Enzymes
Serum alkaline phosphatase
Alkaline phosphatases are enzymes that catalyze hydrolysis of organic phosphate esters at an alkaline pH. These enzymes are found in many tissues. The serum enzyme is principally derived from three sources: (a) the hepatobiliary system: the bile canalicular surface of the hepatocytes and biliary epithelium, (b) bone: the osteoblasts, and (c) the intestinal tract: the brush border of the intestinal mucosal cells (10% of the total serum enzyme). The enzyme has a half-life of about 7 days in the body.
The alkaline phosphatase activity in individuals 18 to 60 years of age is higher in men than in women. After 60 years of age, the level increases in both genders and may be higher in women than men. It is much higher in children than in adults and corresponds to bone growth and osteoblastic activity. In pregnancy, placental phosphatase may cause doubling of serum alkaline phosphatase, especially in the third trimester.
In a patient with elevated alkaline phosphatase, the hepatobiliary system, bone, and occasionally the small intestine and kidney may be the source of the increased enzyme activity. To determine the source of the elevated enzyme, several approaches may be used. Polyacrylamide gel electrophoretic separation of isoenzymes is the most accurate. The heat susceptibility of the enzyme from different sources seems to be different. The heat stability at 56В°C for 15 minutes decreases in the following order: the placenta, liver, and bone. Unfortunately, there is overlap, and results are difficult to interpret and not diagnostically useful.
The preferred approach to differentiate hepatobiliary enzyme from others is to measure the serum activity of another, similar enzyme elevated in liver dysfunction, such as 5’-nucleotidase, Оі-glutamyltanspeptidase, or leucine aminopeptidase. However, lack of elevation of these enzymes in the setting of elevated alkaline phosphatase does not rule out liver disease, because these enzymes do not necessarily rise in parallel with alkaline phosphatase.
Hepatobiliary alkaline phosphatase synthesis and leakage into the circulation seem to be mediated by bile acids. In intra- and extrahepatic biliary obstruction, alkaline phosphatase is elevated before jaundice develops. Values may be three to ten times normal with a minimal rise in the transaminases. In hepatocellular diseases that primarily affect the liver parenchyma (cirrhosis, hepatitis), the alkaline phosphatase may not rise or may rise minimally with a concomitant large rise in transaminases.
Serum alkaline phosphatase elevation (2-10 times normal) is also helpful in the early diagnosis of infiltrative diseases of the liver, including granulomatous involvement with tuberculosis, sarcoidosis, fungal infection, tumors (primary or metastatic), and abscesses.
In summary, the major value of the serum alkaline phosphatase measurement is in differentiation of hepatocellular liver disease from obstructive liver diseases such as bile duct obstruction with stones, tumors, strictures, or granulomas within or outside the liver parenchyma (Table CAUSES OF AN ISOLATED ALKALINE PHOSPHATASE ELEVATION).
5’-Nucleotidase
This enzyme is another phosphatase found in many tissues but primarily located in the liver in the canaliculi and sinusoidal membranes. Serum values are elevated (normal range, 0.3-3.2 Bodansky units) in hepatobiliary diseases with a spectrum of abnormality similar to that found for alkaline phosphatase. It is specific for the liver and is not influenced by gender or race, but values increase with age, reaching a plateau after age 50. It does not rise in bone disease or in pregnancy. Serum 5’-nucleotidase may be particularly helpful in the diagnosis of liver disease in childhood and in pregnancy.
5’-Nucleotidase and alkaline phosphatase are both valuable in the diagnosis of biliary obstruction or hepatic infiltrative or space-occupying lesions. Even though the correlation between the two enzymes is high, the values may not rise proportionately in individual patients, and rarely 5’-nucleotidase may be normal in the presence of elevated hepatic alkaline phosphatase. Like alkaline phosphatase, 5’-nucleotidase may be used to screen for liver metastases and to follow their evolution. In screening for liver metastases, it seems to have a higher predictive value and lower rate of false-positives than Оі-glutamyl transpeptidase, alkaline phosphatase, or a combination of the three.
| TABLE. CAUSES OF AN ISOLATED ALKALINE PHOSPHATASE ELEVATION | ||||||||||||||
|
Leucine aminopeptidase
This protease has been demonstrated in all human tissues but especially in the liver in the biliary epithelium. It is not elevated in bone disease, and the values are similar in both adults and children. In pregnancy, however, the values rise progressively, reaching a peak at term.
