Approach to Jaundice and Abnormal Liver Function Test Results

Bilirubin

Bilirubin ( Table 7-1 ) is the end product of heme catabolism. Bilirubin gives bile its characteristic color and accounts for the yellow discoloration observed in jaundice patients. More than 80% of bilirubin is derived from the breakdown of red blood cells in the mononuclear phagocyte (reticuloendothelial) system. The remaining bilirubin is derived from ineffective erythropoiesis in the bone marrow and other heme-containing proteins (hemoproteins), such as myoglobin, cytochrome, catalase, and endothelial nitric oxide synthase.

Heme (iron protoporphyrin IX) belongs to the highly conserved superfamily of tetrapyrrolic compounds and serves as an oxygen-binding prosthetic group for the protein hemoglobin. After the breakdown of red blood cells, heme, which is released from hemoglobin, is metabolized within macrophages. Heme is converted to biliverdin by heme oxygenase and subsequently catalyzed to bilirubin by biliverdin reductase. In its unconjugated form, bilirubin is water insoluble and is transported bound to albumin. Bilirubin is transferred into hepatocytes by passive and active transport and conjugated with glucuronic acid in the endoplasmic reticulum to water-soluble bilirubin monoglu­curonides and diglucuronides. Conjugation is catalyzed by the enzyme uridine diphosphate glycosyltransferase (UGT), which is encoded by the gene UGT1A1 , located on chromosome 2. The excretion of conjugated bilirubin into bile is mediated by the adenosine triphosphate–dependent transporter multidrug resistance–associated protein 2 (MRP2; also known as canalicular multispecific organic anion transporter ), which is expressed at the apical (canalicular) hepatocyte membrane. In the absence of apical MRP2, homologues of MRP2 are upregulated at the basolateral membrane, thereby facilitating the excretion of conjugated bilirubin into sinusoidal blood. Even under physiologic conditions, a substantial amount of conjugated bilirubin is excreted across the sinusoidal membrane and subsequently taken up by hepatocytes. This action results in the intralobular transfer of conjugated bilirubin and other endogenous and exogenous substrates from the periportal to the centrilobular zone, thereby minimizing the concentrations of potentially toxic substrates in periportal hepatocytes.

Mutations in the UGT1A1 gene, which are transmitted as autosomal recessive traits, result in various degrees of UGT deficiency, which cause hereditary unconjugated hyperbilirubinemia. The severest form is Crigler-Najjar syndrome type 1, which is associated with the near complete absence of UGT activity. Crigler-Najjar syndrome type 1 presents in newborns with marked jaundice and kernicterus. Without treatment, it results in severe neurologic injury or death within the first 2 years of life. Although symptoms are mitigated by phototherapy, liver transplantation remains the definitive treatment. Crigler-Najjar syndome type 2 is associated with a lesser degree of UGT deficiency and hyperbilirubinemia and is rarely associated with kernicterus or death. The two types also differ in their response to phenobarbital, a UGT inducer, with no effect on bilirubin levels in Crigler-Najjar syndome type 1 (compared with significant reductions in Crigler-Najjar syndome type 2). Another enzymatic cause of unconjugated hyperbilirubinemia is Gilbert syndrome, which results from reduced UGT activity to approximately 25% to 30% of normal levels. Gilbert syndrome affects up to 10% of the Caucasian population. Individuals with Gilbert syndrome present with isolated jaundice that is aggravated by intercurrent infections, fasting, or specific medications. The levels of serum bilirubin, largely unconjugated, are commonly elevated to slightly less than 5 mg/%.

