Introduction

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, they 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.[1] Alkaline phosphatase is present in decreasing concentrations in the placenta, ileal mucosa, kidney, bone, and liver. The majority of alkaline phosphatase in serum (more than 80%) is released from the liver and bone, and in small amounts from the intestine. Even though alkaline phosphatases are present in many tissues throughout the body, their precise physiological function remains largely unknown.[2]

Alkaline phosphatases are classified as tissue-specific and tissue-nonspecific types. Alkaline phosphatases found in the intestine, placenta, and germinal tissue are tissue-specific.[3] This means they are found only in the tissues where they are expressed in physiological conditions. They may also contribute to the circulating pool of serum alkaline phosphatase under specific situations when there is increased stimulation of their production. The tissue-nonspecific alkaline phosphatases form most of the fraction circulating in serum and, therefore, are of clinical interest.[4] A single gene encodes it 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 (produced in excess amounts in Hodgkin lymphoma).[5] All tissue-nonspecific alkaline phosphatases have the same amino acid sequence but different carbohydrate and lipid side chains; post-translational modifications confer their unique physicochemical properties.[6]

Etiology and Epidemiology

Serum alkaline phosphatase levels will vary with age in normal individuals. Levels are high during childhood and puberty due to bone growth and development. The decrease in level in the 15 to the 50-year age group is slightly higher in men than in women.[1] These levels rise again in old age (significant difference in gender distribution). The reasons for these normal variations are not known. Research has shown a positive correlation between body weight and smoking, and there is an inverse correlation with height.[7]

In healthy individuals, the circulating enzyme is primarily derived from the liver and bone. In some individuals, this enzyme comes from the intestinal tract to a minimal extent.[8] 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, the recommendation is to check the serum enzyme levels in a fasting state.[9]

Pathophysiology

The human alkaline phosphatases (hALP) are found anchored on the cell membrane by glycosylphosphatidylinositol. They are released in the serum by the action of specific phospholipase.[10] It has a half-life of 7 days, and clearance from serum is independent of bile duct patency or functional capacity of the liver. However, the site of degradation of alkaline phosphatase is not known. Serum alkaline phosphatase levels may remain elevated for up to 1 week after the resolution of biliary obstruction.[1] The liver is the source in most patients with elevated enzyme levels. Increased osteoblast 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 the late third trimester contributes to the rise in pregnant women.[11]

The mechanism of the increase in alkaline phosphatase in hepatobiliary disorders has been a matter of debate. Research has convincingly shown that it is due to increased enzyme synthesis and not to reduced hepatobiliary excretion of the enzyme.[11] Increased hepatic enzyme activity demonstrably parallels the rise in serum alkaline phosphatase 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 that precipitates its release into circulation has not been elucidated.[1] Studies report 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.[12]

Specimen Requirements and Procedure

Blood should be drawn after a fast of at least 8 hours. Serum and heparinized plasma give the same results. Slight hemolysis is tolerable, but gross hemolysis should be avoided. Certain sample storage conditions tend to increase serum ALP. There is a significant increase in activity after the warming of previously refrigerated or frozen sera.[13] The ALP activity in fresh serum increases by up to 2% in 6 hours at 25°C. Increases of up to 30% of ALP activity occur after the frozen serum is thawed and in lyophilized specimens after reconstitution. These increases may be due to the release of ALP from complexes with lipoproteins or because the non-complexed enzyme has greater activity. It is best to analyze ALP specimens the same day they are drawn.[14] The specimen can be drawn either in a plain red-top tube or a speckled-red-top tube with gel as a serum separator, employing normal phlebotomy procedures. To avoid contamination, do not draw tubes containing anticoagulants before the red-top/speckled-red-top tube.[15]

Diagnostic Tests

There are several clinical methods for the 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.[16] In the most widely used international method, p-nitrophenol phosphate serves as the substrate, while amino alcohol is used as a buffer. The rate of release of p-nitrophenol phosphate from the substrate is measurable as a marker of alkaline phosphatase activity and results are reported in international units per liter (IU/L).[17] 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.[18]

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 more 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.[19] 

Testing Procedures

The alkaline phosphatase test is performed on semi-automatic or fully automated analyzers which are based on the principle of photometry. Photometry is the measurement of light absorbed in the ultraviolet (UV) to visible (VIS) to infrared (IR) range. This measurement is used to determine the amount of an analyte in a solution or liquid. Photometers utilize a specific light source and detectors that convert light passed through a sample solution into a proportional electrical signal. These detectors may be photodiodes, photoresistors, or photomultipliers.[20] Photometry uses Beer–Lambert’s law to calculate coefficients obtained from the transmittance measurement. A correlation between absorbance and analyte concentration is then established by a test-specific calibration function to achieve highly accurate measurements.[21] The electrophoretic technique is most commonly used for differentiating the ALP isozymes and isoforms. However, because there may still be some degree of overlap between the fractions, electrophoresis in combination with another separation technique may provide the most reliable information.[22] A direct immunochemical method for the measurement of bone-related ALP is now available.[23]

