
Key points of the reaction:
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The reaction takes place in an alkaline buffer environment.
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Magnesium and zinc ions act as enzyme activators.
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The product p-nitrophenol forms a yellow color in alkaline medium.
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The intensity of yellow color increases with reaction progress.
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Absorbance is measured at 405 nm or 415 nm.
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The rate of increase in absorbance is proportional to ALP activity in the sample.
Sample
The ALP test can be performed using the following biological samples.
| Sample Type | Description |
|---|---|
| Serum | Most commonly used sample |
| Plasma | Heparinized or EDTA plasma may also be used |
Sample Stability
| Storage Condition | Stability |
|---|---|
| Room temperature (20–25°C) | 4 hours |
| Refrigerated (4–8°C) | 3 days |
| Frozen (-20°C) | 2 months |
Contaminated samples should be discarded.
Reagents
The reagent kit generally contains two main reagents.
| Reagent | Composition | Function |
|---|---|---|
| R1 | AMP buffer (pH 10.4), magnesium acetate, zinc sulfate, HEDTA | Maintains alkaline pH and activates enzyme |
| R2 | p-Nitrophenyl phosphate | Substrate for the enzymatic reaction |
Storage Conditions
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Reagents are usually ready to use.
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Store at 2–8°C.
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Avoid exposure to atmospheric carbon dioxide which may reduce reagent stability.
Procedure
Reaction Setup
| Reagent / Sample | Blank (B) | Standard / Calibrator (S) | Test / Sample (T) |
|---|---|---|---|
| Reagent R1 | 0.80 ml | 0.80 ml | 0.80 ml |
| Distilled Water | 0.02 ml | — | — |
| Standard / Calibrator | — | 0.02 ml | — |
| Serum Sample | — | — | 0.02 ml |
Mix and incubate at 37°C for 5 minutes.
Second Step
| Reagent | Blank (B) | Standard (S) | Test (T) |
|---|---|---|---|
| Reagent R2 | 0.20 ml | 0.20 ml | 0.20 ml |
Procedure
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Mix the reaction mixture.
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Incubate at 37°C for 1 minute.
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Measure absorbance of Standard and Test against Blank.
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Take readings at 1, 2, and 3 minutes.
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Calculate the change in absorbance per minute (ΔA/min).
Calculation
Formula
ALP Activity (U/L) = Absorbance of Test (T) / Absorbance of Standard (S) × Concentration of Standard
Most automated analyzers calculate ALP activity automatically.
Unit Conversion
| Unit | Conversion |
|---|---|
| U/L | Standard reporting unit |
| μkat/L | U/L × 0.017 |
Reference Values
Normal values vary according to age and sex.
| Age Group | Female (U/L) | Male (U/L) |
|---|---|---|
| Children / Adolescents | 54 – 369 | 54 – 369 |
| Adults (20–50 years) | 42 – 98 | 53 – 128 |
| Older adults (≥60 years) | 53 – 141 | 56 – 119 |
Each laboratory should establish its own reference ranges based on its population.
Clinical Significance
Measurement of ALP activity helps in diagnosing many diseases.
Increased ALP Levels
Elevated ALP levels may occur in the following conditions.
Liver Diseases
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Cholestasis
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Hepatitis
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Cirrhosis
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Biliary obstruction
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Liver tumors
Bone Diseases
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Rickets
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Osteomalacia
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Paget’s disease
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Osteogenic sarcoma
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Bone metastasis
Other Conditions
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Pregnancy
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Rapid bone growth in children
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Hyperparathyroidism with bone involvement
In liver disorders, ALP is often elevated along with Gamma-glutamyl transferase (GGT).
Decreased ALP Levels
Low ALP levels may be seen in:
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Hypophosphatasia
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Severe malnutrition
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Conditions causing reduced bone formation
Interfering Substances
Certain substances do not significantly affect the ALP test within the following limits.
| Substance | Acceptable Level |
|---|---|
| Hemoglobin | up to 5 g/L |
| Bilirubin | up to 40 mg/dL |
| Triglycerides | up to 2000 mg/dL |
Quality Control
To ensure accurate results, laboratories should follow proper quality control procedures:
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Calibration using standard calibrators
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Use of normal and pathological control sera
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Regular instrument maintenance
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Running internal quality control samples
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Participation in external quality assurance programs
