APPROACH TO ABNORMAL TESTS

Mitchell L Shiffman, MD Liver Institute of Virginia Bon Secours Health System Richmond and Newport News, VA

CATAGORIES OF LIVER FUNCTION TESTS: What are commonly referred to as liver function tests (LFTs) do not actually measure liver function. Liver transaminases and are utilized to assess injury to the liver but can still be normal in patients with liver disease. True liver function tests measure the metabolic and synthetic function of the liver. Other “non-liver” tests can be utilized to estimate hepatic fibrosis and mortality in patients with advanced liver disease.

Liver chemistries: Aspartate aminotransferase Reflect injury to the liver Alanine aminotransferase Alkaline phosphatase Gamma glutamyl transferase 5’ nucleotidase Tests which assess liver function Albumin INR (factor 7) Tests which assess hepatic metabolism Total Direct and indirect bilirubin Non-Liver function tests Platelet count Serum creatinine

LIVER TRANSAMINASES: Transaminases are enzymes that convert amino acids to alpha-keto acids. During this reaction an amino acid is metabolized to its alpha-keto acid by removing the amino group and leaving an alpha-keto group. Alpha-keto-acids are produced from amino acids by the reverse reaction. The liver contains several transaminases. The two that are typically measured to assess for the presence of liver cell injury are alanine aminotransferase (ALT) and aspartate aminotransferase (AST). ALT is a cytoplasmic enzyme. AST is a mitochondrial based enzyme. Injury to liver cells release ALT into serum in higher concentrations than AST. For this reason, ALT is always higher than AST in all forms of chronic liver disease except for those diseases which selectively injure the mitochondria. This most commonly occurs in patients with either alcohol induced liver injury or non-alcoholic steatohepatitis (NASH).

What is the normal value for ALT: There is no defined WHO (World Health Organization) normal value for serum ALT. In the past the upper normal for ALT was defined as 2 standard deviations above the mean for that population. However, with the epidemic of HCV, obesity and NAFLD it was noted that the upper normal for serum ALT increased between 1970-2000 in many locations and tertiary care medical centers; especially in those centers that specialized in liver disorders. Several studies have attempted to define the true upper limit of normal for ALT. All of these studies included Liver Function Tests

only healthy persons taking no medications and with normal body weight. Patients with a serologic positive test for any liver disorder, persons on any medications and those with an elevated BMI were excluded. Some of these studies were performed on healthy volunteers others were performed on blood donors. In this population the upper normal for serum ALT (defined as two standard deviations above the mean) was approximately 19 for females and 30 for males. African Americans had slightly higher values for ALT then Caucasians.

Ratio of AST and ALT: Although ALT is generally higher than AST in most forms of chronic liver disease the ratio of these enzymes is affected by the degree of fibrosis. As fibrosis progresses and patients develop cirrhosis the ratio of AST/ALT increases and becomes greater than 1 once cirrhosis has developed. This “rule” is true about 80% of the time can be utilized with other laboratory tests to help determine that a patient with chronic liver disease has developed cirrhosis.

ALT is found in high concentrations within the liver and in very low concentrations in other tissues. In contrast, AST is found in many other tissues with high metabolic rates including muscle (skeletal, cardiac and smooth muscle), kidney and brain. Injury to any off these tissues can cause elevations in AST but will not significantly affect ALT. Thus, patients with elevations in serum transaminases where the ratio of AST/ALT exceeds 5 are unlikely to have liver injury, especially if the ALT is normal.

Using liver tests to assess hepatic fibrosis: Since the AST/ALT ratio is affected by the degree of fibrosis several fibrosis scoring systems have been developed to estimate hepatic fibrosis and possibly replace liver biopsy.

APRI Index: The APRI (aspartate-platelet ratio index) is simple and utilizes only 2 commonly ordered laboratory values; AST and platelet count. The APRI is calculated as follows:

APRI = AST/(AST ULN)/(platelet tcount) x 100.

Since AST increases and the platelet count declines with increasing fibrosis, increasing fibrosis is associated with a rise in APRI. The positive predictive value for APRI values greater than 1.5 with cirrhosis (Metavir fibrosis stage 4) is about 80%. The positive predictive value for APRI values less than 0.5 with either none or mild fibrosis is also about 80%. Although APRI values between 0.5-1.5 are suggestive of intermediate stages of fibrosis (Metavir stages 2 and 3) the positive predictive value for APRI between this range is only about 60%.

FIB-4: The FIB-4 was originally developed in a cohort of patients with HCV and HIV co-infection but has now been applied to several populations including patients with NAFLD. The formula is simple to calculate and utilizes 3 commonly performed laboratory tests and the patient’s age. The FIB-4 is calculated as follows:

FIB-4 = ((Age in years) x AST)) / (Platelet count x (ALT/2).

