Enzymatic measures of hepatic cholestasis (alkaline phosphatase, 5'-nucleotidase, gamma-glutamyl transpeptidase)

INTRODUCTION — The liver contains thousands of enzymes, some of which are also present in serum in very low concentrations [1-3]. Elevation of serum enzyme tests can be grouped into two categories:

●Enzymes that reflect generalized damage to hepatocytes

●Enzymes that reflect cholestasis

This topic review will discuss the serum tests that reflect cholestasis. The other categories of liver tests and enzymes that reflect generalized damage to hepatocytes and the evaluation of the patient with abnormal liver tests are discussed separately. (See "Overview of liver biochemical tests" and "Approach to the patient with abnormal liver biochemical and function tests", section on 'Elevated alkaline phosphatase'.)

ALKALINE PHOSPHATASE

Physiologic function — Alkaline phosphatase refers to a group of enzymes that catalyze the hydrolysis of a large number of organic phosphate esters at an alkaline pH optimum. Zinc is an important cofactor. Although alkaline phosphatase is found in many locations throughout the body, its precise function is not known [4]. It appears to have an active role in down-regulating the secretory activities of the intrahepatic biliary epithelium and hydrolyzing phosphate esters to generate inorganic phosphate for uptake by various tissues [5,6]. Intestinal alkaline phosphatase detoxifies lipopolysaccharide and promotes commensal bacterial colonization of the intestine, tight junction integrity, and barrier function, and its absence is associated with increased intestinal inflammation, dysbiosis, bacterial translocation, and systemic inflammation [7,8]. Genetic mutations of the intestinal alkaline phosphatase gene are thought to contribute to some forms of chronic inflammatory diseases [8]. In bone, the enzyme is involved with calcification. At other sites, it may participate in transport processes.

At least three separate genes code for the different alkaline phosphatases that exist in multiple isoenzyme forms [9,10]. Although these alkaline phosphatase isoenzymes catalyze the same reactions, they have different physicochemical properties, which are conferred by carbohydrate and lipid side chains added during posttranslational modification [4,10].

●Most of the alkaline phosphatase isoenzymes of clinical interest are derived from the liver, bone, first trimester placenta, and kidneys, and are coded for by one gene. They are called tissue nonspecific alkaline phosphatase since they have the same immunologic properties and amino acid sequence.

●A second alkaline phosphatase gene codes for third trimester placental and intestinal alkaline phosphatase [9].

●The third gene codes for a second type of intestinal alkaline phosphatase.

Sources — Alkaline phosphatase activity in serum is derived primarily from three sources: liver (more than 80 percent), bone, and, in some patients, the intestinal tract [4,11]. The intestinal contribution (about 10 to 20 percent) is of importance primarily in people with blood groups O and B who are secretors of the ABH red blood cell antigen [12]; it is enhanced by consumption of a fatty meal [13,14].

Circulating alkaline phosphatase appears to behave like other serum proteins [15]. Its half-life is seven days, and its clearance from serum is independent of the functional capacity of the liver or the patency of the bile ducts [15]. Sites of degradation of alkaline phosphatase are unknown.

Measurement

●Assay — The most widely used procedure for determination of serum alkaline phosphatase activity involves measuring the release of p-nitrophenol or phosphate from p-nitrophenylphosphate under specified conditions. The results are expressed in international units (IU/L), which is the activity of alkaline phosphatase that releases 1 mmol of chromogen or inorganic phosphate per minute. Other colorimetric and fluorescent strategies for determining alkaline phosphatase activity and kinetics have been developed [16,17].

●Fractionating serum alkaline phosphatase — Fractionating serum alkaline phosphatase to identify the source of the elevated isoenzyme is helpful if an elevated serum alkaline phosphatase is the only abnormal finding in an apparently healthy person or if the degree of elevation is higher than expected in the clinical setting. In one series of 317 patients evaluated for an increased serum alkaline phosphatase level, the source of elevation was the liver and bone in 80 and 18 percent, respectively [18]. The remaining patients had either a mixed or an intestinal source.

