Alkaline Phosphatase Test of Milk – Principle, Procedure, Result, Uses

Alkaline phosphatase (ALP) is an enzyme which is naturally present in raw milk and also found in different tissues of human body such as liver bone and kidney. It is the enzyme that helps in removing phosphate group from organic molecules and this activity occurs best in alkaline pH condition. In medical field alkaline phosphatase test is generally referred to as a blood test which is done to estimate the level of ALP enzyme and it is used as a biomarker for diagnosis of liver disorders bone related diseases and some metabolic conditions. Increase or decrease in ALP level is considered as an indicator of abnormal physiological condition.

The alkaline phosphatase test is based on the principle that the enzyme catalyzes the hydrolysis of phosphate esters producing inorganic phosphate and alcohol. The reaction rate depends on the amount of enzyme present in the sample. Thus the estimation of enzyme activity helps in understanding the functional condition of organs where this enzyme is produced in higher amount.

Alkaline phosphatase test of milk is a quality control test which is used to check whether the milk has been properly pasteurized or not. It is the most commonly used test in dairy industry to ensure safety of milk for human consumption. Raw milk always contains alkaline phosphatase enzyme and this enzyme is destroyed when milk is heated at proper pasteurization temperature for a specific time. Hence the absence of this enzyme indicates that pasteurization has been done effectively.

This test is based on the fact that alkaline phosphatase enzyme is more heat resistant than most pathogenic bacteria present in milk. Therefore if the enzyme is completely inactivated it is assumed that harmful non spore forming bacteria such as Mycobacterium tuberculosis are also destroyed. In this test a suitable substrate is added to milk sample and if active enzyme is present it breaks the substrate producing color or fluorescence. If no color change is observed it indicates negative result showing proper pasteurization whereas appearance of color indicates presence of enzyme suggesting insufficient heating or post pasteurization contamination of milk.

Principle of Alkaline Phosphatase test

The alkaline phosphatase test is based on the ability of alkaline phosphatase enzyme to catalyze the hydrolysis of phosphate group from organic phosphate esters under alkaline condition. It is the process in which the enzyme acts optimally at an alkaline pH usually between 8 to 10 and the reaction is carried out at a temperature of about 37°C. When active alkaline phosphatase is present in the given sample the substrate used in the test is broken down into phosphate radical and a specific end product.

In this reaction substrates such as disodium phenyl phosphate or p-nitrophenyl phosphate is commonly used. The enzyme splits the phosphate group from the substrate and as a result phenol or p-nitrophenol is released. This is referred to as enzymatic hydrolysis. The liberated product produces a visible colour change or measurable signal and the intensity of the colour formed is directly proportional to the amount of active enzyme present in the sample.

Alkaline phosphatase is naturally present in raw milk and these enzyme is more heat resistant than most non spore forming pathogenic bacteria. During proper pasteurization the enzyme is completely inactivated. Therefore absence of alkaline phosphatase activity indicates effective pasteurization of milk while presence of enzyme activity indicates improper heat treatment.

Objectives of Alkaline Phosphatase test

• To check whether milk has been properly pasteurized by detecting the presence or absence of alkaline phosphatase enzyme.
• To ensure safety of milk and milk products by confirming destruction of heat resistant non spore forming pathogenic bacteria.
• To detect contamination of pasteurized milk with raw milk after pasteurization due to leakage or improper handling.
• To maintain quality control in dairy industry by monitoring efficiency of pasteurization process.
• To meet regulatory and legal standards prescribed for pasteurized milk before distribution to consumers.
• To identify faults in pasteurization equipment such as cracked plates or temperature failure during processing.
• To prevent economic loss by early detection of improper pasteurization and avoiding product recall.
• To estimate alkaline phosphatase level in blood for diagnosis of liver disorders such as hepatitis bile duct obstruction and cirrhosis.
• To help in diagnosis of bone related disorders including rickets osteomalacia and increased bone activity conditions.
• To use alkaline phosphatase enzyme in laboratory research for dephosphorylation of DNA and RNA during molecular biology techniques.
• To employ alkaline phosphatase as a marker enzyme in immunological tests such as ELISA and western blotting.
• To identify undifferentiated stem cells based on high alkaline phosphatase activity present on cell surface.