Dubin-Johnson syndrome and Rotor syndrome are examples of hereditary conjugated hyperbilirubinemia due to mutations in bilirubin excretory pathways. Dubin-Johnson syndrome develops because of deficiency or absence of MRP2, which alters the transport of conjugated bilirubin and other anionic substrates, including antibiotics, chemotherapeutic agents, toxins, and heavy metals. Patients present in young adulthood with fluctuating hyperbilirubinemia and are otherwise asymptomatic. Bromsulphalein clearance is normal at 45 minutes, followed by a delayed increase at 90 minutes; in contrast, a hepatobiliary iminodiacetic acid scan shows absent or late filling of the gallbladder. The liver appears dark on histologic examination because of lysosomal accumulation of melanin-like pigment. Hyperbilirubinemia is exacerbated in pregnancy and with oral estrogen use. Rotor syndrome is characterized by mild, conjugated hyperbilirubinemia without hepatic accumulation of dark pigment. Anionic substrates exhibit delayed excretion without later uptake. Rotor syndrome is associated with the complete absence of the organic anion–transporting polypeptides 1B1 and 1B3. Organic anion–transporting polypeptides are sinusoidal transporters that facilitate the sodium-independent uptake of organic anions.

Conjugated hyperbilirubinemia is a hallmark of cholestasis and results in jaundice. Extrahepatic cholestasis results from the obstruction of the biliary tree, which is commonly due to choledocholithiasis or a pancreatic or biliary neoplasm. Among young children the most common cause is biliary atresia. Intrahepatic cholestasis can result from sepsis or drug hepatotoxicity. In cholestasis the canalicular transportation of organic anions remains unaffected, whereas transporters at the basolateral membrane are downregulated. Reduced MRP2 expression is followed by the upregulation of MRP2 homologues at the basolateral membrane, which promotes the excretion of bilirubin into the bloodstream and may operate as a protective mechanism to minimize solute accumulation in hepatocytes.

Moderately elevated serum bilirubin levels may be beneficial because bilirubin has strong antioxidant effects and acts against atherogenesis and cancer development.

Urinary Bilirubin and Urobilinogen

Unconjugated hyperbilirubinemia occurs without an increase in urinary bilirubin levels because it is water insoluble and circulates bound to albumin. Jaundice is therefore termed acholuric because urine is not darkened. In contrast, dark urine is a prominent symptom of conjugated hyperbilirubinemia that results from urinary excretion of water-soluble bilirubin. Assessment of bilirubin in urine may help identify bilirubinuria when the total serum bilirubin level is normal or only slightly elevated. In contrast, the absence of bilirubinuria, in patients with jaundice, may dictate that conjugated bilirubin is covalently bound to albumin, which often occurs during recovery from acute hepatitis.

Conjugated bilirubin excreted in bile is metabolized to urobilinogen by intestinal bacteria. Urobilinogen is primarily excreted in the feces; however, a small amount is absorbed via enterohepatic circulation, extracted by the liver, and excreted in bile. Only a small amount of urobilinogen escapes hepatic uptake to be excreted in urine (<4 mg/day). Urinary urobilinogen levels are elevated when bilirubin is overproduced, as in hemolytic states, and are decreased during extrahepatic obstruction, when con­jugated bilirubin does not reach the gut. Hepatocellular dys­function results in impaired hepatobiliary urobilinogen excretion and mildly elevated levels of urinary urobilinogen. In practice, however, the detection and quantification of urinary urobilinogen provides no diagnostic information beyond that provided by standard LFTs.

Bile Acids

Bile acids are a group of chemically similar molecules that have diverse physical and biologic properties. They facilitate the emulsion and absorption of dietary fats and lipid-soluble vitamins. The secretion of bile salts into the canaliculi generates an osmotic gradient that promotes bile secretion. Bile salts form mixed micelles with biliary phospholipids, enabling the solubilization of cholesterol and other lipid-soluble compounds. This process promotes the emulsion and subsequent absorption of dietary fats and fat-soluble vitamins. Bile acids also facilitate intestinal calcium absorption and regulate pancreatic enzyme secretion and cholecystokinin release. Bile acids function as signaling molecules through farnesoid X receptor, a specific nuclear receptor, and TGR5, a G protein–coupled bile acid receptor. The activation of these receptors alters gene expression in multiple tissues, leading to changes not only in bile acid metabolism but also in glucose homeostasis, lipid and lipoprotein metabolism, energy expenditure, intestinal motility, bacterial growth, inflammation, and the liver-gut axis.