Serum leucine aminopeptidase is at least as sensitive as alkaline phosphatase and 5’-nucleotidase in the diagnosis of biliary obstructive, space-occupying, and infiltrative diseases of the liver. There is controversy regarding its specificity for these disorders versus hepatic parenchymal disease. However, values greater than 450 U/L are rarely seen in patients with hepatocellular disease such as cirrhosis or hepatitis.
Оі-Glutamyl transpeptidase
This enzyme is particularly found in the liver, pancreas, and kidney. Values are comparable in both genders after age 4. Serum activity does not rise due to pregnancy or bone diseases.
Elevated values are found in diseases of the liver, biliary tract, and pancreas. The degree of elevation of serum Glutamyl transpeptidase is comparable in various liver diseases; thus, it has limited value in the differential diagnosis of jaundice. Glutamyl transpeptidase values are elevated in individuals who have alcoholic liver disease or who ingest large quantities of alcohol and use barbiturates or phenytoin. A Glutamyl transpeptidase/alkaline phosphatase value greater than 2.5 is highly suggestive of alcohol abuse. Its serum values are diminished by female sex hormones, including those in birth control pills.
Serum bilirubin reflects the capacity of the liver to transport organic anions and to metabolize drugs. The formation of bilirubin from heme is essential for mammalian life, because it provides the body with the main means of elimination of heme. Eighty percent of the circulating bilirubin is derived from heme of hemoglobin from senescent red blood cells destroyed in the reticuloendothelium of the bone marrow, spleen, and liver. Ten to twenty percent of the bilirubin comes from other sources such as myoglobin, cytochromes, and other heme-containing proteins processed in the liver. Initially, heme is oxidized at the alpha position to the green pigment biliverdin, which is then reduced at the gamma position to bilirubin.
Bilirubin is virtually insoluble in aqueous solutions. In blood it is reversibly but tightly bound to plasma albumin at a 1:1 ratio. Unbound bilirubin diffuses into tissues and can cross the blood- brain barrier; it may cause kernicterus and jaundice in infants. Sulfonamides, salicylates, free fatty acids, x-ray contrast media, diuretics, hypoxia, and acidosis have the ability to displace bilirubin from its binding site on albumin.
Processing of serum bilirubin by the hepatocyte
Newly formed bilirubin is removed from the circulation very rapidly by the liver. Normally, the plasma bilirubin concentration is less than 1 mg/dL. The processing of the serum bilirubin load by the hepatocyte occurs in four steps. These are uptake, cytosolic binding, conjugation, and secretion.
Hepatic uptake of bilirubin occurs with the dissociation of the albumin-bilirubin complex facilitated by plasma membrane proteins with subsequent translocation of bilirubin into the hepatocyte through a saturable protein carrier, which also binds other organic anions but not bile salts.
The hepatic uptake system operates well below saturation, and uptake does not limit bilirubin excretion. Approximately 40% of the bilirubin taken up by the hepatocyte after a single pass refluxes unchanged back to the plasma. This reflux may increase in hyperbilirubinemia.
Cytosolic binding
In the hepatocyte, bilirubin binds to two cytosolic proteins: ligandin and Z protein. The binding limits the reflux of bilirubin back to the plasma and delivers it to the endoplasmic reticulum for conjugation.
Conjugation of bilirubin involves its esterification with glucuronic acid to form, first, a monoglucuronide, then a diglucuronide. The principal enzyme involved is uridine diphosphate -glucuronyl transferase. Administration of microsomal enzyme inducers such as phenobarbital, glutethimide, and clofibrate causes increased activity of this enzyme. Conjugation renders bilirubin water-soluble and is essential for its elimination from the body in bile and urine. Most of the conjugated bilirubin excreted into bile in humans is diglucuronide with a lesser amount of monoglucuronide.
