LIVER FUNCTION TESTS AND LIVER DISEASES

Prof. Fang Zheng Department of Laboratory Medicine School of Medicine, Wuhan University

Content
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Anatomy of Liver Functions of Liver Tests of Liver Function Liver Diseases How to use biochemical tests of liver functions?

INTRODUCTION
• The liver is divided into four lobes.

THE STRUCTURE OF LIVER

LOBES VASCULAR AND BILIARY SYSTEMS

The portal vein carries blood that has already passed through the capillary bed of the gastrointestinal tract. The hepatic artery carries welloxygenated blood to the liver. Both circulations mix in a vast network of hepatic sinusoids and leave the liver via the hepatic vein. Sinusoids are lined by endothelial and Kupffer’s cells.

LOBES VASCULAR AND BILIARY SYSTEMS
•In the liver the ductules merge into bile ducts, hepatic ducts, and eventually the common hepatic ducts. •Their bile drains into the right and left hepatic ducts.

Liver Function
1. Synthesis secretion plasma proteins, bile 2. Metabolism of carbohydrate lipid, protein vitamins, hormone bile acids, bile pigment, drug and toxins 3. Detoxification (biotransformation)

FUNCTION

TESTS OF LIVER

1.Tests of Protein Metabolism 2. Tests of Bilirublin Metabolism 3. Dye intake and Excretion 4. Biochemical Serum Enzyme Tests 5. Tests of Viral Hepatitis 6. Other tests

Tests of Protein Metabolism
1. Serum total protein (TP), albumin (A) and globulin (G) 2. Serum protein electrophoresis 3. Hepatic neoplasm markers

Serum total protein (TP), albumin (A) and globulin (G)
Albumin Albumin is quantitatively the most important plasma protein synthesized by the liver and thus is a useful indicator of hepatic function. Albumin has a fairly long half-life in serum (20 days) and hence is not a good indicator of hepatic protein synthesis in acute liver disease. Serum albumin levels are depressed in alcoholic cirrhosis. A crude measure of the liver’s synthetic capacity hypoalbuminaemia: advanced chronic liver

Serum total protein (TP), albumin (A) and globulin (G)

Globulin: The main proteins in serum. Total serum globin: the severity of liver disease

Reference Value
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Alb 40-55g/L TP 60-80g/L Globulin 20-30g/L

Serum protein electrophoresis

Densitometric scan of a normal serum protein electrophoresis pattern showing the relative position of the albumin,α1,α2,β and γregions

Hepatic neoplasm markers

alpha-fetoprotein(AFP): primary hepatocellular carcinoma Carcinoembryonic antigen(CEA): ↑liver metastatic carcinoma or other carcinomas of the gastrointestinal system.

Reference Value (AFP)
Normal < 25ng/mL Hepatitis 25~200ng/mL carcinoma >400ng/mL

OTHER SERUM PROTEINS PRODUCED BY THE LIVER
Coagulation factors (prothrombin time ) 1) The liver synthesizes six coagulation factors: factors(fibrinogen),II(prothrombin),V,VII,and X. Only substantial impairment in the liver’s ability to synthesize these proteins can result in clotting abnormalities 2) Prolongation of the prothrombin time is not specific for liver disease.

Tests of Bilirublin Metabolism
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Serum total bilirubin(STB) Conjugated bilirubin Urobilinogen

Bilirubin Metabolism
1) Bilirubin is the principal degradation product of heme. 2) Bilirubin transport and conjugation in the hepatocyte. 3) The generally accepted reference range for normal plasma bilirubin concentrations in healthy persons, which includes 95 per cent of all individuals’ values, is 1.7 to 17.1umol/L.

Bilirubin metabolis m

Bilirubin
Bilirubin is derived from the tetrapyrrole prosthetic group found in haemoglobin and the cytochrome. It is normally conjugated with glucuronic acid to make it more soluble, and excreted in the bile. Both conjugated bilirubin and unconjugated bilirubin may be present in plasma. Conjugated bilirubin is water soluble, unconjugated bilirubin is not, and binds to albumin. Bilirubin is neurotoxic, and if its levels rise too high in neonates, permanent brain damage can occur.

