11.CLINICAL BIOCHEMISTRY OF THE LIVER

11.

CLINICAL BIOCHEMISTRY OF THE LIVER

Neil McIntyre INTRODUCTION All clinical biochemical laboratories use a small battery of blood tests to detect and manage liver disease. It usually includes total bilirubin, aspartate and/or alanine aminotransferase, alkaline phosphatase and albumin; other tests such as gamma glutamyltransferase (GGT) may be added (Table 1). Although called “liver function tests” they are not specific for liver disease and are of little use for assessing liver function. Some functions of the liver, such as galactose elimination, bile acid clearance and removal of some dyes, can be quantified but the methods, discussed later, are inconvenient and costly. Urine tests for bilirubin and urobilinogen are easily performed in the ward or clinic. Prothrombin and partial thromboplastin times, useful markers of the severity of liver disease, are measured in the hematology laboratory; although valuable in managing liver disease they are not usually considered as “liver function tests” and will not be considered in this chapter.

SERUM BILIRUBIN (McIntyre & Rosalki, 1999; Ostrow, 1986) Bilirubin is formed from the heme of hemoglobin, myoglobin and other heme proteins. Insoluble in water, it binds strongly to albumin before uptake by the The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 291–316 Copyright © 2004 by Elsevier Ltd. All rights of reproduction in any form reserved ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15011-3

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Table 1. Liver “Function” Tests.a Serum bilirubin – total – unconjugated, conjugated and bili-proteins Urinary bilirubin and urobilinogen Serum aminotransferases – aspartate, alanine Glutathione-S-transferase Lactate dehydrogenase Serum alkaline phosphatase 5
-Nucleotidase Leucine aminopeptidase Gammaglutamyl transferase Serum albumin Pre-albumin Ceruloplasmin ␣-1-antitrypsin ␣-fetoprotein Cholinesterase Serum bile acids Serum cholesterol Triglyceride Lecithin-cholesterol acyltransferase a Commonly

used tests are in bold face.

liver where it is esterified with glucuronic acid. The water soluble mono- and di-glucuronides are efficiently excreted in bile, little entering the blood in healthy people. They are hydrolyzed by ileal and colonic bacteria; the bilirubin is degraded to “urobilinogen” (a mixture of isomers), most of which is excreted in the feces. Some urobilinogen is absorbed, removed from portal blood by the liver, and excreted in bile; a small amount escapes hepatic uptake and is lost in the urine. Bilirubin esters react directly with Ehrlich’s diazo reagent to give a violet color; unesterified bilirubin requires ethanol or another accelerator for color development. Total bilirubin is measured by the depth of color produced when diazo reagent and an accelerator are added to serum; “direct” bilirubin is the result in the absence of accelerator. “Indirect” bilirubin is the difference between total and “direct” measurements. “Direct” and “indirect” bilirubin are inaccurate measures of esterified and unesterified bilirubin, as some of the latter reacts “directly.” “Direct” bilirubin therefore overestimates esters at relatively low bilirubin levels, but still allows identification of the “unconjugated hyperbilirubinemia” of Gilbert’s syndrome and hemolysis. “Direct” bilirubin is of little value at high bilirubin levels when it tends to underestimate esters.

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The “normal” range for total serum bilirubin is usually taken as 3–17 umol/l. The mean level is 3–4 umol/l higher in men than in women; as Gilbert’s syndrome, a completely benign condition, is often suspected when the serum bilirubin is just above 17 umol/l this explains, in part, why it appears more prevalent in men. The upper limit for “direct reacting” bilirubin is usually taken as about 3 umol/l (about 20% of the total); levels above 5 umol/l, with a normal total bilirubin, suggest a conjugated rather than an unconjugated hyperbilirubinemia, but only if measured in a good laboratory (on a good day!). Accurate methods for measuring bilirubin and its conjugates (e.g. alkaline methanolysis and high performance liquid chromatography), rarely used routinely, show that conjugated bilirubin normally accounts for only 4–5% (<1 umol/l) of total bilirubin. They are the most sensitive marker of liver disease, which causes an increase in bilirubin esters; they also allow discrimination between the “unconjugated hyperbilirubinemia” of Gilbert’s syndrome and that of hemolysis, as the proportion of esters (as a percent of total bilirubin) is normal with hemolysis but low (<1.7%) in Gilbert’s syndrome, a disorder of conjugation. Total bilirubin increases with fasting or a low fat intake; this is an important factor when interpreting bilirubin results. Bilirubin is affected by light; serum or plasma samples should therefore be kept in the dark, preferably in a refrigerator, before measurements are made. An elevated serum total bilirubin reflects increased production, reduced hepatic uptake and/or conjugation, impaired transport of bilirubin esters into bile (with parenchymal liver diseases), or their regurgitation into the blood from biliary canaliculi (with biliary obstruction). Bilirubin itself is insoluble in water and is not excreted in urine. Bilirubin esters are excreted in urine when their plasma level increases; the urine becomes brown in color. Bilirubinuria establishes the presence of a liver disorder. Other compounds cause dark urine. Bilirubinuria should be confirmed using test strips impregnated with a diazo reagent; these detect as little as 1–2 umol of bilirubin per liter; they are underused. Bilirubinuria usually precedes jaundice and may be found when the total serum bilirubin is normal or only slightly elevated. When frank jaundice is present bilirubinuria simply confirms an increase in plasma bilirubin esters. When it is absent in a jaundiced patient, two possibilities are suggested. A simple unconjugated hyperbilirubinemia (due to hemolysis, Gilbert’s syndrome or the rare Crigler-Najjar syndromes) is likely if other liver tests are normal (see Table 2). However, conjugated pigment (detectable as “direct” hyperbilirubinemia) may become covalently bound to serum albumin and other proteins, forming bili-alb or bili-proteins, thus escaping urinary excretion. This fraction may constitute a major fraction of serum total bilirubin, particularly during recovery from jaundice. In

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Table 2. Causes of an Isolated Increase in Serum Unconjugated (“Indirect”) Bilirubin. Hereditary disturbances of bilirubin conjugation Gilbert’s syndrome Crigler-Najjar syndromes (types 1 and 2) Hemolytic disorders Congenital Hereditary spherocytosis, elliptocytosis, G-6-PD deficiency and other enzyme disorders, and sickle cell disease. Acquired Erythroctye fragmentation syndromes, drugs and toxins, and autoimmune hemolytic anemias.

the late stage of an acute hepatitis bilirubin may be absent from the urine even at serum bilirubin levels as high as 170 umol/liter; at the onset bilirubinuria may be found before jaundice appears. When other liver function tests are abnormal serum bilirubin levels above 17 umol/l usually indicate liver disease of some kind and bilirubin esters are elevated. The actual level of bilirubin is rarely of diagnostic value. In acute liver diseases, the serum level is also of little prognostic value; complete recovery usually occurs, even after deep jaundice, with resolution of conditions such as acute viral hepatitis or biliary obstruction. With chronic liver diseases, however, a gradual and pronounced increase in serum bilirubin without obvious cause (such as blood transfusion, which increases the heme load, or the administration of certain drugs) is an ominous prognostic sign. In primary biliary cirrhosis a level of 100 umol/l has been used to trigger consideration of liver transplantation. In bile duct obstruction, even if complete, serum bilirubin tends to plateau between 170 and 500 umol/l; the major pathway for removal of bilirubin in this situation is urinary excretion, but bilirubin also breaks down to unidentified compounds. Extreme hyperbilirubinuria (up to 1500 umol/l or greater) only occurs in severe parenchymal liver disease associated with renal failure and/or with hemolysis (due to sickle cell disease, G-6-PD deficiency or blood transfusion).