Secretion of conjugated bilirubin from the hepatocyte to the bile canaliculi involves a specific carrier and occurs against a concentration gradient. The carrier is shared by other anions including anabolic steroids and cholecystographic agents but not bile acids. In fact, bile acids facilitate bilirubin secretion. Secretion is the rate-limiting step in the transfer of bilirubin from plasma to bile.
Conjugated bilirubin is excreted in bile as a micellar complex with cholesterol, phospholipids, and bile salts. Bacteria in the colon deconjugate and convert it to a large number of urobilinogens. A minor portion of these pigments is absorbed into plasma through the enterohepatic circulation and is excreted in the urine; the rest is excreted in the stool.
Urinary bilirubin
Unconjugated bilirubin is not excreted by the kidney. Conjugated bilirubin may be excreted in the urine in considerable amounts when there is conjugated hyperbilirubinemia. Only the conjugated bilirubin not bound to albumin can be filtered through the glomerulus and appear in the urine. Compounds such as salicylates, sulfisoxazole, and bile salts that displace bilirubin from its binding site on serum albumin augment its renal excretion.
Detection of bilirubin in the urine implies the presence of hepatobiliary disease. Absence of bilirubin in the urine of a jaundiced patient suggests unconjugated hyperbilirubinemia.
Serum bilirubin
Measurement
Serum bilirubin is measured by van den Bergh’s diazo reaction. Unconjugated bilirubin requires the presence of alcohol for the diazo reaction and gives an indirect van den Bergh’s reaction. Conjugated bilirubin reacts directly without alcohol. Total serum bilirubin is measured with the diazo reaction carried out in alcohol, where both the conjugated and the unconjugated bilirubin react with the reagent. The conjugated bilirubin is then measured from the diazo reaction carried out without alcohol. The difference represents the concentration of the unconjugated bilirubin.
The serum concentration of bilirubin depends on a balance between the rate of production and hepatic removal of bilirubin.
Increased bilirubin levels may result from overproduction of bilirubin; impaired hepatic uptake, binding, conjugation, or secretion; and leakage of the bilirubin from damaged cells or bile ducts. Hyperbilirubinemia results in clinical jaundice at a serum bilirubin concentration greater than 2.0 to 2.5 mg/dL.
Unconjugated hyperbilirubinemia results from overproduction or defective hepatic uptake or conjugation, whereas conjugated hyperbilirubinemia results from decreased hepatic secretion (excretion) or leakage of the conjugated bilirubin due to diffuse liver injury or impairment of biliary flow at the canalicular or bile duct level.
| TABLE. CAUSES OF HYPERBILIRUBINEMIA | ||||
|
Unconjugated hyperbilirubinemia is present when the total serum bilirubin is greater than 1.2 mg/dL and the direct fraction is less than 20% of the total serum bilirubin. Causes of hyperbilirubinemia are summarized in Table CAUSES OF HYPERBILIRUBINEMIA .
Unconjugated hyperbilirubinemia
Hemolysis causes increased production of bilirubin. When the excretory capacity of the liver is exceeded, serum levels of unconjugated bilirubin rise. Reduction of red cell survival to one-half normal does not cause elevation of serum bilirubin. A sixfold increase in red cell destruction results in serum bilirubin elevation to less than 5 mg/dL.
In hemolytic hyperbilirubinemia, there may be a concomitant increase in conjugated bilirubin levels. However, if conjugated bilirubin is in excess of 15% of the total bilirubin level, hepatic dysfunction must also be present.
Ineffective erythropoiesis
Patients with hematologic disorders characterized by abnormalities of heme biosynthesis have increased bilirubin turnover without increased extramedullary red cell destruction. These disorders include iron-deficiency anemia, pernicious anemia, thalassemia, sideroblastic anemia, lead poisoning, and erythropoietic porphyria.
