Alkaline phosphatase

Use in research

By changing the amino acids of the wild-type alkaline phosphatase enzyme produced by Escherichia coli, a mutant alkaline phosphatase is created which not only has a 36-fold increase in enzyme activity, but also retains thermal stability. Typical uses in the lab for alkaline phosphatases include removing phosphate monoesters to prevent self-ligation, which is undesirable during plasmid DNA cloning.

Common alkaline phosphatases used in research include:

• Shrimp alkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalus borealis). This phosphatase is easily inactivated by spiking up the temperature, a useful feature in some applications.
• Calf-intestinal alkaline phosphatase (CIP) typically useful in molecular cloning, where DNA undergoes recombination and no new DNA is made. In addition, it is useful in detoxifying mediated lipopolysaccharide inflammatory responses thus a drug for lipopolysaccharide mediated disease.
• Placental alkaline phosphatase (PLAP) and its C terminally truncated version that lacks the last 24 amino acids (constituting the domain that targets for GPI membrane anchoring) – the secreted alkaline phosphatase (SEAP). It presents certain characteristics like heat stability, substrate specificity, and resistance to chemical inactivation.
• Human-intestinal alkaline phosphatase. The human body has multiple types of alkaline phosphatase present, which are determined by a minimum of three gene loci. Each one of these three loci controls a different kind of alkaline phosphatase isozyme. However, the development of this enzyme can be strictly regulated by other factors such as thermostability, electrophoresis, inhibition, or immunology.

Human-intestinal alkaline phosphatase shows around 80% homology with the bovine intestinal enzyme which holds true their shared evolutionary origins. That same bovine enzyme has more than 70% homology with human placental enzyme. However, the human liver enzyme and the placental enzyme only share 20% homology despite their structural similarities.

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), thereby keeping DNA molecules linear until the next step of the process for which they are 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 due to the elevated catalytic activity of ALP enzyme.

Undifferentiated pluripotent stem cells have elevated levels of alkaline phosphatase on their cell membrane, therefore alkaline phosphatase staining is used to detect these cells and to test pluripotency (i.e., embryonic stem cells or embryonal carcinoma cells).

There is a positive correlation between serum bone alkaline phosphatase levels (= BAP) and bone formation in humans, although its use as a biomarker in clinical practice is not recommended.

Dairy industry

Alkaline phosphatase is commonly used in the dairy industry as an indicator of successful pasteurization. This is because the most heat stable bacterium found in milk, Mycobacterium paratuberculosis, is destroyed by temperatures lower than those required to denature the enzyme. Therefore, its presence is ideal for indicating failed pasteurization.

Pasteurization verification is typically performed by measuring the fluorescence of a solution which becomes fluorescent when exposed to active alkaline phosphatase. Fluorimetry assays are required by milk producers in the UK to prove alkaline phosphatase has been denatured, as p-Nitrophenylphosphate tests are not considered accurate enough to meet health standards.

Alternatively the color change of p-nitrophenylphosphate as substrate in a buffered solution (Aschaffenburg Mullen Test) can be used. Raw milk would typically produce a yellow coloration within a couple of minutes, whereas properly pasteurised milk should show no change. There are exceptions to this, as in the case of heat-stable alkaline phosphatases produced by some bacteria, but these bacteria should not be present in milk.

In addition, the Scharer Rapid Phosphatase test investigates the presence and the absence of ALP enzymes in raw milk. This test is advantageous since a full and complete pasteurization inactivates the ALP enzymes, resulting in no color change. However, positive test results in changing the raw milk color into blue, thus indicating the presence of ALP enzymes. The test works by cleaving off a phosphate group from the substrate disodium phenyl phosphate, causing a release of phenol group and indicating positive test results observed visually and by spectrophotometer.

Inhibitors

All mammalian alkaline phosphatase isoenzymes except placental (PALP and SEAP) are inhibited by homoarginine, and, in similar manner, all except the intestinal and placental ones are blocked by levamisole. Phosphate is another inhibitor which competitively inhibits alkaline phosphatase.

Another known example of an alkaline phosphatase inhibitor is [(4-Nitrophenyl)methyl]phosphonic acid.

In metal contaminated soil, alkaline phosphatase are inhibited by Cd (Cadmium). In addition, temperature enhances the inhibition of Cd on the enzyme activity, which is shown in the increasing values of K_m.