In humans the two primary bile acids are cholic acid and chenodeoxycholic acid. These acids are nearly completely conjugated to either glycine (75%) or taurine (25%), which accounts for their water solubility. These conjugated bile acids are called bile salts . Bile acids are synthesized from cholesterol via either the classic pathway or the alternative pathway. The classic, or neutral, pathway is exclusive to the liver and results in the synthesis of the two primary bile acids. The sterol nucleus of cholesterol is modified through a series of enzymatic reactions. Cholesterol 7α-hydroxylase (encoded by CYP7A1 ), a microsomal cytochrome P450 enzyme, is the rate-limiting enzyme of this pathway. The molecule is transported to peroxisomes, where the side chain is truncated by β-oxidation and subsequently conjugated to glycine or taurine. In the alternative, or acidic, pathway, the oxidation of the cholesterol side chain occurs first, resulting in acidic intermediates, followed by sterol ring modifications. This pathway contributes to approximately 10% of the bile acid pool and primarily results in the formation of chenodeoxycholic acid. Bile acid synthesis is regulated by negative feedback from bile acids themselves and is mediated by farnesoid X receptor. Cholesterol modulates its own conversion to bile acid by upregulating cholesterol 7α-hydroxylase. Insulin (and drugs such as phenobarbital and rifampin) suppresses bile acid synthesis by inhibiting CYP7A1 transcription.

Nearly 95% of bile acids are reabsorbed via active uptake at the apical membrane of intestinal cells, primarily in the ileum, and are carried back to the liver through portal blood and to a lesser extent the hepatic artery. The conjugated bile acids are taken up at the sinusoidal membrane in a sodium-dependent manner facilitated by sodium taurocholate cotransporter polypeptide. This process results in the enterohepatic circulation of bile acids. A small fraction of bile acids escapes into the colon, where the bile acids are modified by bacterial flora to secondary bile acids: deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid. Some of these bile acids are deconjugated and returned to the liver via the portal vein. The rate-limiting step in overall bile acid transport is transfer across the hepatocyte canalicular membrane, which is mediated by the adenosine triphosphate–dependent bile salt export pump (encoded by ABCB11 ). The hydrophilicity of bile acids varies, with the naturally occurring ursodeoxycholic acid being the most soluble, followed by cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid. Liver injury can occur when a high proportion of strongly detergent (least hydrophilic) bile salts is perfused through the liver. The more detergent bile salts are unconjugated or glycine conjugated, whereas the lesser ones are taurine conjugated and sulfated. A high proportion of bile salts is observed in several conditions of liver injury, including those characterized by enhanced intestinal bile salt biodegradation, such as chronic inflammatory bowel disease. A normally detergent bile salt pool may become hepatotoxic for liver cells that have been previously injured. Alternatively, increased sulfation, an increased proportion of taurine conjugates, and reduced formation of deoxycholic acid in liver cirrhosis could be regarded as protective mechanisms. Bile acid–induced hepatotoxicity can be prevented by enhancing tauroconjugation, reducing the intestinal degradation of bile salts, or administering poorly detergent bile salts such as ursodeoxycholic acid.

Defects in bile acid synthesis account for 1% to 2% of cholestatic disorders in children and are transmitted in an autosomal recessive pattern. These disorders result in progressive cholestasis and neonatal hepatitis and can lead to liver failure. Defects in bile acid transport result in progressive familial intrahepatic cholestasis types 1-3. Progressive familial intrahepatic cholestasis type 2 results from an ABCB11 mutation that impairs bile acid canalicular secretion. Surgical resection of the small bowel, especially the ilium, disrupts the enterohepatic circulation of bile acids, resulting in their excess concentration in the colon, which manifests itself as diarrhea.