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In general, the higher the value the more likely the patient will have advanced fibrosis or cirrhosis. In patients with chronic HCV values less than 1.45 have a 90% probability for not having advanced fibrosis or cirrhosis; Ishak fibrosis scores 4-6 or Metavir fibrosis score of 3-4. Values greater than 3.25 have a 97% specificity and 65% positive predictive value for having advanced fibrosis or cirrhosis. A Fib-4 of greater than 3.25 has a positive predictive value of about 90% for advanced fibrosis or cirrhosis (Ishak stages 4-6). In patients with non-alcoholic fatty liver/nonalcoholic steatohepatitis values of >2.67 were 90% predictive of advanced fibrosis or cirrhosis and values less than 1.30 were 90% predictive of not having advanced fibrosis or cirrhosis. Cut-off values will vary for other etiologies of liver disease.

Fibrosure: Fibrosure is a proprietary laboratory test which utilizes several liver chemistries to arrive at a value which correlates with the degree of fibrosis in patients with chronic HCV. It may be less reliable in patients with NAFD and other forms of chronic liver disease. The chemistries utilized in fibrosure include: alpha-2-macroglobulin, haptoglobin, gamma-GT, age in years, total bilirubin, apo-A1 and sex. The scale extends from 0-1. Values less than 0.1 have an 80% positive predictive value for none-mild fibrosis (Metavir fibrosis stage 0-1). Values greater than 0.8 have a positive predictive value of about 80% for advanced fibrosis or cirrhosis (Metavir fibrosis stage 3-4). The overlap in fibrosis scores, F1-3, for values between these extremes is significant and therefore, Fibroscore values between 0.1-0.8 do not correlate well with any specific stage of fibrosis.

Assessing fibrosis with elastography: Elastography is a technique which measures how elastic or stiff the liver is. This can be performed with ultrasound or magnetic resonance.

Fibroscan: Fibroscan is a devise which assesses liver stiffness by ultrasound. The Fibroscan devise provides a numerical value for liver stiffness in kilopascals (kPa). The more fibrotic the liver, the more stiff the liver will be in kPa. Fibroscan has been utilized to assess liver fibrosis in many liver disorders. The most data generated has been in patients with HCV. In chronic HCV a liver stiffness value of >12 kPa is about 80% specific for cirrhosis on liver biopsy; and a value of about 4 kPa or less is about 80% specific for none-mild fibrosis. There is significant overlap for values between 4-12 kPa with fibrosis stages F1-F3. In addition, many factors can affect the measurements obtained with Fibroscan. For example, higher values will be obtained after a meal because increased hepatic blood flow increases liver stiffness. Significant amounts of steatosis and severe hepatic inflammation which can be seen in severe autoimmune hepatitis may also lead to higher kPa values.

Shearwave ultrasonography: This is another ultrasound technique to measure liver stiffness. This is performed with a special software package incorporated into a standard ultrasound machine. Instead of reporting the data in kPa these instruments report stiffness as mm/sec. The data accumulated to date using shearwave ultrasound to assess liver fibrosis is currently limited.

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Magnetic resonance elastography: Liver stiffness can also be measured by MR. This requires a specialized MR unit, a coil/probe which is placed over the liver for the scan and a specialized software package. The technique is not yet FDA approved, and MR units that can perform scan and analysis are currently limited. Data is just starting to emerge regarding values that are significant. The cost of MR is also significantly greater in the USA than for Ultrasound, Fibroscan or any non-invasive biochemical marker.

ALKALINE PHOSPHATASE: Alkaline phosphatase (ALP) is an enzyme that is found in many cell types. Its function is to remove a phosphate group from phosphorulated substrates such as phospholipids and sugars. In the liver, the enzyme is located on the canalicular membrane of the hepatocyte and at this location ALP is believed to be involved with the transport of phospholipids into bile. ALP is not present in cells. Serum ALP rises when the canalicular membrane is “disrupted”. This causes translocation of ALP from the canalicular membrane to the basal lateral surface of the hepatocyte where it enters the plasma.