URINARY UROBILINOGEN (McIntyre & Rosalki, 1999) Freshly voided urine containing urobilinogen gives a purple reaction with Ehrlich’s aldehyde reagent. A dipstick containing this reagent allows rough quantification.

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Urinary excretion of urobilinogen is affected by urine pH; its tubular reabsorption increases with increasing urinary acidity which also renders it less stable. Its estimation in acid urine is thus an unreliable index of plasma levels. The peak urinary output of urobilinogen tends to occur between 12 noon and 1600 hours, probably due to the urinary “alkaline tide.” With liver damage hepatic uptake and biliary excretion of urobilinogen falls, and more is excreted in urine. With severe biliary obstruction less bilirubin enters the intestine, less urobilinogen is made and absorbed, and urinary levels fall. However, changes in urinary urobilinogen occur which are unrelated to changes in hepatic function. Intestinal production of urobilinogen increases with constipation or bacterial contamination of the small bowel, or with over-production of bilirubin due to hemolysis. Urobilinogen production and urinary excretion falls with diarrheal states and treatment with antibiotics. Although much emphasis is placed on urobilinogen metabolism in undergraduate texts, the detection of urinary urobilinogen is of little clinical value.

ASPARTATE AND ALANINE AMINOTRANSFERASES (McIntyre & Rosalki, 1999; Moss & Henderson, 1994) Over forty years ago, marked elevations of serum aspartate aminotransferase (AST, glutamic-oxaloacetic transaminase, SGOT) and alanine aminotransferase (ALT, glutamic-pyruvate transaminase, SGPT) were found in viral hepatitis and other hepatic disorders; they were considered an index of liver cell injury. Aspartate aminotransferase catalyzes the reaction: aspartate + alpha-ketoglutarate = oxaloacetate + glutamate Alanine aminotransferase catalyzes the reaction: alanine + alpha-ketoglutarate = pyruvate + glutamate The co-enzyme for both is pyridoxal phosphate, which binds to a lysine residue in the enzyme, forms a transient Schiff base with the relevant aminoacid, receives the amino group and transfers it to the oxoacid. Both enzymes leak from damaged cells, due to increased membrane permeability or cell necrosis. Measurement of these enzymes in serum is done mainly to identify or confirm the presence of liver disorder. Large amounts of AST are found in liver, but also in cardiac and skeletal muscle, kidney, pancreas and red cells, and serum levels may also rise with damage to these tissues. Because ALT was originally found in low concentration in tissues other than liver, a high serum ALT

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was considered relatively specific for hepatic damage but there may be a marked increase in some skeletal muscle disorders. When hepatocytes are damaged there is usually an increased serum level of both AST and ALT. As this occurs in many liver diseases the finding is of limited value for differential diagnosis. We cannot identify the organ source of serum AST but two isoenzymes, one cytoplasmic, the other mitochondrial, can be measured individually. Release of mitochondrial AST from hepatocytes probably implies more severe cellular damage than release of the cytoplasmic isoenzyme or of ALT, which is also confined to the cytoplasm. The ratio of mitochondrial AST to cytoplasmic or total AST has been proposed as a diagnostic test, as it increases with severe hepatocellular necrosis and in alcoholic liver disease, but few, if any, routine laboratories measure mitochondrial isoenzyme activity. Plasma or serum should be separated soon after blood collection to avoid release of erythrocyte AST. Hemolysis causes a small increase in serum AST levels, and gross hemolysis may give misleading results in the measurement of AST. Aminotransferase levels may be spuriously low in uremia, possibly as a result of vitamin B6 deficiency, but this does not appear to cause the low serum levels often seen in patients on long-term hemodialysis. The main value of aminotransferase measurements is to detect hepatocellular damage, and to monitor the patient’s progress; return to normal suggests resolution of the factors causing hepatocellular damage. Liver disease does not always result in aminotransferase elevation; levels may be normal in patients with established but well compensated cirrhosis, with chronic hepatitis C, and in patients with chronic and incomplete biliary obstruction. Furthermore, relatively small increases are sometimes encountered even in severe hepatitis (although the initial increase may then have been missed). Some laboratories measure both enzymes in their battery of liver tests, others only one; there has been debate over their relative value. Those advocating the use of ALT alone do so because they consider it (mistakenly) to be specific for hepatic damage. Those using AST alone do so because it can also be used to detect damage to cardiac and skeletal muscle, and because it is rarely difficult on clinical grounds to decide whether a high AST is due to liver disease. We now realize that both enzymes need to be measured in hepatological practice. With fatty liver or hepatitis C ALT may be high when AST is in the normal range; in alcoholic hepatitis and cirrhosis, AST may rise without an increase in ALT (probably due to a reduced hepatic content of ALT). These conditions may be therefore missed if only one aminotransferase is measured. An obvious increase in the AST:ALT ratio (from <1 to >1) is also a useful indicator of the development of cirrhosis in patients with chronic viral hepatitis (B and C) or primary biliary cirrhosis, but there is overlap in individual cases.

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Table 3. Causes of a Marked Elevation of Serum Aminotransferases. Acute hepatitis Viral and drug induced Autoimmune hepatitis Shock liver Hypotension, acute heart failure Acute biliary obstruction Gallstone obstruction, acute cholangitis

Because they increase in so many conditions even quite marked elevations of aminotransferase levels (up to 500 u/l) are of limited value in differential diagnosis. They have been used to differentiate “hepatocellular” jaundice (when they tend to be high) from “obstructive” jaundice (when they tend to be low). But there are many exceptions to this “rule,” and the classification of jaundice into hepatocellular and obstructive causes on this basis may be misleading. Very high AST and ALT levels are of diagnostic value. Levels more than 20x the upper reference limit (about 1000 units/l) suggest acute hepatitis (due to a virus or drug), “shock liver” (due to hypotension or acute heart failure), or acute extrahepatic biliary obstruction, ascending cholangitis or autoimmune hepatitis (Table 3). Blood should be taken at presentation because in most of these conditions AST and ALT levels may fall rapidly, and are then of little diagnostic value. If previous AST/ALT levels are available they help to establish the cause of a very high aminotransferase. In viral hepatitis, and with most drugs causing acute hepatitis, aminotransferase levels rise gradually for a week or two before the onset of jaundice; normal or only modest elevations of AST/ALT at the onset of symptoms virtually exclude acute hepatitis as a cause. With acute hypotension and heart failure, and with acute biliary obstruction, there is usually an abrupt rise and a rapid fall if the underlying problem can be treated effectively. In rare cases an elevation of serum AST, often marked, results from the presence of “macro AST,” a complex of the enzyme with large molecular weight immunoglobulin (usually IgG, rarely an IgM). This finding, analogous to macroamylasemia, may cause diagnostic confusion. In about 75% of cases of uncomplicated acute viral hepatitis (A and E, and many cases of B and B + D hepatitis) aminotransferase levels fall to normal within 8 weeks. Chronic liver disease often results from infections with hepatitis B and C, and there is usually a persistent (but often modest) increase in AST and ALT levels. In acute hepatitis C, initial aminotransferase levels are much lower than with other types of viral hepatitis (usually less than 1000 iu/l) but many patients