Requirement for Alkaline Phosphatase test

• Milk or milk product sample for testing alkaline phosphatase activity.
• Alkaline phosphatase enzyme present naturally in raw milk which acts as indicator of pasteurization efficiency.
• Alkaline buffered medium to provide suitable pH for enzymatic reaction.
• Specific substrate such as disodium phenyl phosphate or p-nitrophenyl phosphate for detection of enzyme activity.
• Incubation temperature of about 37°C for proper enzymatic reaction.
• Time controlled incubation to allow complete hydrolysis of substrate.
• Standard regulatory limit for pasteurized cow milk where alkaline phosphatase activity should be below 350 mU/L.
• Approved testing method such as fluorometric or chemiluminescent method for high sensitivity detection.
• Proper laboratory instruments like water bath incubator pipette and fluorometer or color comparator.
• Immediate testing of sample after pasteurization to avoid reactivation of enzyme.
• Trained personnel to perform test and interpret result accurately.
• Hygienic handling of samples to prevent post pasteurization contamination.

The procedure of Alkaline Phosphatase test

A. Fluorometric Method (Reference Method)

1. The Fluorophos substrate and buffer is reconstituted as per instruction. The heating block is set at 38°C ± 1°C.
2. Calibration of instrument is done using calibrators A B and C. It is done separately for different milk products like whole milk cream etc.
3. About 2.0 mL of working substrate is taken in a clean cuvette. The cuvette is kept in heating block for 15 minutes to attain 38°C.
4. After incubation 75 µL of well mixed milk sample is added to the cuvette. The contents is mixed properly by gentle inversion or vortexing.
5. The cuvette is immediately placed inside fluorometer. The fluorescence is measured for total 3 minutes.
6. The instrument measures increase in fluorescence with time. Result is displayed in milliunits per liter (mU/L).

B. Colorimetric Method (Traditional Method)

1. Buffer substrate solution is prepared using phenyl phosphate or p-nitrophenyl phosphate.
2. About 5 mL of buffer is taken in test tube and 1 mL of milk sample is added. The mixture is mixed gently.
3. A control tube is prepared using boiled milk sample. This is used as blank for comparison.
4. The tubes are incubated in water bath at 37°C. Incubation time is usually 1 to 2 hours.
5. After incubation colour development is observed. In phenyl phosphate method reagent is added to produce blue colour.
6. In p-nitrophenyl phosphate method yellow colour is produced directly. Colour intensity indicates enzyme activity.
7. The colour is compared with control or standard disc. Darker colour shows higher alkaline phosphatase activity.

C. Rapid Chemiluminescent Method

1. A fixed volume of milk sample is added to test device.
2. The device is activated to release detection reagent.
3. The sample is incubated for short time at about 37°C.
4. The device is placed in luminometer for reading.
   Result is obtained in relative light units and converted to mU/L.

Interpretation of Results of Alkaline Phosphatase test

The interpretation of alkaline phosphatase test is done to assess the effectiveness of pasteurization of milk and milk products.

Negative Result (Pass)

• A negative result indicates that milk is properly pasteurized and safe for consumption.
• In cow’s milk alkaline phosphatase activity less than 350 mU/L is considered negative.
• This level shows that sufficient heat treatment was applied to destroy enzyme and pathogenic bacteria.
• For goat milk a value less than 300 mU/L is taken as negative due to low basal enzyme level.
• For sheep milk alkaline phosphatase activity below 500 mU/L is considered acceptable.
• In older Scharer colorimetric method less than 1 µg phenol per mL of milk indicates proper pasteurization.

Positive Result (Fail)

• A value higher than the permissible limit indicates a positive result.
• In cow milk alkaline phosphatase activity more than 350 mU/L shows pasteurization failure.
• It may indicate that milk was not heated to required temperature or holding time was insufficient.
• It may also indicate contamination of pasteurized milk with raw milk after heating.
• Sometimes positive result may be due to microbial phosphatase or reactivation of enzyme.