Differentiating liver from non-liver ALP: The most common organs which produce ALP include the liver, bone, intestine, kidney and placenta. Certain types of tumors (ovarian and lung adenocarcinoma) also secrete ALP. Elevations in serum ALP may therefore not reflect injury to the liver or biliary tree. Placental ALP increases during the third trimester and immediately post-partum. Bone diseases associate with rapid bone growth such as Padget’s Disease is associated with a marked elevation in bone derived ALP. Bone ALP is affected by age. In children and adolescents marked and rapid bone growth cause bone ALP to be elevated. The total ALP then declines by the early 20s but starts to increase again in the elderly reflecting osteoporosis and bone breakdown. In the absence of overt liver disease it is often important to determine if the elevation in serum ALP is from the liver or another organ. One way this can be done is to fractionate the ALP. ALP from different cell types are not identical, migrate differently with electrophoresis and can be separated. Unfortunately, the total ALP often reflects a mixture of ALP from these different organs and fractionation typically yields a result which provides the relative proportions of several ALPs in the serum and does not identify the specific organ source for the elevation. For this reason measuring an alternate liver enzyme which is also affected in cholestatic liver disease is frequently more helpful. The most common enzyme which is measured is gamma glutamyl transferase (GGT). 5’nucleotidase can also be measured.

GGT: GGT is an enzyme which transfers the gamma-glutamyl moiety of glutathione to water forming glutamate or to an amino acid. GGT is present in many cell types and organs including the liver, bile duct cells, gall bladder, pancreas, kidneys, spleen, heart, brain and seminal vesicles. In the liver GGT is more sensitive than ALP in detecting injury to the bile ducts or liver. GGT is particularly sensitive to ethanol and is therefore frequently utilized to detect alcohol use in abstinence programs. Patients where the ALP is elevated but have a normal GGT do not typically have liver disease. The exceptions to this are in patients with genetic alterations in bile salt synthesis and transport such as familial intrahepatic cholestasis (FIC) and benign recurrent intrahepatic cholestasis (BRIC) where the GGT is normal despite elevations in AST, ALT and/or Liver Function Tests

ALP.

Cholestasis: Cholestasis is a pathophysiologic process in which the liver does not secrete bile. The biochemical hallmark of cholestasis is an elevation in ALP. Cholestasis can be divided into 2 causes: obstructive and intrahepatic cholestasis.

Obstructive jaundice: Is characterized by elevated ALP, total bilirubin, and imaging which demonstrates bile duct dilation. Since the canalicular membrane is the acinus of the biliary tree bile duct obstruction damages the canalicular membrane and releases ALP into the serum. Obstructive jaundice is most commonly seen in patients with pancreatic cancer, cholangiocarcinoma, choledocholithiasis, and can be seen in Primary Sclerosing Cholangitis (PSC).

Histologic features of obstructive jaundice include accumulation of bile salts and bile pigments within the liver leading to bilirubin stasis in zone 1 and bile plugs within canaliculi. Lakes of bile may form in the periportal zone. Toxic bile salts accumulate adjacent to the portal tracts and injure hepatocytes which take on a foamy appearance referred to as feathery degeneration.

Another histologic feature of obstructive jaundice is bile duct proliferation. Normally the portal tract contains a branch of the portal vein, hepatic artery and a bile duct in equal proportions. Bile duct proliferation is present when the number of bile ducts within the portal area exceeds the number of vascular structures. In reality this is not due to an absolute increase in the number of bile ducts, but and increase in the length of same bile duct which becomes tortuous and thus appears numerous times in the histologic section.

Intrahepatic cholestasis: Is characterized by an elevation in ALP with either a normal or elevated total bilirubin. The common bile duct is normal on imaging studies. This is most often caused by medications. Intrahepatic cholestasis can also be caused by injury to small intra-hepatic bile ducts, as seen in Primary Biliary Cirrhosis (PBC), PSC, infiltrative disorders of the liver and alcohol.

When severe and associated with jaundice, bile lakes, bilirubin stasis and bile plugs may be present. However, bile duct proliferation is typically absent and the ducts appear histologically intact. An exception to this occurs in PBC (see lecture on PBC).

Infiltrative disorders: Five diseases which "infiltrate" within the liver all present as intrahepatic cholestasis. These include sarcoidosis, tuberculosis, amyloidosis, lymphoma and hepatic metastasis. Infiltrating tumors such as oat cell carcinoma and lymphoma may not be visualized on CT or ultrasound scans. The diagnosis of these disorders is made by liver biopsy.

Alcoholic hepatitis: The cholestatic form of alcoholic hepatitis presents with a marked elevation in serum ALP. The histologic hallmark of this cholestatic form of alcoholic hepatitis is peri-sinusoidal fibrosis. The liver biopsy is diagnostic. The prognosis for this type of alcoholic hepatitis is very poor with a 1 Liver Function Tests

year survival of under 50%. Abstinence from alcohol does not typically improve the prognosis of this form of alcohol induced liver injury.

