Alkaline phosphatases are a group of isoenzymes, located on the outer layer of the cell membrane; they catalyze the hydrolysis of organic phosphate esters present in the extracellular space. Zinc and magnesium are important co-factors of this enzyme. Although alkaline phosphatases are present in different body tissues and have different physiochemical properties, these are true isoenzymes because they catalyze the same reaction. In the liver, alkaline phosphatase is cytosolic and present in the canalicular membrane of the hepatocyte. Alkaline phosphatase is present in decreasing concentrations in placenta, ileal mucosa, kidney, bone, and liver, but the majority of the alkaline phosphatase in serum (more than 80%) is released from liver and bone, and in small amounts from the intestine. Even though alkaline phosphatases are found in many tissues throughout the body, their precise physiological function remains largely unknown.
Alkaline phosphatases are classified as tissue-specific and tissue nonspecific types. Alkaline phosphatases found in the intestine, placenta, and germinal tissue are tissue-specific, which means they are found only in the tissues where they are expressed in physiological conditions but may contribute to the circulating pool of serum alkaline phosphatase under specific situations when there is increased stimulation of their production. The tissue-nonspecific alkaline phosphatase forms the majority of the fraction circulating in serum and therefore, is of clinical interest. It is encoded by a single gene and is expressed in the liver, bone, and kidneys. Intestinal alkaline phosphatase is coded by a separate gene, which is different from the gene that codes for placental alkaline phosphatase and the Regan isoenzyme (which is produced in excess amounts in Hodgkin lymphoma). All tissue-nonspecific AP have the same amino acid sequence but different carbohydrate and lipid side chains; post-translational modifications confer their unique physicochemical properties.
Serum alkaline phosphatase levels vary with age in normal individuals. During childhood and puberty, due to bone growth and development, levels are high. The levels decrease in the 15 to 50-year age group, are slightly higher in men than in women, and rise again in old age (significant difference in gender distribution). The reasons for these normal variations are not known. A positive correlation has been described with body weight and smoking, and there is an inverse correlation with height.
In healthy individuals, the circulating enzyme is primarily derived from liver and bone, and in some individuals to a minimal extent from the intestinal tract. In individuals with blood groups O and B, serum alkaline phosphatase levels increase after consuming a fatty meal, due to contribution from the intestinal tract. As this elevation can persist for up to 12 hours in the serum, it is recommended to check the serum enzyme levels in a fasting state.
Alkaline phosphatase behaves like any other serum protein. It has a half-life of 7 days, and clearance from serum is independent of bile duct patency or functional capacity of the liver. Tthe site of degradation of alkaline phosphatase is not known. Serum alkaline phosphatase level may remain elevated for up to 1 week after resolution of biliary obstruction. The liver is the source in most patients with elevated enzyme levels. Increased osteoblastic activity seen in disorders of the bone or normally during periods of growth is the next likely contributor. The influx of placental alkaline phosphatase in late third trimester contributes to the rise in pregnant women.
The mechanism of the increase in alkaline phosphatase in hepatobiliary disorders had been a matter of debate. It has been shown convincingly that it is due to increased enzyme synthesis and not to reduced hepatobiliary excretion of the enzyme. Increased hepatic enzyme activity has been demonstrated to parallel the rise in serum AP activity. This occurs primarily due to increased translation of the mRNA of alkaline phosphatase (mediated by the rising bile acid concentration) and increased secretion of alkaline phosphatase into serum via canalicular leakage into the hepatic sinusoid. The mechanism by which it is released into the circulation has not been elucidated. It has been reported that vesicles containing alkaline phosphatase, and many such enzymes bound to the sinusoidal membranes, are found in the serum of patients with cholestasis. Because alkaline phosphatase is newly synthesized in response to biliary obstruction, its serum level may be normal in the early phase of acute biliary obstruction even when the serum aminotransferases are already at their peak.
There are a number of clinical methods for determination of serum alkaline phosphatase levels. They differ by substrate used, ph of the alkaline buffer, and the “normal” values generated. The tests in principle rely on the ability of the enzyme to hydrolyze phosphate esters. In the most widely used international method, p-nitrophenyl phosphate is used as the substrate, and an amino alcohol is used as a buffer. The rate of release of p-nitrophenol or phosphate from the substrate is measured as a marker of alkaline phosphatase activity and results reported in international units per liter. The different methods appear equally effective in the detection of abnormal values in various clinical diseases. Using multiples of the upper limit of normal is a simple way of comparing results obtained via different tests.
Electrophoresis does not reliably differentiate the isoenzymes as the electrophoretic mobility of bone and liver isoenzymes is only slightly different. Electrophoresis on cellulose acetate, with the addition of heat inactivation, is a much reliable test than electrophoresis alone. Polyacrylamide gel slab based separation provides accurate identification of the liver, bone, intestinal and placental isoenzymes; this test is, however, not widely available.
Patients with blood group O and B may be instructed to fast before the test to avoid contribution from the intestinal isoenzyme if there is an unexplained elevation of alkaline phosphatase on routine tests. A gold-top serum separator tube containing a clot activator and serum gel separator is used to collect the blood for analysis.
There are many potential analytic sources of error. Factors such as concentrations of phosphate, magnesium, citrate, type, and concentration of buffer maintenance of the correct temperature may affect the final result.