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develop chronic hepatitis. With hepatitis A, which has no long term sequelae, aminotransferase levels may take many months to return to normal, and during this time levels may rise again due to exacerbation of the disease. More modest aminotransferase increases, up to about 10 × normal, occur in many liver diseases, e.g. chronic hepatitis, cirrhosis, biliary obstruction, hepatic infiltration and neoplasia. ALT is more frequently increased, except in cirrhosis and infiltrative disease. In developed countries the most common cause of ALT elevation in the general population is fatty liver (associated with obesity, diabetes, hyperlipidemia and alcohol abuse); in endemic areas it is hepatitis C. In both conditions a modest elevation of ALT is usually accompanied by a similar elevation of gammaglutamyltransferase. Minor increases in AST and ALT are occasionally found when there is no evidence of significant liver disease on liver biopsy. This is particularly true for ALT, which rises in many conditions (and with many drugs), and for AST after short periods of binge drinking in healthy subjects. Serum AST activity increases in diseases other than those affecting the liver, e.g. myocardial infarction, myocarditis and pulmonary embolism. AST and ALT both rise with some myopathies (e.g. Duchene dystrophy, active polymyositis and hypothyroidism) and with trauma (even intramuscular injections). Serum creatine kinase (CK) levels help to identify AST and ALT elevations due to muscle disease, but quite marked rises of CK may occur without obvious cause in Afro-Caribbean patients; the normal upper limit for CK in this population is twice that for Caucasians. There may be confusion when diseases of skeletal muscle occur with liver disease. In chronic alcoholics painless chronic myopathy is frequent, and acute myopathy can follow a drinking bout; these conditions may contribute to the serum aminotransferase elevations seen in such patients.

OTHER TESTS Glutathione-S-Transferase (Beckett & Hayes, 1987) Glutathione-S-transferases (GST) are widely distributed detoxification enzymes. The bilirubin binding protein, ligandin, is a GST found in liver; its serum activity is a highly sensitive index of hepatocellular damage but is difficult to measure. It has a short plasma half life, allowing early recognition of cessation of active cellular damage. It rises with acute hepatitis of viral or drug origin, massive increases being found with paracetamol toxicity and fulminant hepatitis, with primary and secondary hepatic malignancies, and in untreated hyperthyroidism (presumably

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due to sub-clinical liver damage). It may be high in chronic hepatitis C when AST and ALT levels are normal, and increases occur with alcoholic liver disease, particularly after binge drinking.

Lactate Dehydrogenase and the LD5 Iso-Enzyme Modest elevations of serum lactate dehydrogenase (LD) activity are found early in acute viral hepatitis; LD activity seems to be greater in hepatitis due to paracetamol and ischemia. In most other liver diseases normal (or near normal) values of LD are usual. With hepatic malignancy LD increases in up to 80% of patients, the frequency depending on the extent of metastatic disease; high levels may be found. Although total serum LD is raised in many conditions, a preferential increase in the LD5 iso-enzyme is found only with liver disease, malignancy and muscle disorders. The plasma concentrations of other substances rise with hepatocellular damage. Some are enzymes, presumably released from damaged hepatocytes. The pattern of response does not necessarily mirror that of aminotransferases; in acute viral hepatitis, lactate dehydrogenase and sorbitol dehydrogenase return to normal much more quickly. Serum glutamate dehydrogenase tends to rise with large duct obstruction; its activity in liver tissue, where it is located centrizonally, may also rise with biliary obstruction. Not surprisingly, many of these enzyme measurements have been advocated as useful liver function tests in their own right, or as contributors to patterns of abnormality that might help in differential diagnosis. They have not been widely employed and so it is difficult to assess their relative merits; they seem to have little advantage over aminotransferase estimations.

ALKALINE PHOSPHATASES (McIntyre & Rosalki, 1999; Moss & Henderson, 1994) Alkaline phosphatases, a family of zinc metallo-enzymes with a serine at the active centre, release inorganic phosphate from organic orthophosphates and are present in nearly all tissues. In liver, alkaline phosphatase is found histochemically in the microvilli of bile canaliculi and on the sinusoidal surface of hepatocytes. Alkaline phosphatases from liver, bone and kidney are transcribed from the same gene; alkaline phosphatases from intestine and placenta are coded by other, different genes.

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Total alkaline phosphatase activity should be measured in fresh, unhemolyzed serum or heparinized plasma. Citrate, oxalate and EDTA complex with the zinc in the phosphatase and inactivate it. The international “reference” method uses p-nitrophenol phosphate as substrate in an alkaline transphosphorylating buffer such as 2-amino-2-methyl-1-propanol. The various isoenzymes in serum can be identified and measured by several methods. For routine analysis electrophoresis, which depends mainly on charge, is the method of choice. This allows identification of placental and intestinal isoenzymes, tumor isoenzymes, a high molecular mass “biliary” (or fast liver) isoenzyme, and one or more unusual hepatic isoenzymes. Liver (“slow” liver band) and bone isoenzymes, found in similar proportions in normal sera, are difficult to distinguish with this method; this is unfortunate as the usual question posed by clinicians is whether an increased alkaline phosphatase level is due to liver or bone isoenzyme. These can be identified and quantified using wheat-germ lectin affinity electrophoresis. Other methods can also be used to distinguish between liver and bone alkaline phosphatases. Urea and guanidine inhibit them differently, but the differences are small, determination errors large, and inhibitors of intestinal alkaline phosphatase must be used to compensate for a possible contribution from this fraction. Assay based on heat stability is tedious and subject to considerable inaccuracy. Monoclonal antibodies can identify bone and liver isoenzymes, with some crossreactions but without interference from alkaline phosphatase from other tissues; convenient clinical assays are not yet available. The reference range (and units) for serum total alkaline phosphatase activity varies with the method used, the age and gender of the patient, and with other factors. Any one isoenzyme may be increased even when total alkaline phosphatase is normal. Alkaline phosphatase elevation occurs in many diseases other than those involving the liver. Bone and liver isoenzymes account for most of the activity in normal serum. Alkaline phosphatase activity may be above the normal adult range until about 20 years of age, with peaks in the neonatal period and in adolescence (periods of rapid bone growth). Liver phosphatase tends to increase in elderly males, bone phosphatase in post-menopausal females. Intestinal phosphatase may contribute up to 20% of total activity in about 20% of normal subjects; it is associated with blood groups A and O, the ABH red cell antigen and absence of the Lewis antigen, and its serum level tends to rise after a fatty meal. In normal pregnancy alkaline phosphatase activity increases during the third month, rises to twice the usual adult female level in late pregnancy, and can remain high for weeks after delivery. The main source is the placenta. Serum bone-specific alkaline phosphatase often remains higher (for up to six

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Table 4. Causes of an Increase in Serum Alkaline Phosphatase Activity. Liver alkaline phosphatase Obstructive liver disease; extra-hepatic and intra-hepatic Minor increase with age Bone alkaline phosphatase Physiological – childhood, puberty, post-menopausal Paget’s disease, osteomalacia, bone metastases Intestinal alkaline phosphatase Cirrhosis Physiological after fat ingestion in secretors of blood groups O and B Placental alkaline phosphatase Pregnancy Indian childhood cirrhosis Tumor alkaline phosphatase Ovarian, testicular, hepatocellular carcinoma Macro alkaline phosphatase – immunoglobulin bound

months) in women who breast-feed than in those who bottle feed their babies (see Table 4).