Sarcoidosis: The liver is the second most common organ affected by Sarcoidosis. Although these patients have hepatic granulomas only about 10% of patients with hepatic sarcoidosis develop progressive liver injury, fibrosis and cirrhosis. These patients are recognized histology because they have significant inflammation associated with the granulomas. The vast majority of patients with hepatic sarcoidosis are asymptomatic. There is very little reason to treat patients with hepatic sarcoidosis with steroids or other forms of immune suppression. No clinical trials have demonstrated that corticosteroids are helpful in patients with hepatic Sarcoidosis including those patients with significant inflammation who are at greatest of risk of developing fibrosis progression. A small subset of patients with hepatic sarcoidosis develop hepatomegally and abdominal pain or spiking fevers. A short tapering course of prednisone may be helpful in these particular patients. Patients with hepatic sarcoidosis can develop pre-sinusoidal portal hypertension and esophageal varicies with bleeding. Hepatic sarcoidosis is one of the causes of non-cirrhotic portal hypertension. Patients with repeated episodes of bleeding are excellent candidates for TIPS or shunt surgery.

Benign intrahepatic recurrent cholestasis (BRIC): BRIC is a rare autosomal recessive disorder found in persons of Dutch ancestry. It is characterized by repeated episodes of severe intense pruritus, profound elevations in serum ALP and bilirubin with normal or near normal values for serum GGT and normal serum liver transaminases. Attacks last from several weeks to months and resolve spontaneously. Between attacks patients remain asymptomatic for months to years. The disorder does not lead to progressive liver injury and is not fatal. However, the symptoms of cholestasis may be severe and debilitating. Between attacks the liver is histologically normal. During attacks liver histology may be normal or demonstrate features of cholestasis and but devoid of inflammation. The disorder results from a mutation in ATP8B1, a gene which codes for the FIC1 (familial intrahepatic cholestasis) protein. It is believed that this protein plays a role in bile salt secretion, aminophospholid transport and is important in maintaining cell membrane fluidity. Therapy is supportive and aimed at relieving both pruritus and other complications of severe cholestasis until the episode resolves spontaneously. Although the trigger which precipitates each episode is unknown a shift in the bile salt pool to secondary, more toxic, bile salts has led to the use of ursodeoxycholic acid as prophylaxis against additional episodes. It remains unclear just how effective this strategy is. A recent case series has demonstrated that removing the toxic bile acids from the enterohepatic circulation by diverting bile externally via a nasobiliary drain allows the normal bile salt pool to reestablish quicker and can shorten attacks.

COAGULATION FACTORS AS LIVER FUNCTION TESTS: Most coagulation factors are synthesized by the liver. These include Factors I, II, V, VII, IX, X, XII and XIII. The formation of Factors II, VII, IX and X is dependent upon gamma- carboxylation. This reaction requires vitamin K as a co-factor. The prothrombin time (PT) is a measure of the time taken to convert prothrombin to thrombin. Factor VIII is a necessary step in this process. The half-life of Factor VII is only 6 hours and as a result the PT represents the most Liver Function Tests

sensitive test of acute hepatic dysfunction. A number of different factors may cause a prolongation of the PT.

Inadequate dietary intake of vitamin K: Inadequate dietary vitamin K results in decreased gamma-carboxylation of Factors VII, IX, X and II. Causes for this include malnutrition, an inability to absorb vitamin K secondary to intestinal disease, gastric bypass surgery which may lead to poor mixing of fat soluble vitamins with bile salts necessary for absorption and bacterial overgrowth syndrome. In these cases the PT rapidly returns to normal following the administration of subcutaneous vitamin K.

Cholestasis: Bile salts are necessary for the absorption of fat soluble vitamins including vitamin K. Prolonged cholestasis as is seen during the Intrahepatic cholestasis of pregnanacy, BRIC, primary biliary cirrhosis, sclerosing cholangitis, prolonged drug induced cholestasis or obstructive jaundice can lead to a prolongation in the PT. This will readily correct following the administration of subcutaneous vitamin K.

Administration of drugs which inhibit gamma-carboxylation: Several drugs or toxins may interfere with gamma-carboxylation and therefore prolong the PT. The most widely utilized of these drugs is warfarin.

Intrinsic hepatocellular dysfunction: Acute and chronic liver failure will lead to a prolongation of PT. This is secondary to an inability of the liver to synthesize appropriate amounts of this coagulation protein. In patients with severe acute liver failure, an improvement in the PT is one of the first signs that the event is resolving and the liver function will improve. In patients with chronic liver disease the PT (INR) is utilized as an indication of liver disease severity and for calculation of the MELD score to assess priority for liver transplantation. Administering vitamin K to patients with advanced liver disease will not improve the PT unless the patient is also deficient in vitamin K stores for one of the other reasons listed above. Thus, the administration of vitamin K to a patient with cirrhosis can be extremely useful if a patient with cirrhosis has an unexpected prolongation in PT. However, if the PT does not rapidly improve vitamin K should not be continued in such a patient.

