Alkaline phosphatase level (ALP)

Sample

1. It is done on the Serum of the patient.
2. A fasting sample is a better choice.
3. This test can be done on the random sample as well.
   • How to get good serum: Take 3 to 5 ml of blood in the disposable syringe or a vacutainer. Keep the syringe for 15 to 30 minutes at 37 C and then centrifuge for 2 to 4 minutes to get the clear serum.
4. Keep the sample refrigerated as soon as you separate the serum.
   1. Because at room temperature level increases.
   2. The refrigerated sample also increases, but that is slow compared to the room temperature sample.
   3. Testing should be done on the same day.
5. Serum at 0 to 4 °C is stable for 2 to 3 days, and at -25 °C is for one month.
6. Perform the test as soon as possible because ALP activity increases 3 to 10% on standing at 25° C or 4 ° C for several hours.

Precautions

1. Storage At room temperature increases the ALP activity.
2. Avoid EDTA and oxalate anticoagulants, which decreases Alkaline phosphatase activity.
3. Reject sample in oxalate and citrate.
4. If serum left at room temperature:
   • Then there is a 1% increase in 6 hours.
   • 3 to 6% in 1 to 4 days.
   • Even though it may increase if refrigerated the serum, which is 2%/day, the increase is slow.
5. Recent intake of food may increase the value.
   1. Values may be 25% higher after taking the high-fat meal.
6. Drugs that increase the level:
   1. Drugs like allopurinol, antibiotics, colchicine, indomethacin, fluorides, isoniazid (INH), methotrexate, nicotinic acid, methyldopa, phenothiazine, and probenecid can increase the alkaline phosphatase level.
   2. It increases after a fatty meal.
7. Drugs that decrease the level:
   1. Drugs like arsenal, cyanides, nitrofurantoin, and zinc salts may decrease the alkaline phosphatase level.
8. Hemolysis may cause a slight increase in the ALP. ALP is 6 times more in the RBC than the serum.
9. Young children experiencing rapid growth, pregnant women, and post-menopausal females have a physiological high level of alkaline phosphatase.
10. After I/V infusion of albumin, there may be a sometimes marked increase in alkaline phosphatase.

Indications

1. Alkaline phosphatase is estimated to detect diseases of the liver and bone.
2. Alkaline phosphatase is the best marker for obstructive jaundice.
3. Alkaline phosphatase is the marker:
   1. For hepatic metastasis.
   2. In parathyroid diseases.
   3. Vitamin D deficiency.
   4. Acute pain in the abdomen.

Pathophysiology

1. The alkaline phosphatase’s main function is to remove the phosphate group from the proteins and other molecules.
   1. ALP is associated with lipid transport in the intestine.
   2. ALP is also associated with the calcification process of the bone.
   3. Alkaline phosphatase is called Alkaline because its function is seen between a pH of 9 to 10 and best at a pH of 9.0.
      

      Alkaline phosphatase in various tissues and functions

2. Distribution of the alkaline phosphatase:
   1. ALP is found in many tissues, at or in the cell membrane.
   2. ALP is a nonspecific enzyme capable of reacting with many different substrates.
   3. Liver and bone ALP are predominantly more in the serum.
   4. A small amount of the ALP from the intestinal epithelium is found in the sera of blood group B or O, who are secretors of blood group substances.
   5. The highest concentration is found in the liver. Within the liver, ALP is present in the Kupffer cells, and these cells line the biliary collecting system. From there, this enzyme is excreted in the bile.
      

      Alkaline phosphatase in liver cell

   6. ALP is mainly from the liver, biliary tree, nearly up to 50% from the skeleton (osteoblastic cells), found in the intestinal epithelium, renal tubule cells, and lower concentration leucocytes and placenta.
      1. This ALP is age-dependent.
3. Effect of temperature on ALP:
   1. 1. ALP (serum)  is denatured at 56 °C and stable at lower temperatures.
      2. The liver isoenzyme is moderately heated stable at 55° C, but bone isoenzyme is heat-labile.
      3. Placental isoenzyme is stable at 65 °C  but not others.
      4. When serum is kept at room temperature will show increased activity.
         1. This varies from:
            1. 1% when kept for >6 hours.
            2. 3 to 6% >1 to 4 days.
            3. When kept in the fridge, it will still increase slowly to 2% per day.
            4. When frozen, then the activity decreases, which will recover slowly after thawing.
      5. In lyophilized sera used for control as a reference also shows increased activity:
         1. 50 to 100% in 24 hours period.
         2. 10% when stored at 4 °C.
         3. 30% when stored at 20 °C.
   2. ALP enzymes require Magnesium for the reaction.
4. Alkaline phosphatase is produced by the bone, bile ducts, intestine, and placenta.

5. Post-puberty ALP is mainly from the liver.
6. This is an enzyme present in the blood, and its subtypes are present in the liver, intestine, bones, and placenta.
7. The conditions which will stimulate bone cells lead to an increase in the level of ALP.