Human

Physiology

In humans, alkaline phosphatase is present in all tissues throughout the body, but is particularly concentrated in the liver, bile duct, kidney, bone, intestinal mucosa and placenta. In the serum, two types of alkaline phosphatase isozymes predominate: skeletal and liver. During childhood the majority of alkaline phosphatase are of skeletal origin. Humans and most other mammals contain the following alkaline phosphatase isozymes:

• ALPI – intestinal (molecular mass of 150 kDa)
• ALPL – tissue-nonspecific (expressed mainly in liver, bone, and kidney)
• ALPP – placental (Regan isozyme)
• ALPG – germinal tissues

Four genes encode the four isozymes. The gene for tissue-nonspecific alkaline phosphatase is located on chromosome 1, and the genes for the other three isoforms are located on chromosome 2.

Intestinal alkaline phosphatase

Intestinal alkaline phosphatase is secreted by enterocytes, and seems to play a pivotal role in intestinal homeostasis and protection as well as in suppressing inflammation via repression of the downstream Toll-like receptor (TLR)-4-dependent and MyD88-dependent inflammatory cascade. It dephosphorylates toxic/inflammatory microbial ligands like lipopolysaccharides (LPSs), unmethylated cytosine-guanine dinucleotides, flagellin, and extracellular nucleotides such as uridine diphosphate or ATP. Dephosphorylation of LPS by IAP can reduce the severity of Salmonella tryphimurium and Clostridioides difficile infection restoring normal gut microbiota. Thus, altered IAP expression has been implicated in chronic inflammatory diseases such as inflammatory bowel disease (IBD). It also seems to regulate lipid absorption and bicarbonate secretion in the duodenal mucosa, which regulates the surface pH. Since the 1960s intestinal alkaline phosphatase has been utilized in drug delivery. As it cleaves phosphate substructures from drugs, auxiliary agents, and even from the surface of nanocarriers, this enzyme enables the design of drug delivery systems that can alter their properties in the body on demand. The solubility of many drugs can be substantially improved by the design of phosphate prodrugs. On the intestinal mucosa the phosphate substructures are cleaved off by alkaline phosphatase and the drug is absorbed. Furthermore, anionic nanocarriers exhibiting bioinert properties can alter their surface to interactive once having reached the intestinal epithelium as due to an alkaline phosphatase triggered cleavage of anionic phosphate groups from their surface charge converts to cationic improving for instance cellular uptake.

In cancer cells

Studies show that the alkaline phosphatase protein found in cancer cells is similar to that found in nonmalignant body tissues and that the protein originates from the same gene in both. One study compared the enzymes of liver metastases of giant-cell lung carcinoma and nonmalignant placental cells. The two were similar in NH₂-terminal sequence, peptide map, subunit molecular weight, and isoelectronic point.

In a different study in which scientists examined alkaline phosphatase protein presence in a human colon cancer cell line, also known as HT-29, results showed that the enzyme activity was similar to that of the non-malignant intestinal type. However, this study revealed that without the influence of sodium butyrate, alkaline phosphatase activity is fairly low in cancer cells. A study based on sodium butyrate effects on cancer cells conveys that it has an effect on androgen receptor co-regulator expression, transcription activity, and also on histone acetylation in cancer cells. This explains why the addition of sodium butyrate show increased activity of alkaline phosphatase in the cancer cells of the human colon. In addition, this further supports the theory that alkaline phosphatase enzyme activity is actually present in cancer cells.

In another study, choriocarcinoma cells were grown in the presence of 5-bromo-2'-deoxyuridine and results conveyed a 30- to 40-fold increase in alkaline phosphatase activity. This procedure of enhancing the activity of the enzyme is known as enzyme induction. The evidence shows that there is in fact activity of alkaline phosphatase in tumor cells, but it is minimal and needs to be enhanced. Results from this study further indicate that activities of this enzyme vary among the different choriocarcinoma cell lines and that the activity of the alkaline phosphatase protein in these cells is lower than in the non-malignant placenta cells. but levels are significantly higher in children and pregnant women. Blood tests should always be interpreted using the reference range from the laboratory that performed the test. High alkaline phosphatase levels can occur if the bile ducts are obstructed.

Also, the level of alkaline phosphatase increases if there is active bone formation occurring, as the enzyme is a byproduct of osteoblast activity (such as the case in Paget's disease of bone).

The level of alkaline phosphatase is much more elevated in metastatic prostate cancer cells than non-metastatic prostate cancer cells. High levels of ALP in prostate cancer patients is associated with a significant decrease in survival.