The liver fraction migrates the fastest, followed by bone, placental, and intestinal fractions. Because of the similarity between liver and bone phosphatases, there often is not a clear separation between them. Quantitation with the use of a densitometer is sometimes difficult because of the overlap between the two peaks. The liver isoenzyme can actually be divided into two fractions— the major liver band and a smaller fraction called fast liver, or α1 liver, which migrates anodal to the major band and corresponds to the α1 fraction of protein electrophoresis.[19] When total ALP levels are increased, the major liver fraction is the most frequently elevated. Many hepatobiliary conditions cause elevations of this fraction, usually early in the course of the disease. The fast-liver fraction has been reported in metastatic carcinoma of the liver, as well as in other hepatobiliary diseases.[24] Its presence is regarded as a valuable indicator of obstructive liver disease. However, it is occasionally present in the absence of any detectable disease state.[25]

The difference in heat stability is the basis of a second approach used to identify the isoenzyme source of an elevated ALP.[26] Typically, ALP activity is measured before and after heating the serum at 56°C for 10 minutes. If the residual activity after heating is less than 20% of the total activity before heating, then the ALP elevation is assumed to be a result of bone phosphatase. If greater than 20% of the activity remains, the elevation is probably a result of liver phosphatase. These results are based on the finding that placental ALP is the most heat stable of the four major fractions, followed by intestinal, liver, and bone fractions indecreasing order of heat stability. Placental ALP will resist heat denaturation at 65°C for 30 minutes.[11] Heat inactivation is an imprecise method for differentiation because inactivation depends on many factors, such as correct temperature control, timing, and analytic methods sensitive enough to detect small amounts of residual ALP activity. In addition, there is some degree of overlap between heat inactivation of liver and bone fractions in both liver and bone diseases.[27]

The third approach to the identification of ALP isoenzymes is based on selective chemical inhibition. Phenylalanine is one of several inhibitors that have been used. Phenylalanine inhibits intestinal and placental ALP to a much greater extent than liver and bone ALP. With phenylalanine use, however, it is impossible to differentiate placental from intestinal ALP or liver from the bone ALP.[28] In addition to the four major ALP isoenzyme fractions, certain abnormal fractions are associated with neoplasms. The most frequently seen are the Regan and Nagao isoenzymes.[29] They have been referred to as carcinoplacental alkaline phosphatases because of their similarities to the placental isoenzyme. The Regan isoenzyme migrates to the same position as the bone fraction and is the most heat-stable of all ALP isoenzymes, resisting denaturation at 65°C for 30 minutes. Its activity is inhibited by phenylalanine.[30] The Nagao isoenzyme may be considered a variant of the Regan isoenzyme. Its electrophoretic, heat stability and phenylalanine inhibition properties are identical to those of the Regan fraction.[31] However, Nagao also can be inhibited by L-leucine. Its presence has been detected in metastatic carcinoma of pleural surfaces and in adenocarcinoma of the pancreas and bile duct.[32]

Interfering Factors

ALP is inhibited by metal-complexing anticoagulants. They inhibit the enzyme by complexing Mg and Zn and should not be used. Hemolyzed and lipemic specimens should be rejected if they have a high background absorbance. Bilirubin at concentrations of up to 20 mg/dL does not interfere.[33] The purity of transphosphorylation buffer is of great importance. Sources of diethanolamine (DEA) have been found to contain significant concentrations of monoethanolamine (MEA), a potent inhibitor of ALP. Solutions of DEA can deteriorate during storage, with the concomitant formation of MEA.[34] Some commercial p- nitrophenol phosphate (pNPP) substrate preparations contain excessive amounts of pNP or inorganic phosphate, or both, with the former producing high blank absorbance and the latter inhibiting ALP.[35]

Serum ALP levels are affected by many drugs, physical conditions, herbal medicines, food intake, smoking, alcohol intake, and pregnancy. Some 1000 drugs, herbs, and physiological conditions that affect ALP activity have been documented. Clofibrate lowers serum ALP activity, reducing all ALP fractions except the liver; this may be caused by increased biliary clearance of ALP.[10] Azathioprine lowers ALP by an unknown mechanism.[36] Estrogens, alone or in combination with androgens, depress ALP activity, whereas, in other studies, estrogens and androgens increased serum ALP activity. The decrease seen with estrogen therapy may result from a lowered rate of bone turnover.[37]

Verapamil, widely used in the treatment of cardiac arrhythmias, angina pectoris, and essential hypertension, increases serum ALP in hypertensive patients. When verapamil is administered to hypertensive patients for 2 months, total ALP activity increased significantly, and an increase in bone ALP in serum is subsequently observed. These results suggest that verapamil may affect bone metabolism which is secondary to the enhancement of parathyroid hormone secretion.[38] Any drug that is hepatotoxic or induces cholestasis will increase serum ALP, sometimes dramatically.[39]

Alkaline phosphatase assays are susceptible to negative interference because alkali denaturation of hemoglobin may cause a negative offset in absorbance readings. Icterus exerts effects on chemistry tests primarily through spectrophotometric and chemical interferences. Bilirubin absorbs light between 400 and 540 nm, with a peak around 460 nm.[40] Colorimetric assays taking primary or secondary absorbance measurements at these wavelengths may be affected. The unconjugated and conjugated forms of bilirubin may exert different effects on certain assays. Conjugated bilirubin has been found to cause a greater degree of interference with most assays.[41]