Alkaline phosphatase isoenzymes can be determined using a variety of techniques. However, these methods are not widely available, the most practical approach is to measure other enzymes whose elevation reflects liver disease. These include 5’-nucleotidase and gamma-glutamyl transpeptidase. (See '5'-Nucleotidase' below and 'Gamma-glutamyl transpeptidase' below.)

Methods to assess isoenzymes of alkaline phosphatase include:

•Separation using polyacrylamide gel slabs – This is the most reliable method and produces clear-cut separations of the liver, bone, intestinal, and placental isoenzymes. However, this method is not always available. Electrophoresis on cellulose acetate, with the addition of heat inactivation, may accomplish the same purpose [19].

•Evaluating susceptibility to inactivation by heat – This method is based upon the observation that alkaline phosphatases from individual tissues differ in their susceptibility to inactivation by heat or 2 mol urea [20]. Placental alkaline phosphatase and an isoenzyme found in certain cancers, the Regan isoenzyme, are fully heat-stable after exposure to a temperature of 56°C for 15 minutes [21]. Thus, the finding of an elevated serum alkaline phosphatase concentration in a patient in whom all the excess activity is in a heat-stable fraction strongly suggests that the placenta or a tumor is the source of the elevated enzyme in serum. Unfortunately, in unselected patients, this method has many limitations and is not recommended.

Clinical interpretation

Normal range — Normal serum values of alkaline phosphatase vary based upon demographic and clinical circumstances.

●Patients older than 60 have somewhat higher values (up to 1.5 times normal) than younger adults [22,23].

●In adults 15 to 50 years of age, mean serum alkaline phosphatase activity is somewhat higher in males than in females [24]. In comparison, in individuals over age 60, the enzyme activity of females equals or exceeds that of males [22]. The alkaline phosphatase is usually derived from the liver in older males and from bone in postmenopausal females [22].

●In children, serum alkaline phosphatase activity is considerably elevated in both sexes, correlates well with the rate of bone growth, and appears to be accounted for by the influx of enzyme from osteoid tissue [24,25]. Serum alkaline phosphatase in normal adolescent males may reach mean values three times greater than in normal adults without implying the presence of hepatobiliary disease (figure 1) [24,26-28]. Infants and young children occasionally display marked transient elevation of serum alkaline phosphatase activity in the absence of detectable liver or bone disease. (See "Transient hyperphosphatasemia of infancy and early childhood".)

●Enzyme activity in serum may double late in normal pregnancy, primarily because of an influx from the placenta [29]; in rare cases, higher values have been observed [30].

Elevation in alkaline phosphatase — Increased serum alkaline phosphatase is derived from tissues whose metabolism is either functionally disturbed (the obstructed liver) or greatly stimulated (placenta in the third trimester of pregnancy and bone in growing children). However, an elevation in the alkaline phosphatase concentration is a relatively common finding and does not always indicate the presence of hepatobiliary disease. Rarely, an elevated alkaline phosphatase level is seen because of the presence of macro-alkaline phosphatase. Macro-alkaline phosphatase is rare and occurs due to the formation of complexes of alkaline phosphatase with immunoglobulins (usually IgG), which have reduced renal clearance compared with unbound alkaline phosphatase [31]. The clinical significance of these complexes is uncertain. Macro-alkaline phosphatase should be suspected when all other causes of isolated elevations in alkaline phosphatase have been excluded.

The evaluation of a patient with elevated alkaline phosphatase is discussed in detail separately (table 1 and algorithm 1). (See "Approach to the patient with abnormal liver biochemical and function tests", section on 'Evaluation of elevated alkaline phosphatase'.)

