Ehrlich Test- Definition, Principle, Procedure, Result, Uses

Ehrlich test is a biochemical test that detects the presence of tryptophan, an amino acid, in a protein sample. The test is based on the reaction of tryptophan with Ehrlich reagent, which is a solution of p-dimethylamino benzaldehyde in sulfuric acid. The reaction produces a blue-violet condensation product that can be enhanced by adding sodium nitrite. The test is named after Paul Ehrlich, a German Nobel laureate who discovered this reaction while studying typhoid fever and diarrhea. Ehrlich test has various applications in medical and forensic fields, such as:

• Diagnosing diseases that affect the liver, bile ducts, and blood cells, such as hepatic jaundice, hepatitis, hemolytic anemia, and carcinoid syndrome.
• Identifying psychoactive substances that contain indole or benzyl pyrrole rings, such as tryptamines and ergoloids (e.g., LSD).
• Detecting urobilinogen, a breakdown product of hemoglobin, in urine samples.

Ehrlich test is also known as p-dimethylamino benzaldehyde test or specific amino acid test. It is one of the oldest and simplest tests for tryptophan and indoles. However, it has some limitations and sources of error that need to be considered while interpreting the results.

The main objective of the Ehrlich test is to detect the presence of tryptophan in a given sample. Tryptophan is an essential amino acid that is involved in various biological processes, such as protein synthesis, neurotransmitter production, and melatonin secretion. Tryptophan is also a precursor of indole, a heterocyclic compound that has many biological and chemical functions.

The Ehrlich test can be used for different purposes depending on the type of sample and the reagent used. Some of the common objectives of the Ehrlich test are:

• To distinguish between typhoid and diarrhea. Typhoid fever is caused by Salmonella typhi, a bacterium that produces tryptophanase, an enzyme that converts tryptophan to indole. Diarrhea can be caused by other bacteria that do not produce tryptophanase. By adding Ehrlich reagent to a culture of the bacteria, a positive result (blue-violet color) indicates the presence of indole and thus typhoid fever.
• To identify the presence of urobilinogen in a urine sample. Urobilinogen is a colorless compound that is formed by the breakdown of bilirubin in the intestine. Urobilinogen is normally excreted in urine and feces, but it can also accumulate in the blood and tissues in some diseases, such as liver cirrhosis, hemolytic anemia, and bile duct obstruction. By adding Ehrlich reagent to a urine sample, a positive result (red to purple color) indicates the presence of urobilinogen and thus a possible liver or blood disorder.
• To detect the presence of psychoactive compounds and drugs in a sample. Some psychoactive compounds and drugs, such as tryptamines (e.g., psilocybin, DMT) and ergoloids (e.g., LSD), contain an indole ring in their structure. By adding Ehrlich reagent to a sample containing these substances, a positive result (blue-violet color) indicates the presence of indoles and thus a possible psychoactive effect.

The Ehrlich test is based on the principle that under acidic conditions, the Ehrlich reagent reacts with tryptophan to form a blue-violet condensation product. The Ehrlich reagent consists of p-dimethylamino benzaldehyde (DMAB) dissolved in ethanol and sulfuric acid. DMAB is an electrophile that can attack the indole ring of tryptophan and form a substituted benzyl pyrrole. The color of the product depends on the position and number of substitutions on the indole ring. The reaction can be enhanced by adding sodium nitrite (NaNO2), which oxidizes the product and changes its color to blue.

The reaction can be summarized as follows:

Tryptophan + Ehrlich reagent (DMAB in H2SO4) → Blue-violet condensation product

Blue-violet condensation product + NaNO2 → Blue product

The Ehrlich test can also detect other indole derivatives, such as urobilinogen, which is a breakdown product of bilirubin. Urobilinogen is normally present in urine in small amounts, but it can increase in some liver diseases or hemolytic disorders. Urobilinogen also reacts with the Ehrlich reagent to form a red or pink color, which can be distinguished from the blue-violet color of tryptophan.

The Ehrlich test can also be used to identify some psychoactive compounds, such as tryptamines and ergoloids, which have indole structures similar to tryptophan. These compounds also react with the Ehrlich reagent to produce different colors depending on their chemical structure and concentration. For example, lysergic acid diethylamide (LSD) produces a purple color, while psilocybin produces a blue-green color.

The Ehrlich test is a simple and rapid method to detect tryptophan and other indole derivatives in various samples. However, it has some limitations and sources of error that should be considered before interpreting the results.

The Ehrlich test requires the following reagents and materials:

• Ehrlich reagent: Ehrlich reagent is prepared by dissolving about 0.5 to 2.0 grams of p-dimethylamino benzaldehyde in 50 ml of 95% ethanol. To this, 50 ml of 10% H2SO4 is added and mixed well. The reagent should be stored in a dark bottle and used within a month.
• Protein sample: The protein sample to be tested for tryptophan can be obtained from various sources, such as food, blood, urine, etc. The sample should be diluted with distilled water before performing the test.
• Concentrated HCl: Concentrated hydrochloric acid is used to acidify the protein sample and facilitate the reaction with the Ehrlich reagent.
• NaNO2: Sodium nitrite solution is used to enhance the color of the positive result by oxidizing the condensation product formed by the Ehrlich reagent and tryptophan.
• Test tubes: Test tubes are used to hold the sample and the reagents during the test. They should be clean and dry before use.
• Test tube stand: A test tube stand is used to support the test tubes during the test and prevent them from falling or spilling.
• Pipettes: Pipettes are used to transfer measured amounts of the sample and the reagents into the test tubes. They should be rinsed with distilled water before and after use.