When alkaline phosphatase is the only liver biochemical test that is elevated (i.e., when the serum aminotransferases are within normal limits), or when alkaline phosphatase is disproportionately elevated compared to other liver biochemical tests, evaluation of the patient should be geared toward identifying the cause and the source for the isolated or disproportionate alkaline phosphatase elevation. In asymptomatic patients with isolated elevation of serum alkaline phosphatase, it is important to identify the major source of abnormality. As the alkaline phosphatases derived from the liver, bone, placenta, and intestines have different physicochemical properties, fractionation using electrophoresis can provide serum levels of specific isoenzymes. The results using current techniques, however, are often unreliable and inaccurate. Other methods that relied on the heat or urea denaturation of the different isoenzymes have poor sensitivity and specificity.
The current approach utilizes measurement of the activity of those enzymes which increase in concordance with the liver alkaline phosphatase such as 5’-nucleotidase (5NT) and gamma-glutamyl transpeptidase (GGTP). These enzymes are not elevated in disorders of bone and correlate well with hepatobiliary disorders. Serum GGTP is very sensitive for biliary tract disease but is less specific for liver disorders. The 5NT may be elevated in pregnant patients; however, in non-pregnant patients, it is fairly specific for liver disorder and correlates strongly with serum alkaline phosphatase of liver origin. However, lack of an elevated 5NT in the presence of an elevated alkaline phosphatase does not rule out hepatobiliary disease as they do not rise concomitantly in early or mild hepatic injury.
The principal clinical value of measuring serum alkaline phosphatase lies in the diagnosis of cholestatic liver disease. Some of the highest elevations in alkaline phosphatases are seen in patients with cholestasis. Usually, four-fold of the upper limit of normal or greater elevation is seen in up to 75% of the patients with cholestasis, either intrahepatic or extrahepatic. The degree of elevation is not helpful in distinguishing the two types. Similar elevations are seen in biliary obstruction due to cancer (cholangiocarcinoma, pancreatic head adenocarcinoma, or ampullary adenocarcinoma), choledocholithiasis, biliary stricture, sclerosing cholangitis, or causes of intrahepatic cholestasis such as primary biliary cholangitis, drug-induced liver injury, chronic rejection of liver allografts, infiltrative liver disease (sarcoidosis, amyloidosis, tuberculosis and liver metastasis),severe alcoholic hepatitis causing steatonecrosis. Patients with AIDS may also have particularly high levels, either due to cholangiopathy from opportunistic infections such as cytomegalovirus, cryptosporidiosis, or granulomatous involvement of the liver from tuberculosis.
Moderate elevation (up to four times the upper limit of normal) of serum alkaline phosphatase is nonspecific as it can occur in a variety of conditions affecting the liver including cirrhosis, chronic hepatitis, viral hepatitis, congestive heart failure and ischemic cholangiopathy. Disorders that do not primarily involve the liver such as intra-abdominal infections, cholestasis of sepsis, Hodgkin lymphoma, myeloid metaplasia and osteomyelitis can also cause moderate elevation of serum alkaline phosphatase.
Primary or metastatic cancer raises serum alkaline phosphatase levels by local bile duct obstruction and increasing leakage of the liver isoenzyme. Primary extrahepatic cancer does not necessarily have to involve the liver or the bone; rarely some tumors can produce their alkaline phosphatase (Hodgkin lymphoma secreting the Regan isoenzyme) or exert a paraneoplastic effect causing leakage of the hepatic isoenzyme into the circulation (Stauffer syndrome due to renal cell carcinoma).
Abnormally low levels can be useful clinically as they are seen in Wilson disease, especially when presenting in a fulminant form with hemolysis. Zinc is a cofactor of Alkaline phosphatase, which gets displaced by copper in Wilson disease, a disorder of copper overload, thereby leading to low levels. Other causes of low alkaline phosphatase levels are zinc deficiency, pernicious anemia, hypothyroidism, and congenital hypophosphatasia.
An extensive evaluation is often not needed in those patients who have only a mild elevation of serum alkaline phsophatase (less than 50% elevation). Such patients may be observed clinically with periodic monitoring of serum liver biochemical tests. Whenever alkaline phosphatase levels are deemed to be abnormally elevated, further evaluation should be done to determine whether the source is hepatic or non-hepatic. Hepatic source for an elevated alkaline phosphatase level is supported by the concomitant elevation of either GGTP or 5NT. Electrophoresis and other methods such as stability after heating cannot accurately differentiate the isoenzymes. As the hepatic alkaline phosphatase is more heat stable than bone alkaline phosphatase, alkaline phosphatase fractionation based on its heat stability is less reliable than GGTP or 5NT. If the source is deemed to be non-hepatic, then evaluation of underlying undiagnosed disorder is the next step. An elevated bone alkaline phosphatase can occur in bone metastasis, Paget disease, osteogenic sarcoma, healing fractures, hyperparathyroidism, hyperthyroidism, and osteomalacia. Elevated intestinal fraction tends to occur after a fatty meal and runs in families; this does not require additional evaluation. If the liver is suspected to be the source, imaging of the biliary tree is necessary to differentiate between extrahepatic or intrahepatic cholestasis in addition to reviewing the medication list. A right upper quadrant ultrasonography is often the first imaging study that is ordered. If the bile duct is dilated, either endoscopic retrograde cholangiopancreatography (ERCP) or magnetic resonance cholangiopancreatography (MRCP) is done depending on the clinical indication. If the bile duct is not dilated, testing for serum antimitochondrial antibody (AMA) is suggested to evaluate for primary biliary cholangitis (PBC). If serum AMA is normal, evaluation for causes of intrahepatic cholestasis, AMA-negative PBC, sarcoidosis, and various other previously mentioned disorders should be performed. Liver biopsy is often the final test employed in such situations as it helps to identify the etiology of elevated serum alkaline phosphatase.
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