Alkaline phosphatase



Alkaline phosphatase (ALP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment.

Bacterial
In bacteria, alkaline phosphatase is located in the periplasmic space, external to the cell membrane. Since this space is much more subject to environmental variation than the actual interior of the cell, bacterial alkaline phosphatase is comparatively resistant to inactivation, denaturation, and degradation, and also has a higher rate of activity. Although the actual purpose of the enzyme is still not fully understood, the simple hypothesis, that it is a means for the bacteria to generate free phosphate groups for uptake and use, is supported by the fact that alkaline phosphatase is usually only produced by the bacteria during phosphate starvation and not when phosphate is plentiful. However, other possibilities exist; for instance, the presence of phosphate groups usually prevents organic molecules from passing through the membrane, therefore dephosphorylating them may be important for bacterial uptake of organic compounds in the wild. Some complexities of bacterial regulation and metabolism suggest that other, more subtle, purposes for the enzyme may also play a role for the cell. In the laboratory, however, mutant Escherichia coli lacking alkaline phosphatase survive quite well, as do mutants unable to shut off alkaline phosphatase production.

Use in research
The most common alkaline phosphatases used in research are:
 * Bacterial alkaline phosphatase (BAP), from Escherichia coli C4 cells
 * Shrimp alkaline phosphatase (SAP), from a species of arctic shrimp] (Pandalus borealis)
 * Calf intestine alkaline phosphatase (CIAP), from calf intestine
 * Placental alkaline phosphatase (PLAP) and its C terminally truncated version that lacks the last 24 amino acids (constituting the transmembrane domain) - the secreted alkaline phosphatase (SEAP)

Alkaline phosphatase has become a useful tool in molecular biology laboratories, since DNA normally possesses phosphate groups on the 5' end. Removing these phosphates prevents the DNA from ligating (the 5' end attaching to the 3' end of another molecule), thereby preventing DNA degradation until the next step of the process for which it is being prepared; also, removal of the phosphate groups allows radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of the labeled DNA through further steps in the process or experiment. For these purposes, the alkaline phosphatase from shrimp is the most useful, as it is the easiest to inactivate once it has done its job.

Another important use of alkaline phosphatase is as a label for enzyme immunoassays.

One common use in the dairy industry is as a marker of pasteurisation. This molecule is denatured by elevated temperatures found during pasteurisation, and can be tested for via colour change of a para-nitro-phenol phosphate substrate in a buffered solution. (Aschaffenburg Mullen Test)Raw milk would typically produce a yellow colouration within a couple of minutes, whereas properly pasteurised milk should show no change. There are of course exceptions to this in the case of heat stable alkaline phophatases produced by some bacteria.

Inhibitors
All mammalian alkaline phosphatase isoenzymes except placental (PLAP and SEAP) are inhibited by homoarginine and similarly all except the intestinal and placental ones are blocked by levamisole. Heating for ~2 hours at 65oC inactivated most isoenzymes except Placental isoforms (PLAP and SEAP).

Physiology
In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney, bone, and the placenta. The optimal pH for the enzyme activity is pH=10 in standard conditions (298K,1 atm).

Diagnostic use
Blood plasma (serum) levels of ALP are typically 20-70 units per liter in adults (Reference - USMLE), depending on the assay and local normal guidelines. Levels are significantly higher in children and pregnant women.

Lowered levels of ALP are less common than elevated levels.

The following conditions can cause abnormal levels of ALP:

= Differential Diagnosis =

Increased Serum AP

 * Acromegaly
 * Acute pancreatitis
 * Alcoholic hepatitis
 * Benign or malignant liver tumor
 * Bone tumors
 * Bowel perforation
 * Bronchial carcinoma
 * Cholangitis
 * Cholecystitis
 * Cholelithiasis
 * Cholestasis
 * Cirrhosis
 * Diabetes Mellitus
 * Drug induced liver disease
 * Drugs, toxins
 * During growth
 * Fanconi's Syndrome
 * Fatty liver
 * Fracture healing
 * Hepatic cyst
 * Hepatic granuloma
 * Hepatitis
 * Hereditary hyperphosphatasemia
 * Hodgkin's Lymphoma
 * Hypernephroma
 * Hyperparathyroidism
 * Hyperthyroidism
 * In last trimester of pregnancy
 * Liver abscesses
 * Liver metastases
 * Metastatic bone tumors
 * Multiple Myeloma
 * Osteomalacia
 * Osteomyelitis
 * Osteomyelosclerosis
 * Osteonecroses
 * Osteosarcoma
 * Paget's Disease
 * Polyarteritis Nodosa
 * Pregnancy
 * Primary biliary cirrhosis
 * Renal failure
 * Rickets
 * Right ventricular failure
 * Sarcoidosis
 * Ulcerative colitis

Decreased Serum AP

 * Celiac Sprue
 * Fibrate therapy
 * Hypothyroidism
 * Malnutrition
 * Pernicious anemia
 * Vitamin D intoxication

Elevated levels (hyperphosphatasemia)
If it is unclear why alkaline phosphatase is elevated, isoenzyme studies using electrophoresis can confirm the source of the ALP. Heat stability also distinguishes bone and liver isoenzymes ("bone burns, liver lasts").


 * Liver (Liver ALP):
 * Cholestasis, cholecystitis, cholangitis, cirrhosis, hepatitis, fatty liver, liver tumor, liver metastases, drug intoxication
 * N.B. concurrently elevated GGT(gamma glutamyl transpeptidase) helps rule in favor of liver metastasis (rather than bone, kidney, etc.) when assessing spread of cancer.
 * Drugs: e.g. verapamil, carbamazepine, phenytoin, erythromycin, allopurinol, ranitidine
 * Bone disease (Bone ALP):
 * Paget's disease, osteosarcoma, bone metastases of prostatic cancer (High / very high ALP values)
 * Other bone metastases
 * Fractured bone
 * Multiple myeloma (only when associated with fractures)
 * Skeletal involvement of other primary diseases:
 * Osteomalacia, rickets, vitamin D deficiency, (Moderate rise)
 * Malignant tumors (ALP originating from tumor)
 * Renal disease (secondary hyperparathyroidism)
 * Primary hypothyroidism
 * Polycythemia vera
 * Myelofibrosis
 * Leukemoid reaction to infection
 * Women using hormonal contraception
 * Pregnancy
 * Biliary obstruction
 * Transient hyperphosphatasaemia of infancy: benign, often associated with infection

Lowered levels (hypophosphatasemia)

 * Hypophosphatasia, an autosomal recessive disease
 * Postmenopausal women receiving estrogen therapy because of osteoporosis
 * Men with recent heart surgery, malnutrition, magnesium deficiency, hypothyroidism or severe anemia
 * Children with achondroplasia and cretinism
 * Children after a severe enteritis
 * Pernicious anemia
 * Aplastic anemia
 * Chronic myelogenous leukemia

Other notes
Leukocyte alkaline phosphatase (LAP) is found within white blood cells. Blood levels of LAP can help in the diagnosis of chronic myelogenous leukemia (CML) and leukemoid reaction.