Non-hepatic alkaline phosphatase — The bone is the most likely source of isolated plasma alkaline phosphatase that is not of liver origin (in nonpregnant patients). An elevated bone alkaline phosphatase is indicative of high bone turnover, which may be caused by several disorders including healing fractures, osteomalacia, hyperparathyroidism, hyperthyroidism, Paget disease of bone, osteogenic sarcoma, and bone metastases. (See "Bone physiology and biochemical markers of bone turnover", section on 'Markers of bone turnover' and "Clinical manifestations and diagnosis of Paget disease of bone", section on 'Clinical manifestations' and "Clinical manifestations, diagnosis, and treatment of osteomalacia in adults", section on 'Diagnostic and etiologic evaluation'.)

Intestinal alkaline phosphatase has multiple biological functions [6,32]. Elevated levels of intestinal alkaline phosphatase are usually of no clinical significance. They are most often found postprandially after a fatty meal and tend to run in families, suggesting that there may be a genetic basis [33]. Because elevation is nonspecific, it should not prompt evaluation for a particular intestinal disorder. Normalization of the level while fasting can provide reassurance that its origin is intestinal.

Hepatic alkaline phosphatase — The major value of the serum alkaline phosphatase in the diagnosis of liver disorders is in the recognition of cholestatic disease [34]. Increased serum alkaline phosphatase in hepatobiliary disease is due to regurgitation of hepatic alkaline phosphatase back into serum by the damaged liver rather than a failure to excrete the alkaline phosphatase made in bone, the intestines, and the liver. The data supporting this theory include the following:

●Only hepatic alkaline phosphatase is found in the serum of patients with liver disease, particularly cholestasis [18].

●The clearance rates of infused placental alkaline phosphatase are the same in patients with bile duct obstruction and healthy controls [15].

●In experimental bile duct obstruction in rats, the entire increase in serum alkaline phosphatase activity is due to the leakage of hepatic alkaline phosphatase into serum [35]. The increased serum activity is paralleled by a striking elevation in hepatic alkaline phosphatase activity and is not accounted for by biliary retention of alkaline phosphatase.

Thus, the elevation in serum alkaline phosphatase in hepatobiliary disease results from increased de novo synthesis in the liver followed by release into the circulation [36]. Retained bile acids appear to play a central role in this process; they induce the synthesis of the enzyme and may cause it to leak into the circulation, perhaps by disruption of hepatic organelles and solubilization of phosphatase bound to such membranes [37].

The precise manner in which the alkaline phosphatase reaches the circulation is unclear. In some patients with cholestasis, small vesicles that contain many basolateral (sinusoidal) membrane enzymes still bound to these membranes, including alkaline phosphatase, have been found in serum [38].

Increase alkaline phosphatase and liver tests

●Marked elevation — Approximately 75 percent of patients with prolonged cholestasis have serum alkaline phosphatase values increased to four times the upper limit of normal or more. Elevations of this magnitude can occur in a variety of diseases associated with extrahepatic or intrahepatic obstruction (table 1), and the extent of the elevation does not distinguish between the two [39]:

•Obstructive jaundice due to cancer.

•Bile duct stones.

•Sclerosing cholangitis (primary or secondary).

•Bile duct stricture.

•Drug and toxins associated with cholestasis.

•Primary biliary cholangitis.

•Liver allograft rejection.

•Ischemic and infectious (eg, coronavirus disease [COVID]) cholangiopathy [40].

•Infectious hepatobiliary diseases seen in patients with AIDS (eg, cytomegalovirus or microsporidiosis) and in patients with tuberculosis with hepatic involvement.

•Infiltrative liver disease (eg, sarcoidosis, tuberculosis, metastatic malignancy, amyloidosis).

•Familial cholestatic syndromes.

•Alcohol-associated hepatitis (rarely) [41].

●Moderate elevation — Lesser increases in alkaline phosphates activity, up to four times the upper limit of normal, are nonspecific and occur in all types of liver disease, including viral hepatitis, chronic hepatitis, cirrhosis, infiltrative diseases of the liver, and congestive heart failure (table 1) [3,14,39,42,43]. Elevations in hepatic alkaline phosphatase of this magnitude can also occur in disorders that do not directly involve the liver, such as Hodgkin lymphoma, myeloid metaplasia, intra-abdominal infections, and osteomyelitis [18].