Differentiation of Positive Result

• If residual native bovine alkaline phosphatase is present it indicates true pasteurization failure.
• When enzyme activity remains after laboratory repasteurization it indicates microbial alkaline phosphatase.
• If enzyme activity increases in presence of magnesium it indicates reactivated alkaline phosphatase.
• Reactivated enzyme usually occurs in UHT milk stored at higher temperature and does not indicate pasteurization failure.

Interpretation in Cheese

• In cheese products interpretation depends upon the type of cheese.
• For many cheeses alkaline phosphatase value more than 12 µg phenol equivalent per gram is considered unacceptable.
• In some standards a value less than 10 mU/g indicates proper pasteurization of cheese milk.
• Blue veined cheeses are excluded because mould growth contributes to phosphatase activity.

Uses of Alkaline Phosphatase Test

• It is used to check the effectiveness of pasteurization of milk and milk products.
• It is applied as a standard quality control test in dairy industry.
• The test helps in confirming destruction of heat resistant non-spore forming pathogens in milk.
• It is used to detect contamination of pasteurized milk with raw milk after heating.
• The test is useful in routine monitoring of pasteurization equipment and processing faults.
• It is used as a HACCP verification tool in dairy processing plants.
• It helps regulatory authorities to ensure that milk meets legal pasteurization standards.
• The test is also used for pasteurization verification of goat milk and sheep milk.
• In clinical diagnosis it is used to estimate alkaline phosphatase level in blood.
• It is used in diagnosis of liver disorders such as hepatitis and bile duct obstruction.
• It is used to detect bone diseases associated with abnormal bone formation.
• The test is useful in monitoring certain cancers where alkaline phosphatase level is altered.
• It is used during pregnancy as a marker related to placental activity.
• In molecular biology it is used to remove phosphate groups from DNA and RNA.
• It is used to prevent self-ligation of DNA during cloning experiments.
• Alkaline phosphatase is used as an enzyme label in immunoassays like ELISA.
• It is used in protein detection and antibody based assays.
• The test is also used in stem cell research to identify undifferentiated cells.

Limitation of Alkaline Phosphatase 

• It may give false positive result due to reactivation of enzyme after pasteurization especially in UHT or ESL milk products.
• The enzyme can regain activity during storage at higher temperature (above 30°C) or in presence of magnesium salts which affects test accuracy.
• High fat milk products like cream are more prone to ALP reactivation because the membrane-bound enzyme is present in higher amount.
• It is difficult to differentiate between true residual ALP and reactivated ALP without performing additional differential tests.
• Presence of microbial phosphatase produced by bacteria (such as Bacillus species) can cause false positive results.
• Microbial ALP is more heat stable than bovine ALP and may survive pasteurization even when heating is adequate.
• The test cannot chemically distinguish between bovine alkaline phosphatase and microbial phosphatase.
• Chemical inhibitors such as acidic condition in sour milk or preservatives can inhibit enzyme activity leading to false negative result.
• Detergent or sanitizer residues from CIP procedures may interfere with enzyme reaction.
• Flavoured milk and highly turbid samples may interfere with colour development in older colorimetric methods.
• Certain compounds like antibiotics or vanillin derivatives can interfere with enzyme detection.
• The test is mainly designed for cow milk and shows limitation in milk of other species.
• Goat milk has naturally low alkaline phosphatase activity which reduces sensitivity of detection.
• It is unsuitable for camel milk because camel ALP is heat stable and may survive pasteurization.
• Equine milk has very low basal ALP level making the test unreliable.
• A negative ALP test does not ensure complete sterility or absence of all pathogens.
• It does not detect heat resistant spores or post-pasteurization contamination.