International normalization ratio: The INR was developed as a way to standardize the PT across all laboratories for managing anti-coagulation with coumadin. Several studies have demonstrated that neither the PT nor INR may correlate with the degree of anti-coagulation in patients with acute or chronic liver disease. Despite this the INR continues to be utilized as a rough guide to coagulation status in patients with liver disease, as a measure of liver function in patients with cirrhosis and as part of the MELD score to prioritize patients for liver transplantation.

Thrombosis in Patients with Cirrhosis: The PT is a functional test which measures information regarding the presence of clotting proteins in serum. However, the serum also contains anti-coagulant proteins and although immunoassays are available to measure proteins C, S, anti-thrombin III and a genetic test can Liver Function Tests measure for the presence of Liden factor V mutation, no functional assay is currently available to assess anti-coagulation in serum. In normal individuals the presence of clotting and anti-clotting proteins are in balance. In genetic disorders associated with a deficiency of anti-clotting factors (ie. C, S, anti-thrombin III, Leiden V mutation, etc) or an increased synthesis of clotting factors thrombosis may occur in the setting of a normal PT/INR. The treatment is to reduce the concentration of clotting factors. This is accomplished by administering antagonists (warfarin) to reduce the synthesis of vitamin K dependent clotting factors.

In patients with cirrhosis the production of clotting factors and anti-clotting factors are both reduced. However, cirrhosis may not lead to a reduction in both sides of the coagulation system equally. If the synthesis of anti-clotting factors is reduced more than the synthesis of clotting factors spontaneous thrombosis may occur. If the reverse, spontaneous bleeding may occur. As a result, the PT/INR may not be an accurate way to assess the status of the coagulation system in patients with acute or chronic liver failure. This explains why patients with acute liver failure develop spontaneous thrombosis in the setting of a marked prolongation in the PT/INR. This is also why patients with cirrhosis can develop portal vein thrombosis.

BILIRUBIN: Bilirubin is a breakdown product of . The heme is initially cleaved by heme oxygenase into biliverdin, which is subsequently converted to bilirubin. Unconjugated bilirubin is insoluble in an aqueous environment and must be bound to albumin in the circulation. Unconjugated bilirubin is actively transported into the hepatocyte. Within the hepatocyte bilirubin is conjugated by the enzyme UDP-glucuronosyl transferase into a bilirubin-diglucoronide. The conjugated bilirubin is water soluble and secreted into bile.

Excessive bilirubin production: Increased bilirubin production is secondary to increased red blood cell turnover. This is most commonly observed in hemolytic conditions. However, this can also be observed in patients who experience significant intra-abdominal bleeding or bleeding into a limb with the formation of a large clot. As the clot is broken down large amounts of unconjugated bilirubin will be released into the circulation. Examples of hemolytic disorders associated with chronic unconjugated hyperbilirubinemia and jaundice include hereditary spherocytosis and sickle cell disease. In these disorders the serum bilirubin is frequently below 5 mg/dl unless acute massive hemolysis occurs or underlying liver dysfunction is also present.

Decreased hepatic conjugation: Genetic alterations in the UDP-glucuronosyl transferase enzyme lead to either absent or a reduced rate by which bilirubin is conjugated. Certain medications can inhibit this enzyme. Both lead to reduced conjugation of bilirubin and jaundice.

Inherited disorders of bilirubin conjugation: Inherited disorders of bilirubin conjugation are due to deficiencies in the activity of UDP- glucuronosyl transferase. Crigler-Najjar syndrome Type I is characterized by the near complete absence of this enzyme. Such patients develop severe unconjugated-hyperbilirubinemia with Liver Function Tests

unconjugated bilirubin approaching 20 mg/dl. Without treatment unconjugated bilirubin will dissociate from albumin, diffuse across the blood-brain barrier and nearly all involved infants develop kernicterus, permanent neurotoxicity and cognitive changes. The clinical features of kernicterus include lack of muscle tone, abnormal reflexes and progression to atonia and death. Patients who recover from acute bilirubin toxicity may have long-term sequella such as hearing impairment due to cochlear damage, cerebellar abnormalities, and varying degrees of mental retardation.

Crigler-Najjar syndrome type I is a rare, autosomal recessive disorder and often found in consanguineous families. Phototherapy, plasmapharesis and orthotopic liver transplantation at an early age have all been used to treat this life-threatening disorder. More recently transplanting isolated human hepatocytes by infusing these into the native liver has also been shown to lower unconjugated serum bilirubin significantly and may be a viable alternative for the treatment of these children in the near future.