A two- to four-fold elevation in serum alkaline phosphatase levels has been described in several members of a family who had no evidence of bone or liver disease [44]. The elevation appeared to be transmitted as an autosomal dominant trait, but the site of the excess alkaline phosphatase could not be determined.

Isolated elevation — Isolated elevations of hepatic alkaline phosphatase can occur in a number of circumstances including:

●Hepatobiliary causes  

•Partial bile duct obstruction due to gallstones or tumor. The mechanism is unknown but probably represents local areas of bile duct obstruction with induced synthesis of hepatic alkaline phosphatase and leakage of the alkaline phosphatase into the serum.

•Early in the course of some cholestatic liver diseases such as primary sclerosing cholangitis, primary biliary cholangitis, and immunoglobulin G4 (IgG4)-associated cholangitis.

•Infiltrative liver diseases such as amyloidosis, sarcoidosis, hepatic abscesses, tuberculosis, and metastatic carcinoma.

•Ischemic cholangiopathy.

●Extrahepatic diseases

•Nonmalignant causes – Peritonitis, diabetes mellitus, subacute thyroiditis, uncomplicated gastric ulcer, and sepsis. The increase in alkaline phosphatase in these disorders is related to hepatic dysfunction despite the absence of overt liver disease [45]. Vitamin D deficiency has been associated with an isolated elevation of the alkaline phosphatase level [1].

•Malignancy – Myelofibrosis and extrahepatic tumors, including osteosarcomas; lung, gastric, head and neck, renal cell, ovarian, and uterine cancers; and Hodgkin lymphoma, that secrete alkaline phosphatase (often a form known as the Regan isoenzyme) or cause leakage of hepatic alkaline phosphatase into serum by an unknown mechanism [18,46]. Renal cell carcinoma may also be complicated by non-metastatic cholestatic hepatitis, sometimes termed Stauffer's syndrome [47].

●Other causes

•Transient hyperphosphatemia of infancy and childhood – Infants and young children occasionally display marked transient elevation of serum alkaline phosphatase activity in the absence of detectable liver or bone disease. The hyperphosphatasemia typically includes elevations in both bone and liver isoenzymes and appears to be caused by delayed clearance of the enzyme. The clinical presentation and evaluation of an infant or child with this finding is discussed separately. (See "Transient hyperphosphatasemia of infancy and early childhood".)

•Medications – Phenytoin can cause mild-to-moderate elevations in gamma-glutamyl transpeptidase (GGT), which is indicative of hepatic enzyme induction rather than liver injury.

Subnormal values — Extremely low serum alkaline phosphatase concentrations can be seen in patients with acute liver failure due to Wilson disease complicated by hemolysis [48,49]. (See "Wilson disease: Clinical manifestations, diagnosis, and natural history".)

Low values can also occur in patients with hypothyroidism, pernicious anemia, zinc deficiency, hypomagnesemia, malnutrition, certain drugs (eg, oral contraceptives), congenital hypophosphatemia, and certain types of progressive familial intrahepatic cholestasis in children.

5'-NUCLEOTIDASE

Source — 5'-nucleotidase is found in the liver, intestine, brain, heart, blood vessels, and endocrine pancreas but it is only released into serum by hepatobiliary tissue [50]. Although its physiologic function is unknown, 5'-nucleotidase specifically catalyzes hydrolysis of nucleotides such as adenosine 5'-phosphate and inosine 5'-phosphate in which the phosphate is attached to the 5 position of the pentose moiety.