Hereditary unconjugated hyperbilirubinemia
There is no definite evidence that abnormalities of hepatic uptake or cytosolic binding of bilirubin result in hyperbilirubinemia. However, deficiency of hepatic bilirubin uridine diphosphate-glucuronyl transferase is known to result in unconjugated hyperbilirubinemia.
The degree of enzyme deficiency allows this disorder to be divided into three types: Gilbert syndrome and Crigler-Najjar types I and II.
Gilbert syndrome
Gilbert syndrome may be the most common cause of mild unconjugated hyperbilirubinemia. It is present in up to 5% to 7% of white adults in the United States and western Europe. These patients have a partial deficiency in bilirubin glucuronyl transferase. Some patients also have decreased uptake of bilirubin and organic anions and a mild compensated hemolytic state.
The syndrome is inherited as an autosomal dominant trait with incomplete penetrance and is characterized by mild, persistent unconjugated hyperbilirubinemia. The disorder usually does not become obvious until the second decade and often is diagnosed incidentally during a physical or laboratory examination. The serum bilirubin range is 1.3 to 3.0 mg/dL, rarely exceeding 5 mg/dL. The hyperbilirubinemia fluctuates and increases with fasting, surgery, fever, infection, excessive alcohol ingestion, and intravenous administration of glucose solutions. The other liver enzymes and the histologic studies of the liver are normal. Bilirubin monoglucuronide is the dominant pigment in the bile.
The diagnosis of Gilbert syndrome is made in patients with no systemic symptoms, no overt hemolysis, and normal liver serum tests and histologic studies. Patients with this disorder when placed on a 300-kcal diet without lipids for 24 to 48 hours have an elevation of their serum bilirubin by 100% or by 1.5 mg/dL. Also, administration of phenobarbital (180 mg/day in divided doses for 2 weeks) decreases the serum bilirubin levels by enhancing the activity of the glucuronyl transferase.
Crigler-Najjar type I syndrome is an extremely rare disorder with an autosomal recessive inheritance. It appears early in life with hyperbilirubinemia ranging from 24 to 45 mg/dL. In most infants, it is associated with kernicterus and cerebral damage. These patients lack the hepatic bilirubin glucuronyl transferase, and administration of microsomal enzyme inducers such as phenobarbital does not influence bilirubin levels. The patients have normal liver histologic studies and a normal liver enzyme profile but colorless bile. Phototherapy may transiently reduce the bilirubin level, but most affected individuals die within the first year of life with kernicterus. A few patients survive to the second decade, when encephalopathy develops.
Crigler-Najjar type II syndrome
Patients with this disorder have a partial deficiency of the glucuronyl transferase. This disorder is inherited as an autosomal dominant trait with incomplete penetrance. Serum unconjugated bilirubin levels are in the range of 6 to 25 mg/dL. The bile is rich in monoconjugates. Jaundice may not be apparent until adolescence, and neurologic complications are rare.
As in Gilbert syndrome, hyperbilirubinemia increases with fasting or removal of lipid from the diet. Liver histology and serum liver enzymes are normal. These patients also respond to treatment with phenobarbital, with the reduction of their bilirubin to less than 5 mg/dL.
Acquired deficiency of glucuronyl transferase
Neonatal jaundice may be aggravated or prolonged in infants treated with drugs such as chloramphenicol or vitamin K, or exposed to pregnane 3-ОІ-20О±-diol in breast milk due to inhibition of transferase activity. Hypothyroidism delays normal maturation of this enzyme and prolongs neonatal jaundice in these infants.
