Estimation of blood glucose levels by the Folin–Wu method is a classical clinical biochemistry protocol used to measure reducing sugars (mainly glucose) in blood. You can download this protocol using the link provided below.
Blood glucose levels play a vital role in maintaining body homeostasis. Persistently high or low blood sugar can lead to serious health problems, including acute metabolic emergencies and long-term vascular complications.
Glucose is a monosaccharide found in fruits, vegetables, grains, legumes, and many other foods. In humans, glucose is the primary source of energy and is oxidized through cellular respiration to produce ATP. Measuring blood glucose levels helps detect hypoglycemia, hyperglycemia, and diabetes mellitus.
The Folin–Wu reaction is a colorimetric assay for reducing sugars based on their ability to reduce copper in an alkaline medium and subsequently reduce phosphomolybdic acid to form a blue‑colored complex. This method was developed by Otto Folin and H. Wu in the early 20th century.
Advantages of the Folin–Wu Method for Blood Glucose Estimation
Blood glucose estimation plays a central role in diabetes diagnosis and management, helping clinicians decide on insulin therapy and monitor glycemic control. Over time, several methods have been developed, including classical reduction methods (Folin–Wu, Benedict, Somogyi–Nelson) and modern enzymatic assays (glucose oxidase, hexokinase).
Diabetes mellitus is broadly classified into two major types.
- Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic β‑cells, resulting in little or no insulin production; patients require lifelong insulin therapy.
- Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and relative insulin deficiency; management includes lifestyle modification, oral hypoglycemic agents, and sometimes insulin.
Modern practice primarily uses HbA1c, fasting plasma glucose, and oral glucose tolerance tests to assess glycemic control and diagnose diabetes. However, the Folin–Wu method remains important for teaching laboratories and understanding the principles of older reduction‑based methods.
What is the Folin-Wu tube?
The Folin–Wu tube is a specially graduated glass tube used for quantitative blood sugar determination by the Folin–Wu method. The modification used in this protocol includes the following points:
- After color development with the phosphomolybdate–phosphotungstate sugar reagent, the unknown sample is diluted until its color intensity approximately matches that of the standard, rather than to a fixed volume before colorimetric comparison.
- The degree of dilution is included as a variable in the final formula for calculating the concentration of dextrose in the unknown sample.
Estimation of Blood Glucose Levels by the Folin-Wu method
Aim:
To estimate the glucose level in blood by the Folin–Wu method.
Principle:
When glucose or other reducing substances are treated with an alkaline copper solution, cupric ions are reduced to cuprous ions, forming insoluble cuprous oxide. The extent of this reaction depends on temperature, duration of heating, and degree of alkalinity. The cuprous oxide then reacts with phosphomolybdic acid to form a molybdenum blue–colored complex, which can be measured colorimetrically using a red filter at about 680 nm.
Reagents:
- Stock sugar solution (1%):
Dissolve 1 g of glucose in 100 mL of saturated benzoic acid solution. - Working standard glucose solution:
Prepare a working standard containing 2 mg of glucose per 2 mL from the above stock solution. - Alkaline copper reagent:
- Solution A: 2% sodium carbonate in 0.1 N sodium hydroxide. Solution B: 5% copper sulfate in 1% sodium potassium tartrate (Rochelle salt).
- Phosphomolybdic acid reagent:
To 35 g of molybdic acid, add 5 g of sodium tungstate, 200 mL of 10% sodium hydroxide, and 200 mL of water. Boil vigorously for 20–30 minutes to remove all ammonium present in molybdic acid. Cool, dilute to about 350 mL, add 125 mL of 85% orthophosphoric acid, and make up to 500 mL with distilled water. - 10% sodium tungstate solution.
- 2/3 N sulfuric acid (H₂SO₄).
Procedure:
Deproteinization of blood
- Transfer 1 mL of blood into a boiling tube containing 7 mL of distilled water.
- Add 1 mL of 10% sodium tungstate and mix well.
- Add 1 mL of 2/3 N sulfuric acid with shaking.
- Allow the mixture to stand for 10 minutes, then filter.
The clear filtrate, known as tungstic acid blood filtrate, is used as the test sample.
Color development
- Pipette 2 mL of tungstic acid blood filtrate into a Folin–Wu tube graduated at 25 mL.
- Into two similar Folin–Wu tubes, add 2 mL of standard glucose solution (standard) and 2 mL of distilled water (blank), respectively.
- Add 2 mL of alkaline copper reagent to each tube; the liquid level should be at the 4 mL mark.
- Place the Folin–Wu tubes in a boiling water bath for 8 minutes.
- Cool the tubes under running water.
- Add 2 mL of phosphomolybdic acid reagent to each tube and mix.
- After 1 minute, dilute each tube with distilled water up to the 25 mL mark.
- Transfer the contents to suitable cuvettes and read the optical density (OD) at 680 nm against the blank using a colorimeter or spectrophotometer.
Normal values:
- Normal fasting blood glucose ranges approximately from 70 to 110 mg/100 mL of blood in healthy adults.
- In mild diabetes, blood glucose values may range from about 140 to 300 mg/100 mL.
- In severe untreated or poorly controlled diabetes, values can rise markedly above 300 mg/100 mL.
- Low blood sugar values are found in conditions such as excess insulin administration, Addison’s disease, hypoglycemia, and hypopituitarism.
Report:
_ mg of glucose is present in 100 mL of the given blood sample.
Calculation:
mg of glucose per 100 mL of blood = mg of glucose in standard × OD of Test / OD of standard × 100 / 0.2
Here, 0.2 mL is the volume of blood represented in the deproteinized filtrate used for the assay.
Some other protocols for reference and free download:
- Assay of Salivary Amylase enzyme activity
- Effect of Temperature on Amylase activity
- Estimation of Proteins by Lowry method
- Assay of Urease Enzyme Activity
Clinical Significance
- Elevated blood glucose levels are most commonly associated with diabetes mellitus.
- Hyperglycemia may also be seen in conditions such as Cushing’s syndrome, hyperthyroidism, acromegaly or hyperpituitarism, liver disease, acute stress, and infection.
- Decreased blood glucose levels may result from hyperinsulinism, liver disease, and deficiencies of adrenal, thyroid, or pituitary hormones.
Final words
The purpose of this experiment is to estimate a person’s blood glucose level using the Folin–Wu method in a laboratory setting. Once the blood glucose concentration is obtained, it is compared with normal reference ranges, and the results are interpreted in relation to the patient’s clinical condition.
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