Alkaline Phosphatase and Liver Disease It was recognized over 60 years ago that serum alkaline phosphatase activity rose with bile duct obstruction, and that lower but still high levels were found in “toxic, infective, and catarrhal” jaundice; the increase was attributed to regurgitation of bile phosphatase. With the introduction of the King-Armstrong method of measurement in 1934, a phosphatase level over 30 KA units became accepted, erroneously, as a diagnostic criterion for an extrahepatic block. Serum alkaline phosphatase activity goes up in many types of liver disease; the highest levels are seen with either intra- or extra-hepatic obstruction to bile flow, and with intrahepatic space occupying lesions such as primary or metastatic liver tumors. The high phosphatase of liver disease is not simply due to retention of the biliary enzyme. The phosphatase accumulating in plasma is made in the liver, and animal studies show increased hepatic synthesis of alkaline phosphatase after biliary ligation. There are at least two hepatic isoenzymes, one from hepatocytes (“slow” liver), and a high molecular mass biliary phosphatase (“fast” liver) from the canalicular membrane. With biliary obstruction the membrane phosphatase, solubilized by retained bile salts or shed as fragments, reaches plasma by

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paracellular regurgitation or transcellular endocytosis. “Biliary” enzyme is a better marker of biliary obstruction than total serum alkaline phosphatase activity, but is rarely sought in clinical practice. A high serum alkaline phosphatase is not always found in “cholestatic” liver disease. It may be normal or only slightly raised in patients with primary biliary cirrhosis or primary sclerosing cholangitis, with a confirmed extrahepatic block due to tumor or stones, or with bacterial cholangitis, particularly in the early stage. This may cause diagnostic confusion. In acute viral hepatitis, serum alkaline phosphatase is usually either normal or only moderately raised, but up to 40% of patients have levels two and a half times the upper reference limit. Hepatitis A may present an obstructive picture with prolonged itching and marked elevation of alkaline phosphatase, and a very high alkaline phosphatase has been reported in Epstein-Barr virus infection, even with normal bilirubin levels. Serum alkaline phosphatase is increased by drugs which cause cholestatic liver disease; an elevation has also been found with cimetidine, furosemide, phenobarbital and phenytoin. In about 25% of patients with cirrhosis, an intestinal band may be the major alkaline phosphatase in serum (normally it does not exceed 20% of total phosphatase activity). This may be due to destruction of receptors for intestinal alkaline phosphatase on the liver cell surface (causing reduced hepatic uptake), or to diminished hepatic excretion or catabolism. When osteomalacia complicates liver disease, the bone isoenzyme may increase in association with increased osteoblastic activity. An increase in serum alkaline phosphatase with the properties of hepatic phosphatase has been found in Hodgkin’s disease, congestive heart failure, and in infectious and inflammatory diseases not primarily involving the liver (e.g. polymyalgia rheumatica); its origin is not clear. Some tumors secrete specific isoenzymes into plasma. The Regan isoenzyme (a heat-stable placental type isoenzyme) is found with bile duct carcinoma, the Kasahara isoenzyme (a fetal-intestinal type phosphatase) in about 30% of patients with hepatocellular carcinoma; in the latter condition another distinct alkaline phosphatase (heat labile) may be found. Unfortunately, such isoenzymes are of little diagnostic value; they are found in few patients with tumors, and generally at low activity so that sensitive immunological methods need to be used. They help in monitoring anti-tumor therapy; successful treatment results in a fall or disappearance of isoenzyme from plasma. Identification of alkaline phosphatase isoenzymes is of limited value in distinguishing between various kinds of liver disease; “biliary” enzyme is more frequently raised in cholestatic and neoplastic disorders, and intestinal

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alkaline phosphatase in parenchymal disease, but there is considerable overlap. However, isoenzyme studies certainly help to decide whether an elevated alkaline phosphatase activity is due to liver disease or bone disease. When alkaline phosphatase isoenzyme results are not available, clinicians tend to use other markers of biliary obstruction (gamma-glutamyl-transferase or 5
-nucleotidase) in order to confirm the hepatic origin of raised phosphatase levels. Gammaglutamyltransferase has 90% sensitivity and specificity for this purpose, as it is normal with bone disease. When a marked alkaline phosphatase elevation is accompanied by a modest gamma-glutamyltransferase elevation, the possibility of concomitant bone and liver disease should be considered. Occasionally serum liver phosphatase increases due to its binding to serum immunoglobulins which interferes with plasma clearance of the enzyme. This has been reported with auto-immune hepatitis and ulcerative colitis. In rare families serum liver phosphatase activity is increased for no obvious reason.

5
-NUCLEOTIDASE AND LEUCINE AMINOPEPTIDASE (McIntyre & Rosalki, 1999; Moss & Henderson, 1994) 5
-nucleotidase is an alkaline phosphatase acting on nucleotides with a phosphate at the 5
position of the pentose. Although present in all tissues only liver disease appears to cause an increase in its serum activity (normal range 1–15 i.u./l). The highest levels occur with obstruction to bile flow (intra- or extra-hepatic, but moderate elevations are found with chronic hepatocellular disorders. Leucine aminopeptidase hydrolyzes peptides in which an l-leucine residue contains the free amino group. Widely distributed in the body, it is present in bile, bile ducts and canaliculi. Blood levels are highest with intra- or extra-hepatic obstruction to bile flow, but also increase in acute hepatitis, cirrhosis, hepatic malignancy, and in the last trimester of pregnancy. These tests are rarely used in routine practice. Both were used to confirm a hepatic cause of an elevated alkaline phosphatase, but have been superseded by measurement of GGT. However, alkaline phosphatase iso-enzyme studies are better than 5
-nucleotidase, leucine aminopeptidase or GGT for establishing liver disease as the cause of an elevated alkaline phosphatase.

GAMMA-GLUTAMYL TRANSFERASE (GGT) (Moss & Henderson, 1994; Nemezansky, 1986) Gamma-glutamyl transferase is a membrane bound glycoprotein which catalyses the transfer of ␥-glutamyl groups from ␥-glutamyl peptides, particularly