These are the requirements for performing the Ehrlich test. The test should be performed in a well-ventilated area and with proper safety precautions. The reagents and materials should be disposed of properly after use.

The procedure of Ehrlich test is as follows:

• Take about 3-4 ml of diluted protein solution or urine sample in a test tube.
• Boil the liquid with 1 ml of concentrated HCl for a few minutes.
• Add a few drops of the Ehrlich reagent (p-dimethylamino benzaldehyde in H2SO4) to the test tube and shake well.
• Observe the color change in the test tube. If a red to purple or blue-violet color appears, it indicates a positive result for tryptophan.
• To enhance the color, add a few drops of NaNO2 solution to the test tube and observe the change to blue color.

The procedure can be summarized in the following table:

• A positive result in the Ehrlich test is indicated by the appearance of red to purple or blue-violet color on the addition of the Ehrlich reagent. The color then changes to blue with the addition of NaNO2. This indicates that the sample contains tryptophan.
• A negative result in the Ehrlich test is indicated by the absence of blue-violet color on the addition of the Ehrlich reagent. This indicates that the sample doesn`t contain any tryptophan.
• The intensity of the color depends on the concentration of tryptophan in the sample. The higher the concentration, the darker the color.
• The Ehrlich test can also be used to measure the amount of urobilinogen in urine samples. Urobilinogen is a breakdown product of bilirubin, which is a pigment produced by the liver. Urobilinogen reacts with the Ehrlich reagent to produce a pink to red color. The intensity of the color depends on the amount of urobilinogen in urine. The normal range of urobilinogen in urine is 0.1 to 1 mg/dL. Higher levels of urobilinogen may indicate liver diseases or hemolytic anemia. Lower levels of urobilinogen may indicate bile duct obstruction or antibiotic therapy.

• The Ehrlich test is an aldehyde test used for the detection of tryptophan in a protein sample. Tryptophan is an essential amino acid that plays a role in various biological processes, such as protein synthesis, neurotransmitter production, and immune system regulation. The Ehrlich test can help determine the quality and quantity of tryptophan in food products, supplements, and biological samples.
• The test is also used for the diagnosis of diseases like typhoid and other disorders of the hemolytic process or the obstruction of the common bile duct. Typhoid is a bacterial infection caused by Salmonella typhi that affects the intestinal tract and the bloodstream. The Ehrlich test can help differentiate typhoid from simple diarrhea by detecting the presence of tryptophan in the stool sample. The hemolytic process is the breakdown of red blood cells that releases hemoglobin into the blood. The obstruction of the common bile duct is a blockage of the tube that carries bile from the liver to the intestine. Both conditions can result in increased levels of urobilinogen in the urine, which can be detected by the Ehrlich test.
• The test can also be used for the detection of psychoactive compounds and drugs like tryptamines and ergoloids. Tryptamines are a class of compounds that have a similar structure to tryptophan and act on serotonin receptors in the brain. Some examples of tryptamines are psilocybin, DMT, and LSD. Ergoloids are derivatives of ergot, a fungus that grows on rye and other grains. They have various effects on the central nervous system, such as vasoconstriction, hallucination, and stimulation. Some examples of ergoloids are ergotamine, ergine, and lysergic acid. The Ehrlich test can help identify these substances by reacting with their indole ring and producing a blue-violet color.
• The Ehrlich test also detects the presence of indoles and urobilinogen. Indoles are organic compounds that have a benzene ring fused with a pyrrole ring. They are widely distributed in nature and have diverse biological functions. Some examples of indoles are serotonin, melatonin, and indole-3-acetic acid. Urobilinogen is a colorless compound that is formed by the breakdown of bilirubin in the intestine by bacteria. It is normally excreted in urine and feces. The presence of urobilinogen in high concentration in urine sample helps in the diagnosis of hepatic jaundice and hepatitis. Hepatic jaundice is a condition where the liver is unable to process bilirubin properly, resulting in yellowing of the skin and eyes. Hepatitis is an inflammation of the liver caused by viral or bacterial infection, alcohol abuse, or autoimmune disease. The Ehrlich test can help detect urobilinogen by reacting with its aldehyde group and producing a red color.

• The Ehrlich test is not a very sensitive test as it requires a high concentration of tryptophan or urobilinogen to produce a positive result. A low concentration of these substances might not be detected by the test.
• The Ehrlich test is not a very specific test as it can react with other substances that have an indole or benzyl pyrrole ring, such as serotonin, melatonin, and skatole. These substances might interfere with the test and give a false positive result.
• The Ehrlich test can be affected by the pH of the sample. A high pH can reduce the reactivity of the Ehrlich reagent and give a false negative result. A low pH can increase the reactivity of the Ehrlich reagent and give a false positive result.
• The Ehrlich test can be influenced by the presence of other compounds in the sample, such as nitrites, sulfates, and bilirubin. These compounds might react with the Ehrlich reagent or the condensation product and alter the color of the test. For example, nitrites can oxidize urobilinogen to urobilin and give a false negative result. Sulfates can reduce the intensity of the blue-violet color and give a false negative result. Bilirubin can mask the blue-violet color and give a false negative result.
• The Ehrlich test is not a confirmatory test for any disease or disorder. It only indicates the presence or absence of tryptophan or urobilinogen in the sample. A positive result does not necessarily mean that the person has typhoid, carcinoid syndrome, or liver disease. A negative result does not necessarily mean that the person is healthy or free from any disease or disorder. The Ehrlich test should be used in conjunction with other tests and clinical signs and symptoms to make a diagnosis.

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