Advantages of Alkaline Phosphatase 

• It is used as a reliable indicator for checking proper pasteurization of milk and milk products.
• Alkaline phosphatase enzyme is naturally present in raw milk and is destroyed only when sufficient heat treatment is given.
• A negative test result confirms that pasteurization temperature was adequate to destroy harmful non-spore forming pathogens.
• It ensures safety of milk by indicating destruction of organisms like Mycobacterium tuberculosis and Coxiella burnetii.
• The test is highly sensitive and can detect very small amount of raw milk contamination in pasteurized milk.
• Modern fluorometric methods can detect trace levels of alkaline phosphatase with high accuracy.
• It reduces interference caused by fat content or colour present in flavoured milk products.
• The test gives rapid results and can be completed within few minutes.
• It is faster when compared to microbiological culture methods which take longer time.
• It helps in routine quality control and monitoring of pasteurization equipment.
• Minor processing faults like temperature fluctuation or equipment leakage can be detected early.
• It prevents large scale product spoilage and economic loss due to early detection of errors.
• The test is simple to perform and suitable for routine laboratory use.
• In clinical field alkaline phosphatase level is used as diagnostic marker for liver and bone disorders.
• It is widely used in molecular biology for removing phosphate groups from DNA and RNA.
• Due to high catalytic activity it is used as an enzyme label in immunoassays like ELISA.

FAQ

Q1. What is an alkaline phosphatase test?
A.
Alkaline phosphatase test is a laboratory test used to measure the level of alkaline phosphatase enzyme in blood. This enzyme is mainly found in liver bone intestine and placenta. It is commonly used to assess liver function and bone metabolism.

Q2. Why do I need an alkaline phosphatase test?
A.
It is done to check liver health and bone related disorders. The test is advised when there are symptoms of liver disease bile duct obstruction or abnormal bone growth. It is also done as a part of routine liver function test.

Q3. What does high alkaline phosphatase (ALP) mean?
A.
High alkaline phosphatase level usually indicates liver disease or bone disorder. It suggests increased enzyme release due to liver damage bile duct blockage or increased bone formation.

Q4. What does low alkaline phosphatase (ALP) mean?
A.
Low alkaline phosphatase level is less common and may indicate nutritional deficiency genetic disorders or hormonal imbalance. It may also be seen in conditions with reduced bone activity.

Q5. What causes high alkaline phosphatase levels?
A.
High ALP level may be caused by liver diseases such as hepatitis cirrhosis or bile duct obstruction. Bone diseases like rickets Paget’s disease bone tumors and healing fractures can also increase ALP. Pregnancy can also increase ALP level.

Q6. What causes low alkaline phosphatase levels?
A.
Low ALP level may occur due to malnutrition zinc deficiency hypothyroidism or certain inherited disorders. It may also be seen in severe anemia or after blood transfusion.

Q7. What happens during an alkaline phosphatase test?
A.
During the test a small amount of blood is collected from a vein usually from the arm. The sample is then analyzed in laboratory to measure alkaline phosphatase level. The procedure is simple and quick.

Q8. How do I get ready for an alkaline phosphatase test?
A.
Generally no special preparation is required. In some cases fasting for few hours may be advised. Certain medicines should be informed to doctor as they may affect result.

Q9. What is a normal alkaline phosphatase (ALP) level?
A.
Normal alkaline phosphatase level varies with age and sex. In adults it generally ranges between 40 to 130 IU/L. Children and pregnant women may have higher normal values.

Q10. What are symptoms of liver disease that may require an ALP test?
A.
Symptoms include jaundice dark urine pale stool fatigue abdominal pain nausea and itching. These symptoms indicate possible liver or bile duct problem.

Q11. What are symptoms of bone disorders that may require an ALP test?
A.
Bone pain bone deformity frequent fractures and delayed growth are common symptoms. These conditions are associated with abnormal bone metabolism.

Q12. What other tests might I have along with an alkaline phosphatase test?
A.
It is often done with liver function tests like AST ALT bilirubin and GGT. For bone disorders calcium phosphate and vitamin D tests are also advised.

Q13. Should I be concerned if I have high or low alkaline phosphatase (ALP) test results?
A.
Abnormal ALP result alone does not confirm disease. It should be interpreted with symptoms and other test results. Doctor decides further investigation if required.

Q14. What is an ALP isoenzyme test?
A.
ALP isoenzyme test is used to identify the source of alkaline phosphatase. It helps to differentiate whether ALP is coming from liver bone or other tissues.

Q15. What might affect alkaline phosphatase test results?
A.
Age pregnancy medications recent meals and medical conditions can affect ALP level. Improper sample handling may also influence test result.