Folin-Wu Method For Estimation of Blood Glucose
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Biochemistry

Folin-Wu Method For Estimation of Blood Glucose

Learn the Folin-Wu method for blood glucose estimation: step-by-step procedure, sodium tungstate role, spectrophotometric measurement (680 nm), advantages/disadvantages, and modern comparisons. Essential for clinical labs and history.

By Dayyal Dg.
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Blood Sample for Glucose Estimation by Folin-Wu Method
Blood sample for the estimation of blood glucose level by Folin-Wu method. Freepik / @freepik

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.
Folin-Wu Tube Diagram
Folin-Wu tube used for the estimation of blood glucose level.

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

  1. Test Setup: Prepare three Folin-Wu tubes (constricted to prevent oxygen interference):
    ReagentBlankStandardTest
    Distilled Water 1 mL
    Glucose Standard 1 mL
    Protein-Free Filtrate 1 mL
    Alkaline Copper Tartrate 1 mL 1 mL 1 mL
  2. Heating: Place tubes in a boiling water bath for 10 minutes to reduce cupric ions (Cu²⁺) to cuprous oxide (Cu₂O).
  3. 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.
  4. Measurement: Read absorbance at 680 nm using a spectrophotometer. Calibrate using the Blank tube.

Calculation

\[\text{Glucose (mg/dl)} = \left( \frac{\text{OD of Test}}{\text{OD of Standard}} \right) \times \text{Conc. of Standard} \times \left( \frac{100}{\text{Amount of Sample Taken}} \right)\] \[\text{Glucose (mg/dl)} = \left( \frac{\text{OD of Test}}{\text{OD of Standard}} \right) \times 0.1 \times \left( \frac{100}{0.1} \right)\] \[\text{Glucose (mg/dl)} = \left( \frac{\text{OD of Test}}{\text{OD of Standard}} \right) \times 100\]
  • 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.

FAQs

  1. What is the reagent used in Folin-Wu method?

    The primary reagents include sodium tungstate, sulfuric acid, alkaline copper tartrate, and Folin-Ciocalteu reagent.

  2. Why is sodium tungstate used in Folin-Wu method?

    It precipitates proteins to create a clear filtrate, ensuring accurate glucose measurement.

  3. Is the Folin-Wu method still used today?

    While largely replaced by enzymatic methods, it’s still taught for its historical and educational value.

Last Updated:

Reference(s)

  1. J., Ochei., et al. “Medical Laboratory Science: Theory and Practice.”, Tata McGraw-Hill, 2008, isbn: 9780074632239.
  2. Naigaonkar, A. V.. “A Manual Of Medical Laboratory Technology.” 9th ed., Nirali Prakashan, 7 July 2008, isbn: 9788185790299.
  3. Damodaran K, Geetha. “Practical Biochemistry.” 2nd ed., Jaypee Brothers Medical Publishers, 15 June 2010, isbn: 9789351529941.

Cite this page:

Dayyal Dg.. “Folin-Wu Method For Estimation of Blood Glucose.” BioScience. BioScience ISSN 2521-5760, 14 February 2025. <https://www.bioscience.com.pk/en/topics/biochemistry/folin-wu-method-for-estimation-of-blood-glucose>. Dayyal Dg.. (2025, February 14). “Folin-Wu Method For Estimation of Blood Glucose.” BioScience. ISSN 2521-5760. Retrieved February 20, 2025 from https://www.bioscience.com.pk/en/topics/biochemistry/folin-wu-method-for-estimation-of-blood-glucose Dayyal Dg.. “Folin-Wu Method For Estimation of Blood Glucose.” BioScience. ISSN 2521-5760. https://www.bioscience.com.pk/en/topics/biochemistry/folin-wu-method-for-estimation-of-blood-glucose (accessed February 20, 2025).
  • Posted by Dayyal Dg.

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