Evaluation Of Liver Function

Normal Bilirubin Metabolism

Bilirubin is the major metabolite of heme, the iron-binding tetrapyrrole ring found in hemoglobin, myoglobin, and cytochromes. Approximately 250 to 350 mg of bilirubin is produced daily in healthy adults, about 85% of which is derived from turnover of senescent red blood cells ( ; ; ). In macrophages mainly in the spleen, methemoglobin from red cells is split to give free globin chains and heme. The porphyrin ring of heme is oxidized by microsomal heme oxygenase, producing the straight-chain compound biliverdin and releasing iron. In this ring-opening reaction, 1 mole of carbon monoxide is released, which is transported ultimately as carboxyhemoglobin, whose serum levels can be useful in the diagnosis of hemolytic anemia, as discussed in Chapter 9 . Biliverdin is then reduced to bilirubin ( Fig. 22.1 ; ) by the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzyme, biliverdin reductase. Bilirubin, bound mainly to albumin, is then transported mainly in the portal system to the liver, where it enters the hepatocyte through its membrane surface in contact with the sinusoids, as shown in Figure 22.2 . Specific transporters on the sinusoidal (basolateral) plasma membrane of hepatocytes help mediate the transport and uptake of bile acids and non-bile acid organic anions such as bilirubin, sulfobromophthalein (BSP), various drugs, and hormones. The fenestrated sinusoidal endothelium of the liver permits these compounds to have access to hepatocytes. These transporters include organic anion transport proteins (oatps), a family of 12 transmembrane domain glycoproteins and Na+/taurocholate transporting protein (ntcp), a seven transmembrane domain glycoprotein ( ; Russell, 2007).

As free bilirubin enters hepatocytes, additional bilirubin dissociates from albumin. This process is highly efficient; clearance of unconjugated bilirubin at normal values is about 5 mg/kg/day or, for a 75-kg individual, about 400 mg/day ( ). The half-life of unconjugated bilirubin is short; 60% of labeled bilirubin appears within hepatocytes within 5 minutes of injection ( ). The clearance rate increases with an increasing concentration of unconjugated bilirubin up to at least 4 mg/dL ( ).

In its most common isomeric ( trans- ) form, bilirubin is highly insoluble in water, and most of it is transported bound to albumin, with only a small fraction of free bilirubin. Light can cause photoisomerization of bilirubin, from a trans- form to a more compact cis- form, making it much more water soluble and allowing it to be excreted in urine ( ). This forms the basis for phototherapy in the treatment of neonatal (unconjugated) hyperbilirubinemia. The pathway for clearance of bilirubin by the liver is illustrated in Figure 22.2 . Note that unconjugated bilirubin enters the hepatocyte at the membrane surface adjacent to the sinusoids, opposite the face that is in contact with bile canaliculi.

Bilirubin enters hepatocytes by two mechanisms: passive diffusion and receptor-mediated endocytosis. As summarized in Figure 22.2 , once in the hepatocyte, bilirubin is “handed-off” from one protein complex to another in a chain. First, it complexes with the so-called Y and Z proteins; then, it binds sequentially to a protein complex called ligandin . From this complex, it is transported to the smooth endoplasmic reticulum (SER). In the SER, bilirubin becomes the substrate of the enzyme glucuronyl transferase, encoded by the UGT1A1 gene, which catalyzes the esterification of the propionic acid side chains of bilirubin with glucuronic acid (present as uridine diphosphoglucuronic acid) to form mainly the diglucuronide conjugate shown in Figure 22.1 (bottom) ( ). Some monoglucuronide and a small amount of triglucuronide also form. The ratio of monoconjugated to diconjugated pigment in bile is 1:4, whereas the ratio is nearly 1:1 in plasma, suggesting that monoconjugates reflux into plasma more readily.

Both monoconjugated and diconjugated pigments undergo ATP-dependent transport via an ABC cassette that is mediated by multidrug resistance protein (MRP1). Additionally, there is a distinct canalicular isoform (MRP2). Both MRP1 and MRP2 are ATP-dependent export pumps. This transporter function is deficient in Dubin-Johnson syndrome ( ), discussed later.

As shown in Figure 22.2 , an energy-dependent mechanism transports the conjugated bilirubin to the canalicular face of the hepatocyte, at which it is directly secreted into the canaliculi. Only conjugated bilirubin can be directly excreted into the canaliculi; unconjugated bilirubin cannot traverse this membrane.

