Folin-Wu Method For Estimation of Blood Glucose

The Folin-Wu method is a colorimetric assay developed in 1919 by Otto Folin and Hsien Wu to quantify blood glucose. It involves:

• Deproteinization: Removing proteins using sodium tungstate and sulfuric acid.
• Reduction: Glucose converts alkaline copper tartrate to cuprous oxide, reacting with phosphomolybdic acid to produce a measurable blue color.

This method was pivotal in early clinical biochemistry but has been largely replaced by enzymatic assays.

Principle of Folin-Wu Method

The principle of Folin-Wu method involves a two-step biochemical reaction to estimate blood glucose:

1. Deproteinization: Proteins in blood are precipitated using 10% sodium tungstate and 2/3N sulfuric acid, creating a protein-free filtrate.
2. Reduction & Color Development:
   • Step 1: Heating the filtrate in an alkaline medium converts glucose to enediol, which reduces cupric ions (Cu²⁺) to cuprous oxide (Cu₂O).
   • Step 2: Cuprous oxide reacts with phosphomolybdic acid, forming phosphomolybdenum blue (intense blue color).
   • Measurement: The blue color’s intensity, measured at 680 nm, correlates with glucose concentration.

Role of Sodium Tungstate in Folin-Wu Method

Sodium tungstate acts as a precipitating agent. Combined with sulfuric acid, it denatures blood proteins, ensuring they don’t interfere with glucose measurement. This step is critical for achieving a clear, analyzable filtrate.

Features

• Folin-Wu Tubes: Constricted design prevents atmospheric oxygen from reoxidizing cuprous oxide, ensuring accuracy.
• Non-Specificity: Reacts with other reducing sugars (e.g., fructose), yielding slightly higher glucose values than true levels.

What is Folin-Wu Tube?

A Folin-Wu tube is a specialized glass tube designed for the Folin-Wu method, a historic biochemical technique for blood sugar analysis. Its primary uses include:

• Preventing Reoxidation: The tube’s constricted design minimizes contact with atmospheric oxygen, avoiding reoxidation of cuprous oxide (a critical intermediate in glucose estimation).
• Ensuring Precision: Equipped with calibrated markings, it ensures accurate measurement of reagents and filtrates during the glucose reduction process.

Specifications

• Durability: Made from heat-resistant glass to withstand boiling during the reaction.
• Measurement: Final glucose levels are determined by measuring the blue color intensity at 680 nm, proportional to blood sugar concentration.

Requirements

1. Essential Equipment

• Folin-Wu Tubes: Constricted design to prevent reoxidation of cuprous oxide by atmospheric oxygen.
• Colorimeter/Spectrophotometer: Measures absorbance at 680 nm for quantifying phosphomolybdenum blue.
• Centrifuge or Filter Setup: Whatman No. 1 filter paper for obtaining protein-free filtrate.

2. Reagents & Preparation

A. Deproteinization Reagents

1. 2/3N Sulfuric Acid (H₂SO₄): Mix 2 mL concentrated H₂SO₄ with 50 mL distilled water (D/W), then dilute to 100 mL.
2. 10% Sodium Tungstate: Dissolve 10 g sodium tungstate in 100 mL D/W.

B. Reduction & Color Development Reagents

1. Alkaline Copper Tartrate:
   • Solution A: Dissolve 40 g sodium carbonate + 7.5 g tartaric acid in 400 mL D/W.
   • Solution B: Dissolve 4.5 g copper sulfate in 100 mL D/W.
   • Mix A + B and dilute to 1,000 mL with D/W.
2. Phosphomolybdic Acid:
   • Dissolve 35 g molybdic acid + 5 g sodium tungstate in 200 mL 10% NaOH.
   • Boil for 45 minutes to remove ammonia. Add 125 mL 89% phosphoric acid, then dilute to 500 mL with D/W.

C. Standards & Controls

• Stock Glucose Standard (1 g/dL): Dissolve 1 g glucose in 100 mL 0.3% benzoic acid.
• Working Standard (10 mg/dL): Dilute stock 1:100 with benzoic acid.

3. Critical Requirements for Accuracy

• Protein-Free Filtrate: Ensures no interference from blood proteins.
• Standardized Heating: Boiling water bath for 10 minutes to fully reduce cupric ions.
• Calibration: Use a Blank (distilled water) and Standard (glucose solution) to validate results.

Procedure

Step 1: Protein-Free Filtrate Preparation

1. Sample Dilution: Mix 1 mL of whole blood with 7 mL distilled water to lyse cells.
2. Precipitation:
   • Add 1 mL of 10% sodium tungstate.
   • Slowly add 1 mL of 2/3N sulfuric acid (H₂SO₄) while mixing.
3. Filtration: Let the mixture stand for 5 minutes, then centrifuge or filter using Whatman No. 1 filter paper to obtain a clear, protein-free filtrate.

Step 2: Glucose Reduction & Color Development

4. Test Setup: Prepare three Folin-Wu tubes (constricted to prevent oxygen interference):

   Reagent	Blank	Standard	Test
   Distilled Water	1 mL	–	–
   Glucose Standard	–	1 mL	–
   Protein-Free Filtrate	–	–	1 mL
   Alkaline Copper Tartrate	1 mL	1 mL	1 mL

5. Heating: Place tubes in a boiling water bath for 10 minutes to reduce cupric ions (Cu²⁺) to cuprous oxide (Cu₂O).
6. Color Reaction:
   • Cool tubes, then add 1 mL phosphomolybdic acid to each.
   • Shake to eliminate bubbles and dilute to 12.5 mL with distilled water.
7. Measurement: Read absorbance at 680 nm using a spectrophotometer. Calibrate using the Blank tube.

Calculation

• Blood Sample: A 1:10 dilution (1 mL blood + 9 mL diluent) is used. The test employs 1 mL of diluted sample, representing 0.1 mL of actual blood.
• Glucose Standard: 1 mL of working standard (10 mg/dL) contains 0.1 mg glucose, ensuring precise calibration.

Interpreting Folin-Wu Method Results

Results are calculated using a standard calibration curve. Normal fasting blood glucose ranges between 70–110 mg/dL. Values outside this range may indicate hypoglycemia or diabetes. Fasting levels are categorized as:

• Normal: 70–110 mg/dL
• Prediabetes: 111–125 mg/dL
• Diabetes: ≥126 mg/dL

Folin-Wu Method vs. Modern Techniques

While the Folin-Wu method laid the groundwork for glucose estimation, newer methods like glucose oxidase and hexokinase assays offer faster, automated solutions. However, Folin-Wu’s role in historical and educational contexts remains significant.

Folin-Wu Method Advantages and Disadvantages

Advantages

• Specificity: Minimizes interference from non-glucose substances.
• Reliability: Proven consistency in clinical laboratories.
• Cost-Effective: Uses readily available reagents.

Disadvantages

• Time-Consuming: Requires multiple steps and heating.
• Toxic Reagents: Sodium tungstate and sulfuric acid demand careful handling.
• Outdated: Modern methods like glucose oxidase are faster.