Bile acid supplementation has therapeutic value in several conditions, such as primary biliary cholangitis (PBC), cholesterol cholelithiasis, and cholestasis, and the prevention of cholelithiasis and metabolic complications in patients undergoing bariatric surgery. The Food and Drug Administration has also approved its use in mesotherapy to dissolve unwanted fat. In addition, the FLINT trial provided promising evidence for the efficacy of obeticholic acid, a farnesoid X receptor agonist, in improving the histologic features of nonalcoholic steatohepatitis (NASH).

Aminotransferases

The aminotransferases ( Table 7-2 ), ALT and AST, are the most frequently used indicators of hepatocyte injury and are markers of hepatocellular necrosis. Aminotransferases catalyze the transfer of α-amino groups of alanine and aspartate to the α-keto group of ketoglutaric acid, resulting in the formation of pyruvic acid and oxaloacetic acid, respectively. These enzymes play a role in gluconeogenesis by facilitating the synthesis of glucose from noncarbohydrate sources. ALT is located entirely in the cytosol, whereas 20% of the AST is in the cytosol and 80% is in the mitochondria. ALT is primarily expressed in the liver, whereas AST is present in a variety of tissues, including the liver, heart, skeletal muscles, kidney, brain, pancreas, lungs, leukocytes, and erythrocytes. AST levels are therefore elevated in cardiac and skeletal muscle diseases. Pyridoxal 5′-phosphate, the active form of pyridoxine (vitamin B ₆ ), acts as a cofactor for all transamination reactions. Both ALT and AST are present in serum either as apoenzymes or as holoenzymes bound to pyridoxal 5′-phosphate. Rarely, isolated AST level elevation is due to a macroenzyme that is a complex of AST and immunoglobulins. It is important to recognize macro-AST to avoid unnecessary investigations and diagnostic delays.

An investigation of a population at lowest risk for liver disease indicated that the healthy upper limit of serum ALT levels is 30 IU/L for men and 20 IU/L for women. Serum ALT and AST levels are elevated in most liver diseases. The highest elevations are observed in acute viral hepatitis and toxic or ischemic liver injury. Although the degree of elevation may reflect the extent of hepatocellular necrosis, it is not correlated with outcome. One example is acetaminophen hepatotoxicity, which is associated with marked increases in serum ALT and AST levels; however, most patients achieve full recovery. Moderately elevated ALT and AST levels (3- to 20-fold of the upper normal limit) are observed in drug hepatotoxicity, autoimmune hepatitis (AIH), and acute and chronic viral hepatitis. Mild elevations (less than 3 times the upper normal limit) are observed in alcoholic steatohepatitis and NASH, drug hepatotoxicity, and chronic hepatitis C. Serum AST levels (and to a lesser extent) ALT levels may increase because of myositis or muscle injury. Patients with cirrhosis and cholestatic liver diseases may exhibit a mild increase in serum ALT and AST levels. Choledocholithiasis may be associated with a marked increase in serum ALT and AST levels soon after the obstruction develops, and they decline rapidly during the following 24 to 72 hours. Serum transaminase levels have been shown to be useful in screening asymptomatic patients for liver disease. Abnormal levels may lead to the diagnosis of metabolic liver disease or nonhepatic diseases, such as Addison disease, hypothyroidism, and gluten-sensitive enteropathy.

Serum aminotransferase levels are influenced by several factors, including age, body mass index (BMI), muscle mass, lifestyle, and lipid and carbohydrate metabolism. ALT activity may be influenced by accelerated aging and frailty in older adults. Marked decreases in serum AST levels have been reported in patients undergoing long-term hemodialysis. Drinking coffee lowers serum levels of aminotransferases, as well as total protein and albumin levels. Assessment of mitochondrial AST may aid in the diagnosis of alcohol abuse and sobriety monitoring. A higher mitochondrial AST to total AST ratio has a sensitivity of 93% in patients with alcoholic liver disease and 100% in alcoholic patients without liver disease.