8. Effects of Obstruction:
   6. The liver ALP is increased in biliary obstruction when its excretion is impaired.  While in intrahepatic obstruction, there is less increase in ALP level than biliary obstruction, which may go up to 2.5 times.
   7. In extrahepatic obstruction, it may reach 10 to 12 times the upper limit and normal when surgically obstruction is removed.
   8. In the case of infectious hepatitis, there may be moderate elevation or even normal.

9. Effect of ALP on bone:
   1. The stimulus which increases bone activity causes an increase in the ALP level. So elevated levels indicate liver or bone diseases.
   2. Bone alkaline phosphatase is produced by the osteoblast of the bone and not from the osteoclasts.
   3. ALP level rises during active bone formation like in infants, children, and adolescents. So they have raised the level to 3 times of the norm than the adults.
   4. Bone alkaline phosphatase indicates bone-forming activity.
10. Pregnancy can also lead to a raised level.
11. Alkaline phosphatase in urine is from the renal tubule and not from the plasma.
    1. This may be seen in renal lesions as a malignant tumor (carcinoma), nephrosis, nephritis, infarction, and systemic lupus erythematosus.
12. Alkaline phosphatase function:
    1. The ALP’s exact metabolic function is not understood, but this enzyme appears to be associated with the intestine’s lipid transport and the bone’s calcification process.
13. The activator of the ALP enzyme are:
    1. 1. Magnesium, Cobalt, and manganese.
    2. The exact ratio of Mg²/Zn² is important and to avoid the displacement of the Mg².
    3. Zinc is a constituent metal ion.
14. Inhibitors of the ALP enzyme are:
    
    1. Borate, phosphate, cyanide, and oxalate are inhibitors of the enzyme.

Alkaline Phosphatase Isoenzymes are:

1. Liver ALP isoenzyme (ALP1):
   1. This is derived from the epithelial cells of the biliary tract.
   2. The normal route of elimination is its excretion into the intestine as bile.
   3. This fraction moves fastest on electrophoresis, followed by bone, placenta, and an intestinal fraction.
   4. This is heat stable.
   5. This will be raised in liver diseases.
2. Bone ALP isoenzyme (ALP2):
   1. This increases due to osteoblastic activity and is normally elevated in children and older people above 50.
   2. Adults with healing fractures of the bone have raised levels of ALP.
   3. Paget’s disease leads to a very high level.
   4. Deficient levels of this bone ALP is seen in the metabolic disorder of hypophosphatasia.
3. Intestinal ALP isoenzyme (ALP):
   1. This depends upon the blood group and secretor status of people.
   2. Blood group B or O and secretor are more likely to have this isoenzyme.
   3. This is inactivated by heat.
   4. This will be raised in inflammatory bowel diseases like ulcerative colitis and regional enteritis.
   5. This isoenzyme may lead to abnormal values in a non-fasting sample.
4. Placental ALP:
   1. This is usually seen in the blood pregnant mother, which is placental in origin.
   2. This oncofetal form of placental ALP is also referred to as Regan isoenzyme after the patient in whom it was first-time described.
5. Renal ALP:
   1. Renal tubular cells have ALP activity that is normally excreted in the urine.
   2. It does not reach the serum in pathological conditions.
6. Granulocytes ALP:
   1. ALP in the granulocytes does not raise the serum ALP level.
   2. This is helpful as a marker for the granulocytic maturity in leucocytosis.
      

      Alkaline phosphatase isoenzyme

Alkaline phosphatase isoenzymes classification and differentiation:

Basis for classification	Isoenzyme liver	Isoenzyme intestine	Isoenzyme bone	Isoenzyme placenta	Renal isoenzymes
Amount of ALP	Present in the serum	Present in the serum in inflammatory GI tract diseases	Present in the serum	Trace amount found in serum	Excreted in urine
Heat stability	Stable at 56 °C for 30 minutes, more than bone	Disappear at 56 °C within 15 minutes	Disappear at 56 °C within 10 minutes	Stable at 65 °C for 30 minutes (Regan 15 to 15% cases of caners)
Chemical inhibition	By urea but low by L-phenylalanine	Strong by L-phenylalanine	Strong by urea  but low by L-phenylalanine	Resistance to urea  but strong inhibition by L-phenylalanine
Electrophoresis	Most anodic	Cathodic to the bone fraction	Intermediate	Migrate with the placenta or bone fraction	Renal isoenzyme more cathodic and slower
Gene arrangement	Same as liver, the short arm of chromosome 1	Product of unique gene long arm of chromosome 2	Same as liver, the short arm of chromosome 1	Different Chromosome 2	Same as liver, the short arm of chromosome 1

Normal

value varies according to the kit and methodology used.

As ALP exists in the various tissue, in the case of raised levels, isoenzymes may be advised.