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glutathione, to other peptides, to amino acids and to water. Its gene on chromosome 22 codes for a precursor protein of about 61 kD. The enzyme is found mainly in cells with a high rate of secretory or absorptive activity, but also in many other tissues. Large amounts are found in kidneys, pancreas, liver, intestine and prostate. The GGT activity of bile is approximately 100 times greater than that of normal serum. There are several GGT isoenzymes, but without clear evidence of tissue specificity. The heterogeneity appears to be related to the number of sialic acid residues, to the degree of glycosylation, and to binding to lipoproteins. The reference interval for serum GGT is higher in men than in women. Activity is high in neonates and infants up to 1 year, and in subjects above the age of 60. Because about 15% of those screened in some centers have levels over the usually accepted upper limit of normal (50 mU/liter in men, 30 mU/liter in women) a higher upper limit is sometimes used (e.g. 80 in men, and 50 in women). About 4% of men have a level more than 100 mU/liter. Reference levels are lower in lifelong abstainers from alcohol than in the general population. Serum GGT activity rises in most kinds of liver disease, and is high in about 90% of patients with hepatobiliary disease. It is therefore of little value in differentiating between them. It is a sensitive indicator of the presence of liver disease (0.87–0.95) but has limited specificity, because many other conditions, not primarily hepatic, increase serum GGT, possibly as a consequence of mild, clinically insignificant hepatic involvement. The highest levels are found with intrahepatic biliary obstruction or with primary or secondary hepatic malignancies. However, a normal or low GGT is seen occasionally in patients with intrahepatic cholestasis, even with a very high bilirubin and alkaline phosphatase. In infants with idiopathic cholestasis a normal GGT is considered to be of poor prognostic significance. In acute viral hepatitis serum GGT levels reach a peak in the second or third week of the illness but in some patients levels are still up at six weeks. Levels remain high with the development of chronic hepatitis or cirrhosis, and are usually elevated in chronic hepatitis C. GGT levels are of definite, though limited, value in managing alcoholic patients. They may rise, presumable due to enzyme induction, even in the absence of significant liver damage, but there is a poor correlation between alcohol intake and GGT activity. With abstinence GGT falls to normal over 2–5 weeks; if it does not, there may be continued alcohol intake, underlying liver damage, or another reason for the high GGT. Unfortunately, about a third to a half of heavy drinkers show no elevation of GGT in the absence of liver disease, so it is not a sensitive screening test for alcohol abuse. GGT levels do not rise as the result of an alcoholic binge in healthy subjects, but may do so in alcoholics and patients with other liver disorders. An increased GGT level is found in many patients who drink no alcohol or only modest amounts. It is important that such patients are not labeled as

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alcoholics. The most common cause is fatty liver associated with obesity, diabetes or hypertriglyceridemia. About 20% of patients with uncomplicated diabetes mellitus have a high serum GGT activity, but rarely more than three times the upper limit of normal. The enzyme probably comes from the fatty liver often found in diabetics. Activity is also high in acute pancreatitis, and most cases of acute myocardial infarction; as there is no measurable GGT activity in cardiac or skeletal muscle, the rise in the latter may be due to secondary effects on the liver, as high levels are also found in congestive heart failure. Modest increases in serum GGT activity occur with enzyme inducing drugs such as phenobarbitone, phenytoin and other anticonvulsant drugs, paracetamol (aminopyrine), tricyclic antidepressants and glutethimide. Smaller increases occur with anticoagulants, oral contraceptives and antihyperlipidemic drugs. Sometimes very high levels of serum ␥-glutamyl transferase are found (up to 1000 u/l) when there is no detectable cause.

PLASMA PROTEINS The liver makes many circulating plasma proteins. Liver disease affects the plasma concentration of many of them. The effects are complex, depending on changes in protein synthesis, catabolism by various tissues, and the effects of liver disease on the volume and distribution of extra-cellular fluids. There may also be changes in the metabolism of plasma proteins produced outside the liver. Estimating “total plasma protein” is of relatively little value as it may be normal even with marked disturbances of individual components. However, an abnormal result may suggest the need to measure the different fractions. Serum protein electrophoresis gives patterns characteristic of, but not diagnostic for, certain types of liver disease. A low albumin and high gamma globulin are found with non-biliary cirrhosis, a marked rise in gamma globulin with autoimmune hepatitis. With biliary obstruction ␣- and ␤-globulins may increase with accumulation of abnormal lipoproteins; a fall in haptoglobin due to intravascular hemolysis causes a low ␣-2 band. A reduced ␣-1 band suggests ␣1-antitrypsin deficiency. Unfortunately the various electrophoretic abnormalities occur in conditions other than liver disease and are of little diagnostic help in jaundiced patients. Serum or Plasma Albumin (Rothschild, Oratz & Schreiber, 1988) Albumin is the most abundant circulating protein. Its total exchangeable pool is about 3.5–5.0 g/kg body weight, 38–45% being present in plasma in which levels

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are normally between 35 and 50 g/l. Albumin accounts for most of the colloid osmotic pressure of plasma. It has high affinity binding sites for many naturally occurring compounds (including bilirubin) and many drugs. Liver is the only site of synthesis (about 15 g/day in a “normal” 70 kg person). About 1 g is lost daily via the gut; the rest is degraded by an unknown mechanism. Serum albumin is widely used as a test of liver function, because it falls with reduction in hepatic protein synthesis. Unfortunately it also falls with gastrointestinal and renal loss, increased catabolism, increased vascular permeability and over-hydration. Changes in serum albumin concentration should therefore be interpreted with caution. It may take many days before reduced synthesis causes an obvious change in serum albumin because of its long half-life (about 20 days), and because when synthesis falls there is a reduction in the fractional catabolic rate. However, with fever or trauma serum albumin levels tend to drop rapidly, often below 30 g/l, even if there is no liver disease; the albumin half-life may fall to about seven days, suggesting increased albumin removal as well as impaired albumin synthesis. The low serum albumin level often found with severe chronic liver disease is due mainly to reduced synthesis, but also results from expansion of the extra-cellular space, which may contain more albumin than normal despite the low concentration. Although the fractional catabolic rate is low, the absolute rate of degradation and synthesis may be normal or even high. In cirrhotics with ascites, hepatic secretion of albumin is disturbed; some enters the blood stream normally via the sinusoids, but much is released directly into the ascites.

Specific Protein Measurements (McIntyre & Rosalki, 1999) Alpha-1-globulin is composed mainly of ␣-1-antitrypsin and ␣-1 acid glycoprotein (orosomucoid), two acute phase proteins which increase in many inflammatory disorders, orosomucoid being especially responsive in liver disease. Haptoglobin, another acute phase protein, runs as an ␣-2 globulin. Increased clearance of haptoglobin-hemoglobin complexes causes the low haptoglobin level seen with intravascular or severe extravascular hemolysis. In non-biliary cirrhosis the ␤-globulin iron binding protein, transferrin, may be reduced; it is considered a negative acute phase protein because it falls non-specifically in many inflammatory disorders. The immunoglobulins (principally IgG, IgA and IgM) are located within the ␥-globulin fraction. Diffuse (polyclonal) increase of staining in this region is common in chronic liver disease (especially autoimmune hepatitis) and in chronic inflammatory and other auto-immune disorders. IgA runs in the ␤-␥ region; an increase in non-biliary cirrhosis (especially alcoholic) causes beta-gamma “fusion” or “bridging” on electrophoresis.

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Although changes in these proteins may be inferred from electrophoretic appearances, they are better determined by quantitative immunological measurement. Specific proteins whose concentration is of particular interest in liver disease include pre-albumin, ␣-1 anti-trypsin, ␣-fetoprotein, ceruloplasmin, procollagen-III-peptide, and the immunoglobulins.

Pre-Albumin (Transthyretin; Hutchinson et al., 1981) Pre-albumin, a tetramer of four identical subunits, binds iodothyronines; it also binds one molecule of retinol binding protein (RBP) which serves to minimize urinary loss of RBP. The serum pre-albumin level is 0.2–0.3 g/l; that of RBP 0.04–0.05 g/l. Measurement of pre-albumin has been proposed as a liver function test as it often falls in liver disease due to reduced synthesis. Because of its short half-life (1.9 days), changes precede alterations in serum albumin. Reduction in plasma RBP in chronic liver disease may be associated with impaired dark adaptation.