Crigler-Najjar syndrome Type II is a milder form of UDP-glucoronosyl transferase deficiency. The enzyme retains approximately 50% of its activity and serum unconjugated bilirubin typically remains below 5 mg/dl in most persons. Liver histology is normal. Patients are asymptomatic and the disorder is typically associated with no clinically significant sequella. The UDP- glucoronosyl transferase enzyme is inducible by phenobarbital and this can be utilized to lower the unconjugated bilirubin concentration when very high.

Gilbert’s Syndrome: Gilbert’s syndrome is the most common cause of unconjugated hyperbilirubinemia in the Caucasian population. This is an autosomal dominant trait which leads to a slight decline in the activity of UDP-glucoronysl transferase activity. Total serum bilirubin is chronically elevated and typically in the 2-4 mg/dl range. The bilirubin typically doubles with fasting or severe stress to values which approach 7-8 mg/dl. Having a patient fast prior to re-measuring the bilirubin and documenting a near doubling in total bilirubin is virtually diagnostic of the disorder. Liver histology is normal and a liver biopsy does not need to be performed simply to confirm the diagnosis of Gilbert’s syndrome. Patients are asymptomatic and the disorder is not associated with any clinical sequella. The enzyme remains inducible with phenobarbital and low doses of this agent can be utilized to lower the bilirubin concentration to normal in persons concerned about the cosmetic effect of being jaundiced.

Maturation of the UDP-glucuronosyl transferase and kernicterus: The bilirubin glucoronidase enzyme is one of the last of the hepatic enzymes to mature. As a result, unconjugated hyperbilirubinemia is not uncommon in the early neonatal period. Serum levels of unconjuated bilirubin may rise to 6 mg/dl or higher. This is typically a self limited process. The UDP-glucuronosyl transferase enzyme typically matures within days of birth and serum bilirubin rapidly returns to normal. Elevations in unconjugated bilirubin in the newborn for short periods of time is benign and may not require treatment. However, if the level of unconjugated bilirubin is very high or maturation of UDP-glucuronosyl transferase is significantly delayed infants may be treated with phototherapy. Plasma exchange is utilized in severe cases.

Liver Function Tests

Decreased secretion of conjugated bilirubin into bile: In contrast to the above disorders which cause unconjugated hyperbilirubinemia, disorders of bilirubin secretion into bile are associated with an elevation in conjugated bilirubin. Both Rotor’s syndrome and the Dubin-Johnson syndrome are rare inherited disorders of bilirubin secretion into bile. Despite the presence of conjugated hyperbilirubinemia these patients have no signs or symptoms of cholestasis; the ALP and GGT are normal and symptoms such as pruritus are notably absent because the transport of bile acids and other substances into bile is not impaired. On liver biopsy patients with Dubin-Johnson syndrome are found to have a dark, heavily pigmented liver whereas liver histology in the Rotor’s syndrome is normal. The two conditions can also be differentiated by oral . The is visualized in Rotor’s syndrome but not in Dubin-Johnson syndrome. Both conditions have a benign clinical course and are not associated with liver injury or any other sequella.

NON-LIVER TESTS UTILIZED TO ASSESS LIVER FUNCTION: Several non-liver tests can be utilized to assess the severity of chronic liver disease. These include the platelet count and the serum creatinine.

Platelet count: The platelet count declines with advancing fibrosis and cirrhosis. This is secondary to a reduction in the synthesis of throbopoetin by the liver and portal hypertension which leads to sequestration of platelets by the spleen. The single most common cause for thrombocytopenia in the absence of a blood disorder is cirrhosis. A platelet count of under 150,000 is highly suggestive of cirrhosis and platelet count of under 100,000 is virtually diagnostic of cirrhosis. There is little reason to perform a liver biopsy to confirm cirrhosis in patients with a low platelet count.

Serum creatinine: The serum creatinine is an important measure of hepatic function in patients with advanced cirrhosis. The creatinine is utilized as part of the MELD score to predict 30 day mortality in patients with cirrhosis and is given more weight in the calculation than either the total bilirubin or platelet count. A rising creatinine in a patient with cirrhosis increases the MELD score exponentially and is associated with extremely poor prognosis.

REFERENCES: Lee TH, Kim WR, Poterucha JJ. Evaluation of elevated liver enzymes. Clin Liver Dis. 2012 May; 16:183-98.

Prati D, Taioli E, Zanella A, Della Torre E, Butelli S, Del Vecchio E, Vianello L, Zanuso F, Mozzi F, Milani S, Conte D, Colombo M, Sirchia G. Updated definitions of healthy ranges for serum alanine aminotransferase levels. Updated definitions of healthy ranges for serum alanine aminotransferase levels. Ann Intern Med. 2002; 137:1-10.