Conjugated hyperbilirubinemia
In jaundice from liver or biliary tract disease, both conjugated and unconjugated bilirubin levels are elevated, and the urine contains bilirubin. With bile duct obstruction, the level plateaus between 10 and 30 mg/dL. Levels greater than 30 mg/dL are more likely in patients with hepatocellular disease. Urinary excretion is the main means of obtaining bilirubin homeostasis in obstructive jaundice. Intra- or extrahepatic causes of jaundice cannot be differentiated on the basis of total serum bilirubin or the proportion of conjugated bilirubin. Fractionation of plasma bilirubin helps to distinguish predominantly unconjugated from conjugated hyperbilirubinemia. The most common causes of cholestasis are listed in Table CAUSES OF CHOLESTASIS.
| TABLE. CAUSES OF CHOLESTASIS | |||||||||||||||||||||||||||||
|
Familial conjugated hyperbilirubinemia
Dubin-Johnson syndrome. This chronic intermittent form of jaundice is an autosomal recessive disorder with elevation of both the conjugated and the unconjugated serum bilirubin. Most patients are mildly jaundiced, but levels may be as high as 30 mg/dL. Patients exhibit normal liver enzyme profiles; however, histologically a yellow-black pigment is seen in the hepatocytes. This melaninlike substance is found in the lysosomes. The liver may be slightly enlarged and may be tender.
The principal defect in these patients is reduced ability to transport organic anions except for bile salts from the liver cell into the bile. Pregnancy and oral contraceptives, which reduce hepatic excretory function, may unmask Dubin-Johnson syndrome by producing overt jaundice. These patients also have an abnormality in coproporphyrin excretion. The urine of these patients shows greater coproporphyrin I than III levels, which is a reversal of the normal pattern.
Rotor’s syndrome
This benign disorder is inherited as an autosomal recessive trait with defects in hepatic uptake and storage of conjugated bilirubin. There may be an additional excretory defect or a decrease in the intrahepatic binding of bilirubin, allowing conjugated bilirubin to reflux into the plasma. The hyperbilirubinemia is usually less than 10 mg/dL. Other liver enzyme tests and liver histology are normal. There is no pigment in the hepatocytes. The urinary coproporphyrin levels are higher than normal, and coproporphyrin I may be greater than III but not as great as in Dubin-Johnson syndrome. The pattern of coproporphyrin excretion is similar to that seen in other hepatobiliary disorders.
Recurrent jaundice of pregnancy
In a minority of pregnant women, usually after the seventh week of gestation but most commonly in the third trimester, an intrahepatic cholestasis occurs with jaundice and pruritus. The serum bilirubin level remains less than 8 mg/dL but is accompanied by considerably elevated alkaline phosphatase and cholesterol levels and mildly abnormal transaminases. Histologically, there are varying amounts of intrahepatic cholestasis but minor parenchymal cell changes.
These patients have an increased sensitivity to the cholestatic (antiexcretory) effects of estrogenic and progestational hormones. The condition is benign and needs to be differentiated from fatty liver of pregnancy, which can be fatal. Pruritus responds to treatment with cholestyramine. The condition usually recurs with subsequent pregnancies, but all of the abnormalities revert back to normal promptly after delivery.
Drug-induced cholestasis
The use of oral contraceptives in some women may result in intrahepatic cholestasis and jaundice due to the inhibitory effect of these drugs on hepatocellular biliary excretion. The liver function abnormalities resolve after the drug is discontinued, and chronic liver dysfunction does not occur.
Male sex hormone analogs such as methyltestosterone may produce cholestatic jaundice and may result in chronic liver disease and biliary cirrhosis.
Many drugs produce cholestasis and liver cell injury, which may be accompanied by allergic manifestations such as fever, rash, arthralgia, and eosinophilia.
Postoperative jaundice
The cause of postoperative jaundice is multifactorial. Most patients have bilirubin pigment overload from blood transfusion with decreased red cell survival and resorption of blood from hematomas and large ecchymoses. Concurrent use of drugs may cause hepatic dysfunction, injury, or cholestasis. Hypotension, hypoxemia, sepsis, and shock contribute to impaired hepatic function. Renal insufficiency may decrease urinary excretion of conjugated bilirubin and enhance the degree of jaundice.
The hyperbilirubinemia may reach 30 to 40 mg/dL, with most of the bilirubin being conjugated. Serum alkaline phosphatase level may be elevated up to tenfold, but the transaminases are usually only moderately elevated. Liver biopsy in most instances shows intrahepatic cholestasis. The course of the jaundice depends on the general condition of the patient. As all the organ systems recover, the jaundice subsides and liver function returns to normal.