Serum Ceruloplasmin Ceruloplasmin, an intensely blue ␣-2-globulin normally present in plasma at a level of 0.2–0.4 g/l, is synthesized in liver. Its gene is on chromosome 3. It is an oxidase for certain aromatic amines and phenols, for cysteine, ascorbic acid and ferrous ions. Its physiological function is still not entirely clear. However, with hereditary absence of ceruloplasmin there is marked iron overload; this suggests that oxidation of ferrous to ferric ions plays a key role in iron metabolism. It is an acute phase protein, its serum concentration rising in pregnancy, with estrogens, infections, rheumatoid arthritis, some malignancies, active non-Wilson’s liver disease and obstructive jaundice. Serum ceruloplasmin is an important diagnostic marker in Wilson’s disease in which the serum level is usually low. A low ceruloplasmin is also found in neonates, Menkes’ disease, kwashiorkor and marasmus, protein losing enteropathy, nephrotic syndrome, severe hepatic insufficiency, copper deficiency, and in hereditary hypo-ceruloplasminemia and a-ceruloplasminemia.

Procollagen-III-Peptide Procollagen-III-peptide is removed from the N-terminal end of procollagen-III in the production of type III collagen. Its serum concentration increases with hepatic

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fibrosis, but also with inflammation and necrosis. The value of serum procollagenIII-peptide measurements is uncertain, but they may help in monitoring chronic liver disease.

Alpha-1 Antitrypsin Alpha-1-antitrypsin, a glycoprotein of about 54 kD, is synthesized by the liver. It inhibits serine proteinases, especially elastase. Its serum concentration is normally 1–1.6 g/l. An acute phase protein, it increases with inflammatory disorders, pregnancy and with oral contraceptives. Alpha-1-antitrypsin shows genetic polymorphism. Approximately 90% of Caucasian populations are homozygous for the M allele (i.e. MM phenotype); other alleles include F, S, Z and null forms. The phenotype is best determined by iso-electric focusing; allelic variation may be associated with a low plasma concentration and deficient functional (inhibitory) capacity. Plasma levels of alpha-1-antitrypsin are approximately 15% of normal with the ZZ phenotype; 38% with SZ; about 60% with MZ and FZ. The presence of the Z allele, particularly in homozygotes, is associated with defective processing of the protein in the liver. The precursor protein, deficient in sialic acid, is poorly secreted by hepatocytes; its intrahepatic accumulation, which can be demonstrated histochemically, may cause liver damage. Neonatal hepatitis occurs in Pi ZZ homozygotes, less often with MZ and SZ phenotypes. Cirrhosis in adults is found with ZZ, less commonly with MZ, SZ and FZ phenotypes. Hereditary ␣-1-antitrypsin deficiency is often suspected because of a reduced ␣-1 band on electrophoresis; deficiency should be confirmed by quantitative measurement.

Alpha Fetoprotein This protein is measured by radio- or enzyme immunoassay. It is the major protein of fetal plasma in early gestation, but levels are very low subsequently (reference limit 25 ug/l). More than 90% of patients with hepatocellular carcinoma have an increased serum level. This is also found with other liver diseases including chronic hepatitis, and the regeneration phase of viral hepatitis, and in up to 15% of cirrhotics without hepatocellular carcinoma; the increase is generally minor compared with that seen with hepatocellular carcinoma. To improve specificity for hepatocellular carcinoma (but with loss of sensitivity) levels above 400 ug/l have been regarded as a diagnostic prerequisite. At such levels the positive predictive value is 70% or more for hepatocellular carcinoma; less than 5% are false positives, and these are often

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transient elevations. High ␣-fetoprotein levels usually accompany hepatocellular carcinoma in blacks and Chinese. They are less common in white Europeans, and with hepatocellular carcinoma arising in non-cirrhotic liver; ␣-fetoprotein may be undetectable when the tumor is associated with oral contraceptive therapy. Serial determination of ␣-fetoprotein is of particular value in monitoring patients with cirrhosis; a progressive increase one should lead to a rigorous search for hepatocellular carcinoma. With successful therapy there is a fall in ␣-fetoprotein levels.

Cholinesterase (McIntyre & Rosalki, 1999; Moss & Henderson, 1994) Cholinesterase, which hydrolyzes many choline esters, is made in the liver. Serum levels fall with decreased hepatic protein synthesis, the changes paralleling those of serum albumin. Low values due to genetic polymorphism are recognizable by the altered inhibition characteristics of the serum enzyme. In acute hepatitis of infective or toxic origin, plasma cholinesterase falls within days, returning gradually to normal with recovery. Low levels are also found with chronic hepatitis and cirrhosis, and with neoplastic and other infiltrative diseases of the liver. In obstructive jaundice values are normal unless there is concomitant liver disease, but if the obstruction is due to a tumor reduced values may be found. Low enzyme levels, due to impaired enzyme synthesis, can occur with malignant disease, even if it is localized and does not involve the liver. With steatosis levels are normal or somewhat increased. Many drugs appear to cause a reduction in cholinesterase activity. Cholinesterase has been used to assess liver function before and after hepatic transplantation. It is best studied serially and is of greatest value as a prognostic tool. A sudden or marked fall to a quarter of the usual activity is an ominous sign.

SERUM BILE ACIDS (Hoffman, 1989) Bile acids are derived from catabolism of cholesterol. The two main primary bile acids, cholic and chenodeoxycholic acids, are made in the liver, conjugated with glycine or taurine, and excreted in bile where they play a key role in the digestion and absorption of fat and fat-soluble compounds. Bile acids are reabsorbed from the terminal ileum by an efficient active transport mechanism, but some reach the colon where they undergo bacterial deconjugation. Bacteria also dehydroxylate them at the 7-␣-position, and so convert cholic acid to deoxycholic acid, and

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chenodeoxycholic to lithocholic acid. The resulting secondary bile acids are absorbed from the colon. Normal liver removes cholic, chenodeoxycholic and deoxycholic acids very efficiently from portal blood and excretes them rapidly into bile, thus establishing an entero-hepatic circulation (i.e. intestine – portal vein – liver – bile – intestine). Lithocholate is sulphated by the liver and lithocholate sulphate is removed in the feces. Liver disease affects bile salt metabolism in several ways. It may impair primary bile acid synthesis, change the relative proportions of the different bile acids, affect the amount of bile acid which is conjugated, change the taurine:glycine ratio, or lead to the production of unusual bile acids. If less primary bile acid enters the intestine, there will be reduced synthesis of secondary bile acids and a fall in plasma levels of deoxycholate. Impaired liver function, or diversion of portal blood, reduces bile acid removal from portal blood causing an increased level of plasma bile acids, particularly after meals. Plasma bile acids rise with biliary obstruction because they regurgitate from the biliary tree into the blood stream. When plasma bile acid levels are high their urinary excretion increases. For clinical purposes the simplest bile acid test is the total serum bile acid concentration, either fasting or after a meal, but it is rarely available in routine laboratories. The fasting level, normally up to 15 umol/l, is increased in only about two-thirds of patients with a variety of types of liver disease; it is therefore of limited value in screening for liver disease. Levels remain high after eating in almost all patients with significant liver disease; the bile acid level two hours after a meal can thus be used to screen for liver disease. The finding of a high serum bile acid level has high specificity for the detection of liver disease (compared with other individual tests), but its sensitivity is limited. Addition of a bile acid test would not improve the specificity already obtained with the usual batteries of tests.