Results, Reporting, and Critical Findings

When alkaline phosphatase is the only liver biochemical test that presents as 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 focus on 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 essential to identify the primary source of abnormality.[10] Alkaline phosphatases derived from the liver, bone, placenta, and intestines have different physicochemical properties.[42]

One approach utilizes the 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 (GGT). These enzymes are not elevated in disorders of bone and correlate well with hepatobiliary disorders. Serum GGT is very sensitive to biliary tract disease but is less specific for liver disorders.[43] The 5’-nucleotidase ( 5NT) levels may become elevated in pregnant patients; however, in non-pregnant patients, it is relatively specific for liver disorder and correlates strongly with serum alkaline phosphatase of liver origin. However, the 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.[44]

Clinical Significance

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 present in patients with cholestasis. Usually, four-fold of the upper limit of normal or greater elevation occurs in up to 75% of the patients with cholestasis, either intrahepatic or extrahepatic.[10] The degree of elevation does not help distinguish the two types. Similar elevations occur 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.[45] Patients with AIDS may also have particularly high levels, either due to cholangiopathy from opportunistic infections such as cytomegalovirus, cryptosporidiosis, or granulomatous liver involvement from tuberculosis.[46]

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.[47] 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.[10]

Primary or metastatic cancer raises serum alkaline phosphatase levels by local bile duct obstruction and increasing leakage of the liver isoenzyme.[48] Primary extrahepatic cancer does not necessarily have to involve the liver or the bone; rarely, some tumors can produce their own 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).[49]

Abnormally low levels can be useful clinically as they are seen in Wilson's disease, especially when presenting in a fulminant form with hemolysis. Zinc is a cofactor of Alkaline phosphatase, which gets displaced by copper in Wilson's disease, a disorder of copper overload, thereby leading to low levels.[50] Other causes of low alkaline phosphatase levels are zinc deficiency, pernicious anemia, hypothyroidism, and congenital hypophosphatasia.[51]

Transient very high levels of ALP (up to 30 times URL) have been recorded in children, but the clinical significance of this finding is unknown. This has been called transient hyperphosphatasaemia and may be either the bone or the liver isoenzyme.[52] Patients are usually asymptomatic, with unremarkable history, physical exam, and laboratory results. Some patients have had mild viral conditions in the recent past.[53]

An extensive evaluation is often not needed in those patients who have only a mild elevation of serum alkaline phosphatase (less than 50% elevation). Such patients may be observed clinically with periodic monitoring of serum liver biochemical tests. Whenever alkaline phosphatase levels are abnormally elevated, further evaluation should take place to determine whether the source is hepatic or non-hepatic.[54] A hepatic source for an elevated alkaline phosphatase level is supported by the concomitant elevation of either GGT or 5NT. If the source is non-hepatic, then the next step is to evaluate underlying undiagnosed disorders. 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.[55] 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.[45]

Right upper quadrant ultrasonography is often the first imaging study ordered. If the bile duct has become dilated, either endoscopic retrograde cholangiopancreatography (ERCP) or magnetic resonance cholangiopancreatography (MRCP) is done depending on the clinical indication. If the bile duct does not show dilation, testing for serum antimitochondrial antibody (AMA) is the suggested next step to evaluate for primary biliary cholangitis (PBC).[56] If serum AMA level is normal, evaluation for causes of intrahepatic cholestasis, AMA-negative PBC, sarcoidosis, and various other previously mentioned disorders is necessary. Liver biopsy is often the final test employed in such situations as it helps to identify the etiology of elevated serum alkaline phosphatase.[57]

Quality Control and Lab Safety

For non-waived tests, laboratory regulations require, at the minimum, analysis of at least two levels of control materials once every 24 hours. Laboratories can assay QC samples more frequently if deemed necessary to ensure accurate results. Quality control samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance. To minimize QC when performing tests for which manufacturers’ recommendations are less than those required by the regulatory agency (such as once per month),[58] the labs can develop an individualized quality control plan (IQCP) that involves performing a risk assessment of potential sources of error in all phases of testing and putting in place a QC plan to reduce the likelihood of errors.[59] Westgard multi-rules are used to evaluate the quality control runs. In case of any violation of a rule, proper corrective and preventive action should be taken before patient testing is performed.[60]

Acceptable performance (accuracy) for alkaline phosphatase assays according to the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88) is the target value ± 30%, which is also the acceptability criteria used by the College of American Pathologists (CAP) in its surveys.[61] A laboratory must score 80% in each testing event for each analyte and overall in all analytes. Each laboratory must establish its own acceptability criteria for intra-daily and inter-daily variations based on the instrument and quality-control materials.[62]

Enhancing Healthcare Team Outcomes

An alkaline phosphatase test may be ordered by a healthcare provider as part of a routine checkup or if the patient has symptoms of a bone disorder or liver damage. Proper evaluation and classification, by the patient's healthcare team, of any abnormal alkaline phosphatase levels and related findings result in better healthcare outcomes for the patient.