Source 6

Adult Male	25 to 100 U/L
Adult Female	25 to 100 U/L
Children/adult	<2 years  = 85 to 235 U/L
	2 to 8 years  = 65 to 210 U/L
	9 to 15 years =  60 to 300 U/L
	16 to 21 years = 30 to 200 U/L

Source 4

Male

• 1 to 12 years = <350 U/L
• 12 to 14 years = <500 U/L
• >20 years = 25 to 100 U/L

Female

• 1 to 12 years = <350 U/L
• >15 years = 25 to 100 U/L

The principle of Alkaline phosphatase  reaction:

Alkaline phosphatase is an enzyme of the hydrolase class that catalyzes orthophosphate cleavage from orthophosphoric monoesters under alkaline conditions.

There is colorless substrate p-Nitrophenylphosphate and by the action of ALP enzyme converted to p-Nitrophenol, which in alkaline media gives a yellow color.

Alkaline phosphatase in liver diseases:

1. In the absence of bone disease or pregnancy, markedly raised Alkaline phosphatase ( around 10 times ) is the best liver obstructive pathology marker like gallstones obstructing the biliary tract.

2. 1 to 2 times raised level may be seen in various liver parenchymal diseases like Hepatitis and Cirrhosis.
3. In extrahepatic obstruction due to stones of cancer, ALP increases 3 times the normal.
4. In the case of complete obstruction, ALP may be raised 10  to 12 times.
5. In the case of intrahepatic obstruction, ALP rises but less than extrahepatic obstruction.
6. In the case of infectious diseases, ALP rises but <3 times.
7. In pregnancy, the average increase in ALP is 1.5 times the normal limit between 16 to 20 weeks, and it persists till the labor.
   1. After labor, ALP comes to normal within 3 to 6 days.
   2. This may be raised in pregnancy complications like hypertension, Eclampsia, pre-eclampsia, and threatened abortion.
8. To confirm the hepatobiliary origin of ALP, advise γ-GT or 5-nucleotidase.
9. ALP is a sensitive marker for hepatic metastasis.

Its level may be mildly raised in:

1. metastatic diseases of the liver.
2. Hepatocellular carcinoma.
3. Biliary Cirrhosis.
4. Intrahepatic and extrahepatic cholestasis.
5. Gilbert’s syndrome.
6. Chronic alcohol ingestion.
7. Diabetes mellitus and diabetic hepatic lipidosis.

Alkaline phosphatase in bone diseases:

1. It also increased in bone disease like Paget’s disease, healing fractures, Rickets, pregnancy, and childhood.
2. Metastatic bone tumors.
3. Osteogenic sarcoma.
4. Osteomalacia ( while in the osteoporosis ALP is normal ).
5. There is a higher level of  ALP in children, infants, and it is 3 times the adult level.

Alkaline phosphatase in bone diseases:

Disease	ALP increased	Explanation
Paget’s disease	10 to 25 times	Osteoblastic activity increase the ALP
Osteomalacia	Moderate increase	Level lowers with treatment
Ricket’s disease	2 to 4 times	Level lowers with treatment
Osteoporosis	Normal level
Physiologic bone growth	Increased level
Healing fracture	mild, transient raised level
Fanconi’s syndrome	Mild to moderate increase
Primary and secondary hyperparathyroidism	mild to moderate increase
Osteogenic sarcoma	Very high level
Pregnancy third trimester	2 to 3-time increase	This is the placental origin.
Children	1.5 to 2.5-times increase	Growing age
Metastatic tumor-like Ca prostate	Raised level
Solitary bone cyst	Normal

Alkaline phosphatase increased level D/D due to obstruction of various degrees:

Other causes for raised alkaline phosphatase level:

1. It is also raised in old age and pregnancy.
2. Hodgkin’s disease.
3. Sarcoidosis.
4. Amyloidosis.
5. Pulmonary and myocardial infarction.
6. Hyperthyroidism (with a raised level of calcium).
7. Chronic renal failure.
8. Ulcerative colitis.
9. ALP is increased during the last trimester of pregnancy and falls to normal within 3 to 6 months (postpartum).
10. Hyperparathyroidism.

ALP Decreased level seen in:

1. Malnutrition.
2. Hypothyroidism (Cretinism).
3. Milk-alkali syndrome.
4. Celiac sprue.
5. Scurvy (vit. C deficiency).
6. Gross anemia.
7. Deposition of radioactive material in the bone.
8. In hypophosphatemia.
9. Pernicious anemia.
10. Nutritional deficiency of zinc or magnesium.
11. Theophylline therapy.
12. Estrogen therapy in postmenopausal females.
13. Wilson’s disease.
    Alkaline phosphatase raised level in various condition:
    

    Level of raised ALP	Causes of raised ALP
    >5 times of the normal (marked elevation)	Biliary cirrhosis Intrahepatic biliary duct obstruction Extrahepatic biliary duct obstruction Paget’s disease Hyperparathyroidism Osteogenic sarcoma
    3 to 5 times of the normal (Moderate elevation)	Infectious mononucleosis Granulomatous infiltration of the liver Metastatic tumors in bone Rickets Osteomalacia
    Up to 3 times of the normal (Mild elevation)	Cirrhosis Viral hepatitis Pregnancy (placental ALP) Physiologic in children

    

    Alkaline phosphatase interpretation