Alkaline Phosphatase

APs ( Table 7-3 ) are a family of well-conserved isoenzymes that are anchored on the outer layer of the cell membrane. These enzymes act on substrates in the extracellular space at an optimum alkaline pH to catalyze the hydrolysis of phosphate esters. Four isoenzymes have been identified in humans: intestinal AP, placental AP, germinal AP, and tissue-nonspecific AP. The intestinal, placental, and germinal isoenzymes are named after the tissue in which they are expressed and are encoded by the homologous genes ALPI , ALPP , and ALPPL2 , respectively. Tissue-nonspecific AP is primarily expressed in the bone, liver, and kidney and is encoded by the ALPL gene. In the bone, tissue-nonspecific AP facilitates mineralization, and genetic deficiency of this enzyme causes a rare metabolic bone disease called hypophosphatasia . The physiologic role of tissue-nonspecific AP in other organs has not been well defined.

In healthy individuals most circulating AP originates from the liver or bone. In pregnant women, circulating placental AP is also observed. Mean serum AP levels vary with age. Serum AP levels are high during childhood and puberty (related to bone growth and development), decrease in middle age (higher in men than in women), and increase again in old age. Serum AP levels are positively correlated with body weight and smoking levels and are inversely correlated with height. In most individuals with elevated AP levels, the enzyme originates in the liver. However, nearly one third exhibit no evidence of liver disease, and among hospitalized patients, transient nonspecific elevation of AP levels is common. Bone is the next likely contributor to elevated AP levels, followed by placenta in pregnancy. The intestine and kidney are unlikely contributors to elevated serum AP levels. The highest AP levels in liver disease occur in patients with intrahepatic or extrahepatic cholestasis. The degree of elevation does not assist in differentiating between these two types of cholestasis. Elevated levels in cholestasis are due to increased AP synthesis and not reduced excretion. Infiltrative liver disease, such as lymphoma, granulomatous disorders, and amyloidosis, also cause elevated AP levels. Primary hepatic malignancy or metastasis to the liver may cause AP elevation either due to discrete biliary obstruction or due to more diffuse hepatic infiltration. Low AP levels are observed in Wilson disease, hypothyroidism, pernicious anemia, zinc deficiency, and hypophosphatasia.

γ-Glutamyl Transpeptidase

GGTP catalyzes the transfer of the γ-glutamyl peptide group, such as glutathione, to other amino acids (except for proline). GGTP is widely distributed in many tissues, including the liver, kidney, seminal vesicles, pancreas, spleen, heart, and brain. It functions in amino acid transport via the γ-glutamyl cycle. GGTP has been localized in the entire hepatobiliary tree, from hepatocytes to the common bile duct, pancreatic acini, and ductules. The highest concentrations of GGTP are observed in the epithelial cells lining biliary ductules.

Serum GGTP levels vary with age and sex; normal values are higher in men than in women and increase with age in adults. Serum GGTP levels are commonly increased in acute and chronic liver diseases and are also elevated in a variety of nonhepatobiliary diseases, including chronic alcoholism, pancreatic disorders, myocardial infarction, renal failure, chronic obstructive pulmonary disease, and diabetes, as well as by specific enzyme-inducing drugs. Smoking also increases serum GGTP levels and decreases serum protein and albumin levels independently of age, sex, regular medication, BMI, and coffee and alcohol consumption.

In patients with liver disease, serum GGTP levels are well correlated with serum AP levels. Serum GGTP is a sensitive indicator of hepatobiliary disease and the most sensitive indicator of biliary tract disease. However, in some forms of cholestatic syndromes presenting in infants and children, such as progressive familial intrahepatic cholestasis type 1 (Byler disease) and type 2, GGTP levels remain normal. Serum GGTP levels have been widely used as an index of liver disease and marker of high alcohol intake. Because it is not produced by bone, GGTP helps in differentiating between bone origin versus hepatic origin of an elevated AP level. Although GGTP level is a sensitive marker, its utility is limited by its poor specificity.