AMMONIA Arterial ammonia levels tend to be high in chronic liver disease, particularly if there is hepatic encephalopathy or a large amount of portal systemic shunting. They also increase in severe acute hepatitis and fulminant hepatic failure. There is a poor correlation between ammonia levels and the degree of encephalopathy. Measurement of blood ammonia is of little clinical value in liver disease. A high blood ammonia is also seen in hereditary deficiencies of urea cycle enzymes, when the blood ammonia is usually higher than in acquired liver diseases; other liver function tests tend to be normal in these conditions.

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OTHER TESTS WHICH MAY BE USEFUL IN MANAGING LIVER DISEASE Glucose Glucose intolerance (due to insulin resistance) and frank diabetes mellitus (due to an added impairment of insulin secretion) are common in patients with cirrhosis. With fulminant hepatic failure hypoglycemia is common and may persist despite intravenous administration of relatively large amounts of glucose; frequent blood glucose measurements are therefore mandatory in this condition. Hypoglycemia also occurs in acute fatty liver of pregnancy. Cholesterol, Triglyceride, and Lipoproteins (Harry & McIntyre, 1999) Serum total cholesterol level is often elevated in patients with biliary obstruction, due to a rise in free cholesterol, and may reach very high levels in some patients with primary biliary cirrhosis. Lipoprotein electrophoresis usually shows an abnormal pattern, with loss of alpha (HDL) and pre-␤ (VLDL) bands and a prominent beta band. Much of the cholesterol is carried in an abnormal lipoprotein, LP-X, which is rich in free cholesterol and phospholipid (lecithin). In severe parenchymal liver disease, acute or chronic, total cholesterol tends to fall due to a reduction in cholesteryl ester. Electrophoresis reveals a loss of alpha (HDL) and pre-beta (VLDL) bands. In acute hepatitis, there may be a rebound hypercholesterolemia during the recovery phase. The abnormalities underlying the lipoprotein changes of liver disease are complex. Lecithin cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transfer of an acyl group from lecithin to cholesterol with the formation of cholesteryl ester. It is produced in the liver, has a short half life in plasma, and falls in many types of liver disease. Some workers hold that determination of LCAT is the single most sensitive test of hepatocellular dysfunction. The changes in plasma lipids and lipoproteins in liver disease seem to depend mainly on plasma LCAT activity, although regurgitation of biliary lipid contributes to the high cholesterol and phospholipid levels found with biliary obstruction. Serum triglyceride levels increase in various types of liver disease; they tend to show reciprocal changes with total serum cholesterol because when LCAT activity falls insufficient cholesteryl ester is produced to occupy the core of the LDL particles derived from triglyceride rich VLDL.

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QUANTITATIVE TESTS OF LIVER FUNCTION Galactose Elimination Capacity (Tygstrup, 1990) Galactose, a naturally occurring monosaccharide, is removed from plasma only by liver and kidneys. Conversion to galactose-1-phosphate by hepatic galactokinase is the rate-limiting step in its metabolism. At high plasma levels the reaction is saturated and so liver removes galactose at a constant rate (i.e. zero order kinetics). To calculate the hepatic removal rate plasma levels are measured every 5 min for an hour after rapid intravenous injection of a bolus of galactose; urinary galactose is also measured. From 20 to 40 min after injection galactose levels fall linearly with time; with several assumptions, and taking urinary excretion into account, the “galactose elimination capacity” rate of the liver can be calculated. In normal subjects it is about 270+/−40 (S. D.) mg/min per m2 of body surface area, or 6.7+/−1.0 mg/min per kg body mass. Galactose elimination capacity correlates well with other indices of hepatocellular function, like prothrombin time, serum albumin and antipyrine removal rate, but with the advantage of measuring just one aspect of hepatocellular function. There is considerable overlap between normal subjects and patients with a variety of liver diseases. Repeat estimations in the same subject after a short time interval vary by only about 10%. The test is most useful for assessing improvement or deterioration in liver function over a longer period of time.

Aminopyrine Removal and Breath Test (Schoeller et al., 1982; Tygstrup, 1990) Removal and catabolism of drugs are functions of the liver which can be assessed in several ways. After a single injection we can follow plasma levels, or the decay in plasma radioactivity of radio-labeled drug. Production of drug metabolites can be assessed from blood or urine measurements. Several groups of drugs are Ndemethylated; the methyl groups removed are oxidized and their carbon atoms appear in the breath as CO2 . The disappearance rate from plasma of labeled aminopyrine (14 C-dimethyl amino-antipyrine) is easily measured. This compound can also be used in noninvasive breath tests. After oral administration of 14 C-aminopyrine breath CO2 is collected by asking the subject to blow into scintillation vials containing a trap for CO2 ; an indicator changes color when 2 mmol of CO2 has been taken up. From the specific activity of breath CO2 one can calculate the amount of labeled CO2 produced by demethylation, assuming constant endogenous production of CO2 . If the specific activity of CO2 samples is plotted against time the disappearance curve

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over about 12 hours is roughly exponential, and so a decay constant, Kb, can be estimated. This correlates well with the decay constant for plasma disappearance (Kp), with BSP disappearance, and with the galactose elimination capacity. Sampling of breath over many hours is tedious. The test has been modified to last only two hours, either by taking the mean value for CO2 specific activity during the first two hours after ingestion of the drug, or by sampling breath only at two hours; results in patients can be compared with those obtained from normal subjects. Values for patients with hepatocellular dysfunction are generally lower than in normal subjects; normal results may be found with biliary obstruction. The aminopyrine two-hour breath test is not a useful diagnostic test but it allows sequential measurements of one aspect of hepatic function to be made in the same subject over time; the coefficient of variation of duplicate tests is about 6%. There is reluctance to use radioactive material with a long half-life for repeated studies in man, but the radiation dosage of the test is small. 13 C aminopyrine can be used but then mass spectrometry is required for measurement of the isotope.

Hepatic Removal of Bromosulphthalein, Dibromosulphthalein and Indocyanine Green (Schoeller et al., 1982; Tygstrup, 1990) Bromosulphthalein (BSP) was widely used to measure certain aspects of hepatic function because it is easy to measure in blood and bile. It is removed from blood by hepatocytes, bound by intracellular proteins, and excreted in bile either unconjugated (approximately 30%) or conjugated with glutathione. In the USA a simple BSP test was widely used to detect liver disease in non-jaundiced subjects. After an IV bolus of BSP its plasma level was measured on a single blood specimen taken at 45 minutes. Assuming an initial distribution in a plasma volume of 50 ml/kg body mass, and thus a known initial concentration of BSP, retention at 45 minutes is easily calculated. In normal subjects, it is up to 7% of the original dose. Higher values are found with many types of liver disease. This test was not popular in Britain, because it provides limited information and is potentially dangerous. Tissue damage results if BSP extravasates at the site of injection: anaphylaxis has been reported on numerous occasions; and if the BSP is not properly prepared for injection, neurological problems may result from injection of microcrystals. More complex studies of BSP removal have also been used to explore different aspects of the hepatic handling of BSP. After a single IV injection the plasma BSP disappearance curve can be represented by two exponentials. If a two compartment model is assumed (plasma and liver), with loss of BSP only in urine and bile, we can calculate the size of the plasma compartment, hepatic storage, the transfer of