Sepsis from any source in the body may result in conjugated hyperbilirubinemia and mild-to-moderate elevation of the transaminases and alkaline phosphatases.
Hepatocellular disease
The most common disorders associated with jaundice are hepatitis and cirrhosis. With injury to the hepatocytes, all the steps in bilirubin metabolism are affected. Because secretion is the rate-limiting step, most of the bilirubin is conjugated. The hyperbilirubinemia in hepatocellular disease usually does not plateau and may exceed 60 mg/dL.
The major causes of intrahepatic cholestasis are
- Alcohol-related liver disease.
- Drugs (phenothiazines, sulfonylureas, allopurinol, azathioprine, thiazides, acetaminophen, aspirin).
- Viral hepatitis (acute and chronic A, B, non-A, non-B, delta, Epstein-Barr virus, cytomegalovirus, and others).
- Toxic hepatitis.
- Sepsis.
- Infiltrative disorders (sarcoid, lymphoma, tuberculosis, primary or metastatic malignancy, sickle cell disease).
Laboratory studies
In intrahepatic cholestasis, the laboratory tests reflect abnormal liver function (Table PATTERNS OF LABORATORY STUDIES IN CHOLESTATIC DISEASE STATES).
In viral hepatitis, the transaminases may be elevated to 10 to 50 times normal.
In alcoholic liver disease, the alkaline phosphatase usually rises up to about five times normal, serum glutamic-oxaloacetic transaminase (aspartate aminotransferase) is elevated less than ten times normal, and the serum glutamic-pyruvic transaminase (alanine aminotransferase) is lower than the serum glutamic-oxaloacetic transaminase (aspartate aminotransferase). The serum glutamic-oxaloacetic transaminase/ serum glutamic-pyruvic transaminase ratio is usually 2:1 to 3:1.
In drug-induced cholestasis, bilirubin may not be high, but there is usually a dramatic rise in alkaline phosphatase with a slight rise in transaminases.
| TABLE. PATTERNS OF LABORATORY STUDIES IN CHOLESTATIC DISEASE STATES | ||||||||||||||||||||||||||||||||||||||||||
|
||||||||||||||||||||||||||||||||||||||||||
The course of the jaundice depends on the general condition of the patient. As all the organ systems recover, the jaundice subsides and liver function returns to normal.
Extrahepatic cholestasis
Causes
Extrahepatic biliary obstruction due to stones, strictures, lymphadenopathy, or tumors can occur anywhere along the route of the bile ducts from the hilum of the liver to the duodenal papilla. Gallstone disease accounts for most of the benign extrahepatic obstruction. Most of these patients have an abrupt onset of jaundice. The disease ranges from biliary colic to acute cholecystitis and ascending cholangitis, especially with common bile duct stones.
Cancer. Pancreatic cancer, cholangiocarcinoma, adenocarcinoma of the duodenum and ampulla of Vater, metastatic or primary liver tumors, and enlarged nodes at the porta hepatis are common causes of extrahepatic biliary obstruction and jaundice.
Laboratory studies
In general, patients with gallstone disease have less hyperbilirubinemia than those with intrahepatic cholestasis or extrahepatic malignant obstruction. The serum bilirubin is usually less than 20 mg/dL. The alkaline phosphatase may be elevated up to ten times normal. The transaminases may abruptly rise about ten times normal and decrease rapidly once the obstruction is relieved.
In pancreatic cancer and other obstructive cancers, the serum bilirubin may rise to 35 to 40 mg/dL, the alkaline phosphatase may rise up to ten times normal, but the transaminases may remain normal. See Table PATTERNS OF LABORATORY STUDIES IN CHOLESTATIC DISEASE STATES for additional patterns of laboratory studies.
Jennifer is a clinical pharmacologist. She helps doctors of all medical specialties choose medications. Jennifer consults about drug dosage, interactions, and side effects.