Alkaline phosphatase

Introduction

1. Alkaline phosphatase (ALP) is an enzyme that catalyzes the hydrolysis of phosphate esters at an alkaline pH, releasing inorganic phosphate.
2. It is present in various tissues, including the liver, bones, intestines, kidneys, and placenta.
3. Clinically, measuring ALP levels in serum is an important diagnostic marker for liver function, bone metabolism, and certain diseases such as hepatobiliary obstruction and bone disorders.
4. Increased or decreased ALP levels provide insight into underlying pathological conditions.

---

Principle

The ALP assay is based on the hydrolysis of a phosphate ester substrate by the enzyme at an alkaline pH. This reaction releases an inorganic phosphate and a chromogenic product that can be measured spectrophotometrically. A commonly used substrate is p-nitrophenyl phosphate (pNPP), hydrolyzed by ALP to produce p-nitrophenol (yellow color). The reaction rate is directly proportional to the ALP activity in the sample and is quantified by measuring the absorbance at 405 nm.

The chemical reaction is as follows: pNPP+H2O→ ^ALP →p-nitrophenol+inorganic phosphate

---

Methods

There are two main methods for determining ALP activity:

1. Kinetic Method: Measures the change in absorbance at regular intervals over time, reflecting the enzyme activity.
2. End-Point Method: Measures absorbance after the reaction has reached completion.

The kinetic method is preferred for its accuracy and reliability in determining enzyme activity.

---

Requirements

Equipment

1. Spectrophotometer (capable of reading at 405 nm)
2. Test tubes or microplate reader
3. Water bath or incubator (maintained at 37°C)
4. Timer or stopwatch

Other Materials

1. Distilled water
2. Pipettes and micropipette tips
3. Clean and dry glassware

---

Reagents

1. Buffer: Alkaline buffer solution (e.g., glycine buffer, pH 9–10).
2. Substrate: p-nitrophenyl phosphate (pNPP).
3. Standard: A known concentration of p-nitrophenol for calibration.
4. Stop Solution (optional): Sodium hydroxide (NaOH) or sodium carbonate to stop the reaction in the end-point method.
5. Control serum: For quality control and ensuring accuracy of results.

---

Procedure

1. Preparation of Reagents

• Prepare the buffer solution (e.g., glycine buffer, pH 10).
• Dissolve p-nitrophenyl phosphate (pNPP) in the buffer to prepare the substrate solution.
• Pre-warm all reagents to 37°C before starting the assay.

2. Sample Preparation

• Use fresh, non-hemolyzed serum or plasma samples.
• Dilute the sample if required, based on the expected ALP activity.

3. Reaction Setup

• Label test tubes as Blank, Standard, and Test.
• Pipette the following into the tubes:
  • Test Tube: 1 mL substrate solution + 0.1 mL serum sample.
  • Standard Tube: 1 mL substrate solution + 0.1 mL p-nitrophenol standard solution.
  • Blank Tube: 1 mL substrate solution + 0.1 mL distilled water.
• Mix thoroughly.

4. Incubation

• Incubate all tubes at 37°C for 10–15 minutes.

5. Stopping the Reaction (End-Point Method Only)

• Add 2 mL of stop solution (e.g., NaOH) to terminate the reaction.

6. Measurement

• Measure the absorbance of each tube at 405 nm using a spectrophotometer.

7. Calculation

• Determine the enzyme activity (U/L) using the formula:

ALP Activity (U/L) = ΔA×Vt×1000 / ε×d×Vs×T​

Where:

• ΔA: Change in absorbance per minute
• Vt​: Total reaction volume (mL)
• ϵ: Molar absorptivity of p-nitrophenol (18.75 mmol/L.cm at 405 nm)
• $d$: Lightpath (cm)
• Vs​: Sample volume (mL)
• $T$: Time of incubation (minutes)

---

Results

• Normal Reference Range for Serum ALP:
  • Adults: 44–147 U/L
  • Children: Higher values are normal due to bone growth.
• Interpretation of Results:
  • Increased ALP:
    • Bone diseases: Paget’s disease, osteomalacia, rickets, bone metastasis.
    • Hepatobiliary conditions: Biliary obstruction, cholestasis, liver metastasis.
    • Pregnancy: Placental ALP is elevated in the third trimester.
  • Decreased ALP:
    • Hypophosphatasia (a rare genetic condition).
    • Malnutrition or zinc deficiency.

---

Clinical Aspects

1. Diagnostic Significance:
   • ALP is used to evaluate bone and liver function. Elevated levels are common in conditions like cholestasis, bone metastases, or fractures.
   • High ALP levels in children or pregnant women are physiological and should not be considered abnormal.
2. Bone Disorders:
   • Elevated ALP reflects increased osteoblastic activity, commonly seen in Paget’s disease, osteomalacia, and bone fractures.
3. Liver Disorders:
   • ALP increases due to hepatobiliary obstruction, primary biliary cirrhosis, or metastatic liver disease.
4. Differential Diagnosis:
   • Isoenzyme analysis (e.g., electrophoresis) or concurrent measurement of gamma-glutamyl transferase (GGT) is recommended to distinguish between bone and liver sources of elevated ALP. Elevated GGT suggests a hepatic origin.
5. Follow-Up Testing:
   • Further imaging (e.g., ultrasound, X-ray) or biopsy may be required for definitive diagnosis based on ALP results.