Similar to alkaline phosphatase, 5'-nucleotidase has a subcellular location in hepatocytes [51]. It is bound to bile canalicular and sinusoidal membranes and must be solubilized, perhaps via the detergent action of bile acids, to gain access to the circulation [52]. In experimental bile duct obstruction in rats, bile acid concentrations rapidly reach levels sufficient to disrupt plasma membranes and solubilize the enzyme [53]. The same phenomenon may occur in hepatobiliary disorders in which there is any degree of cholestasis [51].

Measurement — Serum 5'-nucleotidase activity is most commonly assayed by measuring the release of inorganic phosphate using 5'-phosphate as a substrate [54]. Serum 5'-nucleotidase concentration is not clearly influenced by sex or race [55,56]. Values are substantially lower in children than in adults, rise gradually in adolescence, and reach a plateau after age 50 [57]. The normal range is 2 to 17 IU/L.

Clinical application — The major value of the 5'-nucleotidase assay is its specificity for hepatobiliary disease. Conflicting data have been reported for serum 5'-nucleotidase activity during normal pregnancy [58,59]. In contrast, most studies show that serum 5'-nucleotidase does not rise in bone disease [60], and in the few instances in which an increase was observed, it was of low magnitude [61].

Elevations in serum 5'-nucleotidase are seen in the same types of hepatobiliary diseases associated with an increased serum alkaline phosphatase. Most studies suggest that serum alkaline phosphatase and 5'-nucleotidase are equally valuable in demonstrating biliary obstruction or hepatic infiltrative and space-occupying lesions [56,62]. Although values of the two enzymes are generally correlated, the concentrations may not rise proportionately in individual patients [55,61]. Thus, in selected patients, one enzyme may be elevated and the other normal.

An increased serum 5'-nucleotidase concentration in a nonpregnant person suggests that a concomitantly increased serum alkaline phosphatase is of hepatic origin. However, because of the occasional dissociation between the two enzymes, a normal serum 5'-nucleotidase does not rule out the liver as the source of an elevated serum alkaline phosphatase. (See "Approach to the patient with abnormal liver biochemical and function tests", section on 'Elevated alkaline phosphatase'.)

GAMMA-GLUTAMYL TRANSPEPTIDASE

Function and source — Gamma-glutamyl transpeptidase (GGT) catalyzes the transfer of the gamma-glutamyl group from gamma-glutamyl peptides such as glutathione to other peptides and to L-amino acids. GGT is present in hepatocytes and biliary epithelial cells and the cell membranes in many tissues, including the kidneys, pancreas, liver, spleen, heart, brain, and seminal vesicles [63]. It is thought to play a role in amino acid transport [64].

Measurement — Enzyme activity is most commonly assayed using gamma-L-glutamyl-r-nitroanilide as a substrate [65]. The liberated product, (chromogen r-nitroaniline) can be measured spectrophotometrically.

Clinical application — Aside from its value in conferring liver specificity to an elevated serum alkaline phosphatase level and its possible use in identifying patients with alcohol use disorder, serum GGT offers no advantage over aminotransferases and alkaline phosphatase. Fluorescent probes to detect GGT have been studied as tools for cancer imaging [66].

Normal range — GGT is present in the serum of healthy individuals. The normal range is 0 to 30 IU/L (0 to 0.5 mkat/L). Most studies have found values to be comparable in males and females [67,68], although some reports have noted higher values in males [69]. Newborn infants have serum GGT activity six to seven times the upper limit of the adult reference range; levels decline and reach adult levels by five to seven months of age [70]. A gradual increase occurs in girls until age 10 and in boys through adolescence [71]. Serum enzyme activity does not rise during the course of normal pregnancy [68].

Elevated GGT and alkaline phophatase — Elevated serum activity is found in diseases of the liver, biliary tract, and pancreas, and reflects the same spectrum of hepatobiliary disease as alkaline phosphatase and 5’-nucleotidase. Serum GGT and alkaline phosphatase correlate reasonably well. There are conflicting data as to whether serum GGT has better sensitivity for hepatobiliary disease than alkaline phosphatase [68,72].