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dye between compartments, urinary excretion, and the rate of biliary excretion – which has a theoretical maximum level, the Tm. Unfortunately, BSP conjugation is neglected and other problems also affect interpretation. Even so, it is a useful method of evaluating changes in a fairly clearly defined function of the liver. Attempts were also made to estimate the Tm and hepatic storage capacity (S) for BSP from plasma levels found during constant infusion of the dye at different rates. Unfortunately, the method was based on false assumptions. It is easily shown that the resulting values for S must be wrong; they depend not only on the liver’s capacity to concentrate BSP but also on blood flow (McIntyre, Mulligan & Carson, 1973). Dibromosulphthalein (DBSP) is handled by the liver like BSP, but has the advantage that it is not conjugated with glutathione; this simplifies the assumptions involved in analysis of plasma disappearance. Indocyanine green (ICG) is also rapidly removed by the liver and excreted into bile without conjugation. It is much safer than BSP (but may be contaminated by small amounts of sodium iodide), is simple to measure, and can be used to study hepatic uptake, storage and transport of an exogenous dye. ICG retention at 20 minutes can be measured and plasma disappearance curves analyzed like BSP disappearance curves (Keiding & Skak, 1988). It is more expensive than BSP, and as it is unstable in plasma levels must be measured soon after withdrawal of the sample. Both BSP and ICG have been used to measure hepatic blood flow by the Fick principle. The plasma level is held constant by continuous infusion of the dye; hepatic blood flow can then be calculated by dividing the rate of infusion of dye by the difference in concentration between arterial and hepatic venous plasma. Unfortunately this measurement calls for hepatic venous catheterization.

Dynamic Studies of Bilirubin Metabolism (Ostrow, 1986) Following injection of radioactive unconjugated bilirubin the label is rapidly removed from plasma; its disappearance curve can be represented as the product of three exponentials. Estimates that can be made from analysis of this curve include the mass of unconjugated bilirubin in the initial pool, its initial volume of distribution, the appearance rate of unlabeled bilirubin, hepatic clearance of bilirubin, and the plasma bilirubin turnover rate. Assuming a three-compartment model one can estimate the size of the compartments and the rate of transfer between them, but the calculations depend on the nature of the assumptions made in constructing the model. Such analyses have helped in the study of disorders of unconjugated bilirubin metabolism, like Gilbert’s syndrome and the Crigler-

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Table 5. Quantitative Tests of Liver Function. Galactose elimination capacity Aminopyrine disappearance and demethylation Bromosulphthalein, Di-bromosulphthalein and indocyanine removal Bilirubin kinetics Bile salt removal and metabolism

Najjar syndrome. The method is expensive and time consuming and does not allow interpretation of findings in conjugated hyperbilirubinemias; it is therefore of little value for studying patients with most types of liver disease.

Dynamic Studies of Bile Acid Metabolism (Hoffman, 1989) Measurements have been made of the fractional disappearance rate of bile acids from plasma after injection of radio-labeled bile acids. They disappear rapidly (within minutes) from plasma; data interpretation is complicated because there may be considerable removal of the label before adequate mixing. Although a reduced removal rate can be demonstrated when there is impaired hepatic function, there are problems in deciding the physiological significance of the changes. The fate of a labeled bile acid can be followed through the various compartments of the total bile acid pool. Mathematical interpretation of the results is extremely complicated because of entero-hepatic re-circulation, conversion of primary to secondary bile acids, and the occurrence of both conjugation and de-conjugation of bile acids. This method is of little value for repeated study of patients with liver disease. Table 5 lists the quantitative tests of liver function.

SUMMARY Biochemical investigations are widely used in the management of patients with disorders of the liver and/or biliary tree. A small number of them are cheap to perform and are therefore used routinely as a small battery of so-called “liver function tests” (LFTs); this usually includes serum bilirubin, aspartate and alanine aminotransferases, alkaline phosphatase, ␥-glutamyl transferase and albumin. LFTs help to detect liver disease and to monitor its course, but are of relatively little use for precise diagnosis. Despite their name, they are also of little value for assessing hepatic function.

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Some biochemical tests are of diagnostic value for certain conditions, but the results may be difficult to interpret and immunological tests, liver biopsy and appropriate imaging techniques remain important tools for the hepatologist. Few methods are available for the assessment of individual functions of the liver. Those that are tend to be relatively complicated to perform and are usually costly, in terms of the materials used, laboratory time and in inconvenience to the patient. They help our understanding of the pathophysiology of liver disease, and are of particular value for assessing the response to new therapies.

REFERENCES Beckett, G. J., & Hayes, J. D. (1987). Plasma glutathione-S-transferase measurements and liver disease in man. Journal of Clinical Biochemistry and Nutrition, 2, 1–24. Harry, D. S., & McIntyre, N. (1999). Plasma lipids and lipoproteins (Chapter 2.12). In: J. Bircher, J.-P. Benhamou, N. McIntyre, M. Rizzetto & J. Rodes (Eds), Oxford Textbook of Clinical Hepatology (2nd ed., pp. 287–302). Oxford: Oxford University Press. Hoffman, A. F. (1989). Enterohepatic circulation of bile acids. In: Handbook of Physiology – The Gastrointestinal System (Vol. III). Baltimore: American Physiological Society. Hutchinson, D. R., Halliwell, R. P., Smith, M. G., & Parke, D. V. (1981). Serum “prealbumin” as an index of liver function in human hepatobiliary disease. Clinica Chimica Acta, 114, 69–74. Keiding, S., & Skak, C. (1988). Methodological limitations of the use of intrinsic hepatic clearance of ICG as a measure of liver cell function. European Journal of Clinical Investigation, 18, 507–511. McIntyre, N., Mulligan, R., & Carson, E. R. (1973). BSP Tm and S: A critical re-evaluation. In: G. Paumgartner & R. Preisig (Eds), The Liver: Quantitative Aspects of Structure and Function (pp. 417–427). Basel: Karger. McIntyre, N., & Rosalki, S. B. (1999). Biochemical investigations in the management of liver disease (Chapter 5.1). In: J. Bircher, J.-P. Benhamou, N. McIntyre, M. Rizzetto & J. Rodes (Eds), Oxford Textbook of Clinical Hepatology (2nd ed., pp. 503–521). Oxford: Oxford University Press. Moss, D. W., & Henderson, A. R. (1994). Enzymes (Chapter 20). In: C. A. Burtis & E. R. Ashwood (Eds), Tietz Textbook of Clinical Chemistry (2nd ed., pp. 735–896). Philadelphia: W. B. Saunders. Nemezansky, E. (1986). Gamma-glutamyltransferase (GGT) (Chapter 9). In: J. A. Lott & P. L. Wolf (Eds), Clinical Enzymology. New York: Field Rich and Assoc, distributed by Year Book Publishers. (NB 1986 NOT 1968!!) Ostrow, J. D. (Ed.) (1986). Bile pigments and jaundice. New York: Marcel Decker. Rothschild, M. A., Oratz, M., & Schreiber, S. S. (1988). Serum albumin. Hepatology, 8, 385–401. Schoeller, D. A., Baker, A. L., Monroe, P. S., Krager, P. S., & Schneider, J. F. (1982). Comparison of different methods of expressing results of the aminopyrine breath test. Hepatology, 2, 455–462. Tygstrup, N. (1990). Assessment of liver function: Principles and practice. Journal of Gastroenterology and Hepatology, 5, 468–482.