Levels are also elevated in people with untreated coeliac disease. Lowered levels of the level of alkaline phosphatase are less common than elevated levels. The source of elevated levels can be deduced by obtaining serum levels of γ-glutamyltransferase. Concomitant increases of alkaline phosphatase with γ-glutamyltransferase should raise the suspicion of hepatobiliary disease.

Some diseases do not affect the levels of alkaline phosphatase, for example, hepatitis C. A high level of this enzyme does not reflect any damage in the liver, even though high alkaline phosphatase levels may result from a blockage of flow in the biliary tract or an increase in the pressure of the liver.

Elevated levels

As of 2020, normal ALP levels were "not well defined", and there tend to be variations by sex and racial background, and by age, with children and adolescents having markedly higher levels.

There are many possible explanations for elevated ALP. When the cause is unclear, isoenzyme studies using electrophoresis can confirm the source of the increase. Skelphosphatase (which is localized in osteoblasts and extracellular layers of newly synthesized matrix) is released into circulation by a yet unclear mechanism. Placental alkaline phosphatase is elevated in seminomas and active forms of rickets, as well as in the following diseases and conditions:

• Biliary obstruction
• Bone conditions
• Osteoblastic bone tumors
• Osteomalacia
• Osteoporosis
• Hepatitis
• Mononucleosis
• Cirrhosis
• Acute cholecystitis
• Myelofibrosis
• Leukemoid reaction
• Congestive heart failure
• Lymphoma
• Paget's disease
• Sarcoidosis
• Hyperthyroidism
• Hyperparathyroidism
• Myocardial infarction
• Cholangitis
• Ischemic cholangiopathy
• Pregnancy
• High doses of estrogens

Lowered levels

The following conditions or diseases may lead to reduced levels of alkaline phosphatase:

• Hypophosphatasia, a genetic disorder
• Women receiving estrogen therapy for menopausal symptoms
• Estrogen-containing oral contraceptives
• Men with recent heart surgery, malnutrition, magnesium deficiency, or severe anemia
• Children with achondroplasia and congenital iodine deficiency
• Children after a severe episode of enteritis
• Pernicious anemia
• Aplastic anemia
• Wilson's disease
• Hypothyroidism
• Zinc deficiency
• Malnutrition
• Steroid treatment
• Colitis
• Thyroid disease

Prognostic uses

Measuring alkaline phosphatase (along with prostate specific antigen) during, and after six months of hormone treated metastatic prostate cancer was shown to predict the survival of patients.

Leukocyte alkaline phosphatase

Leukocyte alkaline phosphatase is found within mature white blood cells. White blood cell levels of LAP can help in the diagnosis of certain conditions.

• Higher than typical levels are seen in the physiological response, the leukemoid reaction, and in pathologies that include mature white blood cells, such as polycythemia vera, essential thrombocytosis, and in primary myelofibrosis.
• Lower than typical levels are found in pathologies that involve undeveloped leukocytes, such as chronic myelogenous leukemia (CML), paroxysmal nocturnal hemoglobinuria and acute myelogenous leukaemia.

Structure and properties

Alkaline phosphatase is homodimeric enzyme, meaning it is formed with two molecules. Three metal ions, two Zn and one Mg, are contained in the catalytic sites, and both types are crucial for enzymatic activity to occur. The enzymes catalyze the hydrolysis of monoesters in phosphoric acid which can additionally catalyze a transphosphorylation reaction with large concentrations of phosphate acceptors. While the main features of the catalytic mechanism and activity are conserved between mammalian and bacterial alkaline phosphate, mammalian alkaline phosphatase has a higher specific activity and K_m values thus a lower affinity, more alkaline pH optimum, lower heat stability, and are typically membrane bound and are inhibited by l-amino acids and peptides via a means of uncompetitive mechanism. These properties noticeably differ between different mammalian alkaline phosphatase isozymes and therefore showcase a difference in in vivo functions.

Alkaline phosphatase has homology in a large number of other enzymes and composes part of a superfamily of enzymes with several overlapping catalytic aspects and substrate traits. This explains why most salient structural features of mammalian alkaline phosphatase are the way they are and reference their substrate specificity and homology to other members of the nucleoside pyrophosphatase / phosphodiesterase family of isozyme. Research has shown a relationship between members of the alkaline phosphatase family with aryl sulfatases. The similarities in structure indicate that these two enzyme families came from a common ancestor. Further analysis has linked alkaline phosphates and aryl sulfatases to a larger superfamily. Some of the common genes found in this superfamily, are ones that encode phosphodiesterases as well as autotoxin.