As with serum 5’-nucleotidase, the major clinical value of serum GGT is in conferring organ specificity to an elevated value for alkaline phosphatase since GGT activity is not increased in patients with bone disease [68].

Isolated elevation or disproportionate elevation — Elevation in serum GGT is not completely specific for hepatobiliary disease. High serum GGT values have been associated with the use of barbiturates or phenytoin [73] or ingestion of large quantities of alcohol [73,74], even when values for other serum enzyme tests and serum bilirubin are normal. In these settings, there is no correlation between the serum GGT and alkaline phosphatase [63]. Rarely, isolated high serum GGT level occurs in families on a genetic basis [75].

An isolated elevation in serum GGT or a GGT elevation out of proportion to that of other enzymes (such as the alkaline phosphatase and alanine aminotransferase) may be an indicator of alcohol use or alcohol-associated liver disease [76]. In one prospective study that included 1040 nonselected inpatients, 13 percent had an elevated serum GGT activity; only 32 percent of them had hepatobiliary disease [77]. In the remaining patients, the elevated serum GGT may have been due to alcohol ingestion or medications, which caused a temporary rise in levels but without liver disease.

The reasons for the elevation in serum GGT with alcohol use are not well understood. Although hepatic microsomal GGT may be induced by alcohol [78], neither elevated serum GGT nor a history or recent alcohol ingestion correlates with hepatic GGT activity in patients with biopsy-proven alcohol-associated liver disease [79,80]. In vitro studies suggest an alternative mechanism: alcohol may cause the leakage of GGT from hepatocytes [78].

SUMMARY AND RECOMMENDATIONS

●Alkaline phosphatase function and sources – Alkaline phosphatase refers to a group of enzymes that catalyze the hydrolysis of a large number of organic phosphate esters at an alkaline pH optimum. Although alkaline phosphatase is found in many locations throughout the body, its precise function is not fully understood. Alkaline phosphatase activity in serum is derived primarily from three sources: liver (more than 80 percent), bone, and in some patients, the intestinal tract. (See 'Sources' above.)

●Causes of alkaline phosphatase elevation – The major value of serum alkaline phosphatase in the diagnosis of liver disorders is in the recognition of cholestatic disease (table 1). However, an elevation in the alkaline phosphatase concentration is a relatively common finding and does not always indicate the presence of hepatobiliary disease. The degree of elevation does not distinguish intra- from extra-hepatic cholestasis. (See 'Clinical interpretation' above and "Approach to the patient with abnormal liver biochemical and function tests", section on 'Elevated serum aminotransferases'.)

●Role of 5'-nucleotidase measurement – Elevations in serum 5'-nucleotidase are seen in the same types of hepatobiliary diseases associated with an increased serum alkaline phosphatase. The value of the 5'-nucleotidase assay is its specificity for hepatobiliary disease. An increased serum 5'-nucleotidase concentration in a nonpregnant person suggests that a concomitantly increased serum alkaline phosphatase is of hepatic origin. However, because of the occasional dissociation between the two enzymes, a normal serum 5'-nucleotidase does not rule out the liver as the source of an elevated serum alkaline phosphatase. (See '5'-Nucleotidase' above.)

●Cause of gamma-glutamyl transpeptidase elevation – Elevated serum levels of gamma-glutamyl transpeptidase (GGT) are found in diseases of the liver, biliary tract, and pancreas, and reflect the same spectrum of hepatobiliary disease as alkaline phosphatase and 5'-nucleotidase. Serum GGT and alkaline phosphatase correlate reasonably well. There are conflicting data as to whether serum GGT has better sensitivity for hepatobiliary disease than alkaline phosphatase. The major clinical value of serum GGT is in conferring organ specificity to an elevated value for alkaline phosphatase since GGT activity is not increased in patients with bone disease. However, an elevation in serum GGT is not specific for hepatobiliary disease and may be elevated due to concurrent medications or alcohol use. (See 'Gamma-glutamyl transpeptidase' above.)