Once bilirubin is excreted into the canaliculi and ultimately into the intestinal tract, it is further metabolized by intestinal bacteria, which effect its deconjugation and oxidation or reduction with the formation of compounds collectively called urobilinogen and urobilin , which can then be reabsorbed from the gut. Most urobilinogen absorbed is reexcreted by the liver. A minor fraction may be excreted in the urine. Larger quantities are found in the urine in conditions leading to hyperbilirubinemia or in conditions in which the liver cannot readily secrete urobilinogen absorbed from the gut. Ultimately, intestinal urobilinogen is converted to stool pigments such as stercobilin. Their absence leads to clay-colored stools, often an early sign of impaired bilirubin metabolism.

When conjugated bilirubin is present in serum, it can become covalently bound to albumin, producing biliprotein or delta-bilirubin ( ; ). Although conjugated bilirubin has a half-life of less than 24 hours, delta-bilirubin has a half-life similar to that for albumin at 17 days ( ), causing prolonged jaundice during recovery from hepatocellular injury ( ) or biliary obstruction ( ). Conjugated bilirubin, being water soluble, can be filtered by the glomerulus and can appear in urine, where it may be detected by dipstick examination. Urobilinogen measurement, however, adds little to standard tests of liver function or injury ( ). Urinary bilirubin is elevated in most patients with increased serum conjugated bilirubin ( ).

Derangements of Bilirubin Metabolism

As shown in Figure 22.2 , in each step in the processing of bilirubin, a possible lesion leads to elevated serum levels of unconjugated or conjugated bilirubin. Each of these is discussed in turn.

Laboratory Tests for Bilirubin

Bilirubin is typically measured using diazotized sulfanilic acid, which forms a conjugated azo compound with the porphyrin rings of bilirubin, resulting in reaction products that absorb strongly at 540 nm (see Chapter 28 ). Because unconjugated bilirubin reacts slowly, accelerants such as caffeine or methanol are used to measure total bilirubin. Deletion of these accelerants allows determination of direct-reacting, or direct, bilirubin.

Until the early 1980s, it was accepted that direct bilirubin was equal to conjugated bilirubin. The introduction of dry-slide technology, using differential spectrophotometry to measure conjugated and unconjugated bilirubin separately, led to the observation that the sum of these two entities did not equal total bilirubin and to the characterization of delta-bilirubin. Approximately 70% to 80% of conjugated bilirubin and delta-bilirubin and a small percentage of unconjugated bilirubin are measured in the direct bilirubin assay ( ; ). Although good data support the measurement of conjugated bilirubin instead of estimating it from direct bilirubin ( ; ), the direct bilirubin assay is still widely used. The accuracy of direct bilirubin assays is dependent on sample handling and reagent composition. Prolonged exposure to light causes photoisomerization, increasing direct-reacting bilirubin ( ; ). Use of wetting agents or incorrect pH buffers increases the amount of unconjugated bilirubin measured as direct bilirubin ( ). Typically, direct bilirubin should measure 0 to 0.1 mg/dL in normal individuals, with rare values of 0.2 mg/dL in the absence of liver or biliary tract disease.

Reference values for total bilirubin are both age and gender dependent. Bilirubin levels typically reach peak values at around ages 14 to 18 years, falling to stable adult levels by age 25 years ( ; ; ). Values are higher in males than in females at all ages ( ; ; ; ; ). Strenuous exercise causes a significant increase in bilirubin values compared with those seen in sedentary individuals or those engaging in chronic exercise ( ; ; ; ). African Americans have bilirubin levels significantly lower than those of other ethnic groups.

Ammonia

This critical and toxic compound is metabolized exclusively in the liver. Ammonia is derived mainly from amino acid and nucleic acid metabolism. Some ammonia is also produced from metabolic reactions such as the action of the enzyme glutaminase on glutamine, resulting in the production of glutamic acid and ammonia. As it happens, ammonia can be metabolized only in the liver because the liver uniquely contains the critical enzymes for the Krebs-Henseleit or urea cycle, in which ammonia, a toxic substance, is ultimately converted into urea, a nontoxic compound that is readily excreted. In this cycle, ammonia, with the enzyme carbamoyl phosphate synthetase, is condensed with carbon dioxide (CO ₂ ) and ATP to form carbamoyl phosphate that then, in the rate-determining step, carboxamidates the delta-amino group of ornithine to form citrulline using the enzyme ornithine carbamoyltransferase (OCT), an enzyme that is unique to the liver. Congenital deficiency of this or other urea cycle enzymes leads to increased levels of ammonia in serum and in cerebrospinal fluid ( ).