Siddique A, Kowdley KV. Approach to a patient with elevated serum alkaline phosphatase. Clin Liver Dis. 2012; 16:199-229. Liver Function Tests

Krier M, Ahmed A. The asymptomatic outpatient with abnormal liver function tests. Clin Liver Dis. 2009; 13:167-77.

Kamath PS, Kim WR. The international normalized ratio of prothrombin time in the model for end-stage liver disease score: a reliable measure. Clin Liver Dis. 2009; 13:63-6.

Luketic VA, Shiffman ML. Benign recurrent intrahepatic cholestasis. Clin Liver Dis 2004; 8:133- 150.

QUESTIONS:

Question 1: A 35 year old male is referred for evaluation of elevated liver enzymes. He went to see his primary care physician 2 weeks ago because of weakness. He was found to have an AST of 250 and ALT of 25 IU/ml. ALP, total bilirubin and albumin were all normal. Serologic studies for all causes of chronic liver disease are normal except for anti-HBcore. What test would be the most likely to lead to the diagnosis in this patient?

A. Liver biopsy B. Ultrasound of the liver C. CPK D. HBV DNA

Question 2: A 77 year old male is referred for a persistent elevation in ALP of 222 IU/ml. The ALP has been elevated for about 1 year. All other liver chemistries have been normal. The CBC and INR are normal. An ultrasound, CT and MRI/MRCP of the liver are all normal. The patient underwent a cholecystectomy 9 months ago when the ALP was noted to be elevated. The ALP has remained elevated. He takes a stain, a calcium channel blocker, ACE inhibitor, and 2 oral agents for diabetes mellitus. Which of the following would you order next?

A. Fractionate the ALP B. Obtain gamma GT C. Liver biopsy D. ERCP

Question 3: You are asked to see a 35 year old male with an ALP of 1100 IU/ml. AST and ALT are normal. The gamma GT is 700 IU/ml. Serologic studies for anti-HCV, HB surface antigen, ANA, AMA and ANCA are all normal. On physical examination there is splenomegaly. An ultrasound of the liver demonstrates hepatomegaly with increased echogenicity. An MRI with MRCP also demonstrated hepatomegaly, but no intrahepatic mass lesions, normal bile ducts and mildly enlarged periportal lymph nodes. Which of the following would you order next?

A. Fractionate the ALP B. Bone scan C. Liver biopsy Liver Function Tests

D. EUS with biopsy of the lymph nodes

Question 4: A 25 year old male returns to see you because of intense itching and jaundice which started 2 weeks ago. You had seen him 2 years ago for similar symptoms which resolved spontaneously after 6 months. Back then serologic studies for all causes of chronic liver disease were negative, US, CT, MRI/MRCP, ERCP were normal, and a liver biopsy demonstrated cholestasis. You attempted to treat the itching with various anti-histamines, rifampin, questran and naltrexone. None was effective. AST and ALT are normal, ALP 875 IU/ml, total bilirubin 11 mg/dl, serum albumin 3.5 gm/dl, INR 1.0, Hemoglobin 13.5 gm/dl and platelet count 255,000. Which of the following is most likely to lead to the diagnosis?

A. Repeat MRCP. B. Repeat liver biopsy. C. Total serum bile acids. D. Gamma GT.

Question 5: You are asked to see a 6 month old female by her pediatrician for persistent jaundice. The AST, ALT, ALP, albumin and CBC were all normal. The total bilirubin was 6 mg/dl and the direct bilirubin 6 mg/dl. An ultrasound of the liver was normal and there is no evidence of bile duct dilation. Which diagnosis are you considering? A. Rotor Syndrome B. Gilberts Syndrome C. Crigler Najjar Type 1 D. Biliary atresia

Liver Function Tests

ANSWERS:

Question 1: CORRECT ANSWER is C. This patient has an elevation in serum AST with normal or near normal ALT. The AST:ALT ratio of 5 is much greater than is observed in patients with alcohol induced liver injury. Since the liver contains more ALT than AST it is not possible for liver injury to present with this type of pattern in liver transaminases. This suggests that the AST is coming from anther organ, the most common of which is muscle. A CPK is very helpful in these situations. Although the anti- HB core is positive the HB surface antigen is negative. This means that this patient likely had prior exposure to HBV but does not have HBV at this time. HBV DNA would therefore not be useful. Performing an ultrasound of the liver would be reasonable to do. However, even if the liver ultrasound was abnormal this would not explain the marked isolated elevation in AST. Since we do not think the patient has live disease a liver biopsy is also not useful.