Other Enzymes

5′-Nucleotidase catalyzes the hydrolysis of nucleotides by releasing phosphate from the 5′-position of the pentose ring. 5′-Nucleotidase is present in the intestines, brain, heart, blood vessels, pancreas, and liver. In the liver, 5′-nucleotidase is primarily associated with canalicular and sinusoidal cell membranes. Elevated serum levels are generally hepatobiliary in origin despite the distribution of the enzyme in other body tissues. Serum levels of 5′-nucleotidase are strongly correlated with serum AP levels and have relative specificity for liver disease. Serum 5′-nucleotidase levels can be used to confirm AP of hepatic origin. For example, in childhood and pregnancy, increased serum AP levels are observed, whereas 5′-nucleotidase levels remain normal.

LDH is commonly included in liver biochemical panels but has poor diagnostic specificity for liver disease. Markedly increased LDH levels are observed in hepatocellular necrosis, shock liver, lymphoma, or hemolysis associated with liver disease.

Albumin

Human albumin is a 67-kDa globular protein that is the principal component of circulating plasma proteins, constituting 50% of the total pool. Albumin is synthesized in hepatocytes and released into the bloodstream, where 30% to 40% remains. Most circulating albumin crosses capillary membranes into the interstitial space, where it returns to the circulation via lymphatics. Albumin is synthesized at a rate of 10 to 15 g/day in healthy individuals and has a half-life of 12 to 19 days. It carries a net negative charge because of its highly acidic amino acid content. Albumin accounts for 75% of plasma oncotic pressure because of its high plasma content and negative charge, which attracts sodium and water. The negative charge also enables albumin to bind and carry an array of molecules, such as bilirubin, bile acids, hormones, anions, fatty acids, metals, drugs, and endotoxin. Albumin provides most extracellular antioxidant activity through an abundance of thiol groups, which are avid scavengers of oxidative and reactive species. Albumin exhibits immunomodulatory activity, such as endotoxin binding and the inhibition of proinflammatory pathways involving tumor necrosis factor (TNF)-α and nuclear factor κB. It stabilizes vascular endothelium and modulates vasodilation and platelet aggregation by binding to nitric acid. Thus hypoalbuminemia is often associated with increased vasodilation and platelet aggregation.

Serum albumin levels reflect hepatic synthetic function and are therefore a component of the commonly used Child-Pugh staging system for cirrhosis. However, because of the relatively long half-life of albumin, serum levels are commonly normal in patients who present with acute liver failure. Hypoalbuminemia has several nonhepatic causes, such as nephrotic syndrome, severe malnutrition, malabsorption, and protein-losing enteropathy. Additionally, hypoalbuminemia may develop during pregnancy because of expanded intravascular volume.

Prothrombin Time and International Normalized Ratio

The coagulation cascade involves the sequential activation of a series of clotting factors. Most clotting factors are produced in the liver, whereas some are released from vascular endothelial cells. Coagulation follows either the intrinsic (contact activation) pathway or the extrinsic (tissue injury) pathway. PT is used to evaluate the extrinsic pathway, which involves prothrombin, factors V, VII, and X, and fibrinogen. This test requires decalcified platelet-rich plasma, to which thromboplastin, phospholipid, and calcium chloride are added. PT is defined as the time needed for a clot to form. Because of the differences in thromboplastin sensitivity, tissue origin, and the preparation method, the normal PT range varies. In 1983 the international sensitivity index was adopted to address these issues. The international sensitivity index indicates the potency of each thromboplastin by comparing it with a reference standard that is assigned a potency of 1.0. Each laboratory follows a standardized method to establish the reference range for PT by testing healthy volunteers. The international normalized ratio standardizes PTs and is calculated as the ratio between the patient PT and the control PT.

Because PT depends on the activity of clotting factors synthesized in the liver, liver dysfunction may result in prolonged PT. Therefore PT serves as an important indicator of liver disease severity and has been incorporated into several prognostic indices, such as the King's College criteria for acute liver failure, Child-Pugh categorization of cirrhosis, and the Model for End-Stage Liver Disease, to allocate donor livers for patients requiring liver transplantation. However, PT can be influenced by factors other than hepatic dysfunction, such as vitamin K deficiency, disseminated intravascular coagulation, and vitamin K antagonists such as warfarin.