A unique feature of liver tissue is its ability to regenerate. To abolish liver tissue function, more than 80% of the liver must be destroyed. If most of the liver is destroyed by such conditions as cirrhosis ( ) or, less commonly, acute fulminant hepatic failure, including Reye syndrome ( ; ), urea cycle enzymes are no longer present. This leads to a toxic buildup of ammonia and some of the amino acid intermediates in the urea cycle, such as arginine, which has known neurotoxic effects. The result is an increase in ammonia and these amino acid intermediates in the circulation and in the central nervous system (CNS), giving rise to hepatic encephalopathy. In addition, in most cirrhotics, intrahepatic portal-systemic shunting occurs, causing ammonia to bypass the liver and resulting in elevated serum ammonia concentrations. Elevated serum levels of ammonia therefore often indicate some form of liver failure, although other conditions can also induce increases in serum ammonia levels.

In patients with cirrhosis or fulminant hepatic failure, there has been some dispute as to whether ammonia itself is the cause of the observed metabolic encephalopathy; possibly other toxins that accumulate as a result of absent hepatic detoxification are the cause. One of the arguments often used is that there is no clear correlation between the severity of the encephalopathy and serum ammonia concentrations ( ). Countering this argument is the finding that, although venous ammonia levels do not correlate with the degree of encephalopathy ( ), arterial levels of ammonia do generally correlate with the degree of encephalopathy. Furthermore, in patients with cirrhosis or fulminant hepatic failure, lowering serum ammonia invariably diminishes the severity of the encephalopathy ( ). Furthermore, idiopathic hyperammonemia, not related to liver disease, also induces lethal encephalopathy ( ; ). An important mechanism by which ammonia can cause toxicity to the CNS is its ability to lower the concentration of γ-aminobutyric acid (GABA), a critically important neurotransmitter in the CNS, by reacting with glutamic acid to form glutamine via reversal of the glutaminase-catalyzed reaction ( ). This depletes glutamic acid in the CNS. However, GABA is formed directly from the decarboxylation of glutamic acid; thus, GABA levels consequently decrease, with potentially serious effects on neurotransmission (see Chapter 24 ). Because ammonia causes accumulation of glutamine in the CNS, there is the suggestion that, at least in valproic acid–induced hyperammonemia, cerebrospinal fluid levels of glutamine can be used in the diagnosis and management of hepatic encephalopathy ( ). More recently, besides evidence that ammonia in the CNS is directly toxic to astrocytes, there is growing recognition that there is a complex and synergistic relationship between ammonia, inflammation, and oxidative stress in the pathogenesis of hepatic encephalopathy. Neutrophil dysfunction results in the generation of reactive oxygen species contributing to oxidative stress and inflammation, with lowered ability of the CNS to block infectious agents. In accordance with these findings, strategies targeting systemic inflammation, such as rifaximin-α and other anti-inflammatory agents being studied, are quickly becoming the new mainstay in the treatment of hepatic encephalopathy ( ; ). One major new finding put forth by these studies is that treatment of hepatic encephalopathy with suitable anti-inflammatory agents may be effective.

At present, most commonly, elevated serum ammonia concentrations in hepatic encephalopathy are reduced by the agent lactulose, which is metabolized by a wide range of gut bacteria to lactic acid ( ). The acid so produced in the intestinal lumen traps ammonia as ammonium ion, which can no longer diffuse across the intestinal membrane and is thus excreted. Ammonia-producing bacteria in the intestine are removed by treatment with antibiotics such as neomycin.

Lipids

Drug Metabolism

Many xenobiotics, such as drugs, are metabolized in the liver, mainly in the microsomes of hepatocytes. Complex series of reactions occur, many of which are dependent on cytochrome P450, which is involved in the oxidation of these compounds. Whether specific exogenous compounds are converted to metabolites depends on the isoforms of cytochrome P450, such as CYP1A and CYP2B (cytochrome P450 1A and 2B, respectively). Often, the conversion of xenobiotics into metabolites using this system involves two phases: Phase I reactions involve oxidations/hydroxylations, and phase II reactions conjugate the metabolite (or parent compound) to polar compounds, such as glucuronic acid, glycine, taurine, and sulfate. In more severe liver disease, which involves microsomal damage, this ability to metabolize xenobiotics is compromised. Thus, the ability of hepatocytes to metabolize drugs can be used to measure liver damage.