Question 2: CORRECT ANSWER B. The patient has had an isolated elevation in serum ALP for about a year and was elevated prior to undergoing cholecystectomy. Imaging of the lvier did not demonstrate ether bile ducts or a retained stone. Performing an ERCP is therefore not likely to be very helpful. The patient is on multiple medications but the ALP has been stable and therefore if this is from a medication it is not causing progressive liver injury. A liver biopsy is not likely to be helpful in this situation. The main question is if the ALP is from liver or another organ. This could be determined by fractionating the ALP. However, this frequently yields a mixed pattern of the different ALPs and does not indicate which organ the excessive ALP is originating from. Another way to assess if the ALP is coming from liver is to measure a liver enzyme that should be elevated when the ALP is elevated. This would be the GGT or 5’-nucleatotidase.

Question 3: THE CORRECT ANSWER IS C. The patient has an isolated elevation in ALP. The elevation in ALP indicates that this is of liver origin. A bone scan will therefore not be helpful. Fractionation of the ALP will also not be helpful since the we already know that the elevation in ALP is of liver origin. Although an EUS with biopsy of lymph nodes could be performed this will likely not clarify why the ALP is elevated. The best approach would be to biopsy the liver. If the liver biopsy does not provide an explanation as to why the lymph node may be enlarged or there is other concern for malignancy the lymph node can be biopsied at a later time.

Question 4: CORRECT ANSWER is D. This patient most likely has benign recurrent intrahepatic cholestasis. This is a rare genetic disorder of the FIC gene family. The defect alters the fluidity of the canalicular membrane which impairs bile salt transport. The disease is characterized by repeated attacks of cholestasis with severe itching which is refractory to almost all treatments and jaundice. The ALP is very elevated but liver transaminases are normal. Attacks generally last several weeks to several months and sometimes over a year. Between attacks the ALP and total bilirubin return to Liver Function Tests

normal. Imaging of the liver and bile ducts are always normal. The liver histology demonstrates cholestasis but no inflammation or fibrosis. The diagnostic hallmark of the disorder is a normal gamma-GT, which is otherwise always elevated in patients with bile duct cholestatic diseases. The diagnosis is almost never made during the first episode, but should be recognized during the second episode. Repeating imaging studies or the liver biopsy would not likely provide any new information compared to 2 years ago. Total serum bile salts will be elevated but this would not be helpful in establishing the diagnosis.

Question 5: CORRECT ANSWER is D. This patient has intrahepatic cholestasis and hepatomegaly. The ALP is of liver origin because the GGT is elevated. Fractionating the ALP would not be helpful. There is no evidence of bile duct dilation on the ultrasound. MRCP would not likely yield a diagnosis and is not likely to demonstrate any bile duct disease with a normal ultrasound and such a great elevation in ALP. HCV does not lead to an isolated marked elevation in ALP and is not necessary to perform. The diagnosis here is most likely lymphoma which has infiltrated the liver causing hepatomegaly. In these patients the ultrasound and CT are typically normal or show hepatomegaly because the lymphoma infiltrates through the liver and does not cause a mass. Sarcoidosis is also a possibility but is less likely because of the normal ACE level. A liver biopsy would lead to the diagnosis.

Question 4: CORRECT ANSWER is B. This patient has laboratory evidence of cirrhosis. The AST/ALT ratio is greater than 1 and the platelet count is low. The ultrasound is consistent with chronic liver disease. A liver biopsy is therefore not necessary since the patient is highly likely to have cirrhosis. If the biopsy did not demonstrate cirrhosis the validity of the sample would be suspected and the patient would still be considered to have cirrhosis. The better test to confirm cirrhosis in an older patient with biochemical evidence of cirrhosis is upper endoscopy to look for evidence of portal hypertension including esophageal varices and portal hypertensive gastropathy. AFP is no longer recommended as a screening test for HCC. The proper way to screen for HCC is with a liver ultrasound. The Fibrosure can be utilized to assess fibrosis but the assay was developed in patients with chronic HCV and may not be reliable in patients with another etiology for the liver disease.

Question 5: CORRECT ANSWER is A. The patient has a persistent elevation in conjugated bilirubin for 6 months. The ALP and all liver chemistries are normal and so bile duct disease such as biliary atresia is not possible. The direct bilirubin is elevated, the indirect or unconjugated bilirubin normal. Both Gilbets syndrome and Grigler-Najjar syndrome are genetic disorders of unconjugated bilirubin. Since it is the direct bilirubin that is elevated this must be a genetic disorder of indirect hyperbilirubinemia. The would be either Rotor syndrome or Dubin-Johnson syndrome.