This is generally accomplished by administering a known dose of isotopically labeled (usually ¹³ C-labeled) drug and measuring the ¹³ CO ₂ exhaled over time in a patient’s breath. Two categories of breath tests have been developed based on the rate-limiting step in metabolism. The first group includes drugs such as aminopyrine, caffeine, and diazepam, which are metabolized at rates that are independent of hepatic blood flow to the liver and depend only on the enzymatic activity of different cytochromes P450 (e.g., CYP1A). The second group is composed of drugs such as methacetin, phenacetin, and erythromycin, whose rates of metabolism depend on the rate of blood flow (i.e., their rates of metabolism are fast compared with their rates of delivery to the liver). These types of dynamic tests appear not to be so useful in the initial diagnosis of hepatic disease; rather, they are more useful in estimating the extent of liver damage in known liver disease ( ).

Some interferences that complicate interpretation of the results of these tests include dependence of the demethylation of aminopyrine (the methyl group is oxidized to CO ₂ ) on vitamin B ₁₂ . In cases of vitamin B ₁₂ deficiency, less than normal amounts of ¹³ CO ₂ will be exhaled because of low B ₁₂ levels, not necessarily because of liver damage. Rates of caffeine metabolism generally decrease with increasing age but are increased by smoking; these findings can complicate interpretation of test results.

Determination of Serum Protein Levels

This is based usually on the biuret method as described in Chapter 28 . This method reflects the ability of the peptide backbone C=O groups of proteins to form color complexes with copper that absorb strongly at 540 nm. Some methods utilize a dye-binding method in which the proteins form a complex with the dye Coomassie blue. Albumin forms a unique color complex with the dyes bromocresol green and bromocresol purple, such that they absorb maximally at slightly different wavelengths, thus allowing direct spectrophotometric quantitation ( ; ; ; ). Bromocresol purple tends to react more exclusively with albumin than does bromocresol green (which reacts to a minor extent with some globulins); thus, serum albumin levels may be slightly lower when determined with bromocresol purple. The reference range for total serum protein levels is generally in the 6.0 to 7.8 g/dL range. At least 60% of this should be albumin, the normal range for which is about 3.5 to 5.0 g/dL.

Serum protein electrophoresis and quantitative Ig may reveal characteristic changes in liver disease, as discussed in Chapter 20 . Typically, in cirrhosis, albumin is significantly decreased, as are the α-1, α-2, and β (principally transferrin) bands. However, a polyclonal increase in Ig, which is seen frequently, produces the characteristic β-γ bridging pattern, as discussed in Chapter 20 . In autoimmune hepatitis, albumin is typically decreased; this is accompanied by a marked polyclonal increase in IgG. Primary biliary cirrhosis, an autoimmune disease, is accompanied by a polyclonal increase in IgM. However, as noted later, this polyclonal IgM does not react with the mitochondrial antigens against which the autoimmune antibodies in this disease do react ( ).

Albumin

Albumin is the major protein produced by the liver. Its synthesis is increased by low plasma oncotic pressure and is decreased by cytokines, particularly interleukin-6. Although normal albumin synthesis occurs at about 120 mg/kg/day, the rate of synthesis can approximately double with low oncotic pressure. A decrease in albumin is one of the major prognostic features in patients with cirrhosis. Albumin measurements were discussed previously in Chapter 20 and are further discussed in Chapter 28 .

Albumin is a transport protein for many substances, both endogenous (e.g., bilirubin, thyroid hormone) and exogenous (e.g., drugs). Low serum albumin levels due to liver disease are almost always caused by massive destruction of liver tissue and are seen primarily in cirrhosis, most often secondary to alcoholism. The diminution in albumin is paralleled by a fall in total serum protein. Because albumin is the osmotically active intravascular colloid, hypoalbuminemia often results in edema. In cirrhosis, in which increased resistance to blood flow in the sinusoids causes portal hypertension, the combined effect of elevated hydrostatic pressure in the portal system and low colloid osmotic pressure results in ascites, a frequent finding in cirrhosis. These same changes may also be seen acutely in fulminant hepatic failure ( ; ).

Other Serum Proteins

Although most of the proteins discussed in Chapter 20 are produced by the liver, two bear special importance in the detection of congenital liver disorders.