BILIRUBIN
Bilirubin metabolites are responsible for the brown coloration of faeces. If bilirubin does not reach the gut, stools become pale in color. Bilirubin in the gut is metabolized by bacteria to produce stercobilinogen, which is partly reabsorbed and reexcreted in the urine as urobilinogen. When high levels of conjugated bilirubin are being excreted , urine may be a deep orange colour.

High bilirubin level
•Haemolysis (Haemolytic)The increased haemoglobin breakdown produces bilirubin which overloads the conjugating mechanism. Unconjugated hyperbilirubinaemia is commonly encountered in babies. •Liver disorder (Hepatocellular) Failure of the conjugating mechanism within the hepatocyte. •Obstruction in the biliary system (Cholestatic) including extrahepatic biliary obstruction, Intrahepatic biliary obstruction is much more difficult to diagnose

Jaundice

What’s Jaundice? Jaundice is a yellow discoloration of the skin or sclera. Due to the bilirubin concentration in plasma is much greater than normal (greater than about 40 µ mol/L)

Causes of jaundice

Jaundice indicates that there is an elevated concentration of bilirubin in serum. In neonates it is important to determine the concentration of unconjugated bilirubin in order to decide what treatment is required. In adults, the most common cause of jaundice is obstruction, and this is confirmed by the elevation of both bilirubin and alkaline phosphatase

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Jaundice/More than 34.2µ mol/L(2.0mg/100mL) Latent jaundice /17.2 ~ 34.2 µ mol/L Normal/ Less than 1.70~17.2 µ mol/L 80% is unconjugated bilirubin and 20% is conjugated bilirubin

Serum bilirubin

Urine Bilirubin

Because only conjugated bilirubin is excreted in urine, it is indirect test for increased concentration of conjugated bilirubin in serum. A fresh urine specimen is required since bilirubin is very unstable when exposed in light and room temperature. The chemstrip test for bilirubin in urine.

Haemolytic Featur 1.Bilirubin usually< es

Cholestatic

Hepatocellular
1.AST+ALT ↑ ↑ 2.Bilirubin ↑ later 3.Bilirubin in urine 4. ALP↑ later

1.Bilirubin maybe ↑↑↑ 75µ mol/l. 2.Bilirubin in 2.No bilirubin in urine urine. 3.ALP Usually>3× 3.Reticulocytosis upper limit of 4.haemoglobin↓ reference range. 4.AST, ALT+LDH 5. haptoglobin↓( 触 usually modestly ↑ 珠蛋白 ) 6. LDH may↑

DIFFERENTIAL DIAGNOSIS OF JAUNDICE
Unconjugated hyperbilirubinemia Increased heme catabolism Hemolytic anemia Hematoma Impaired hepatic conjugation Neonatal jaundice Conjugated hyperbilirubinemia Impaired hepatic excretion Hepatocellular disease Posthepatic obstruction

Dye intake and Excretion

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Indocyanine green retention rate (ICGR): 15min<10% Chronic hepatitis: 15-20% Cirrhosis: 35%

SERUM ENZYME TESTS
Aminotransferase 1) AST (Aspartate) and ALT (Alanine) is present in a wide variety of tissues-including heart, skeletal muscle, kidney, and brain in addition to liver. 2) Serum levels of AST and ALT are elevated to some extent in almost all liver diseases (viral hepatitis, obstructive jaundice and liver cirrhosis). 3) The elevations of the enzyme levels do not correlate with eventual outcome, even though they may reflect the extent of hepatocellular necrosis.

SERUM ENZYME TESTS
Aminotransferase Clinical value of AST and ALT The ratio of AST to ALT Reference Value AST and ALT: 5~40U/L

SERUM ENZYME TESTS
MARKERS OF CHOLESTASIS ——Alkaline phosphatase (ALP) An indices of a blockage of bile flow Cholestasis, which maybe intra- or extra-hepatic disease (tumor, cirrhosis ) ADULT 25~90U/L CHILD 50~350U/L

Plasma alkaline phosphatase activity as a function of age and sex(—men ; …… , women). Horizontal lines refer to multiples of the adult upper reference limit.

SERUM ENZYME TESTS
1) The reference range for ALP is dependent on age. 2) In the human body, ALP has been identified in liver, bone, intestine, kidney and leukocytes. ALPs are a heterogeneous group of enzymes. For patients with high ALP levels, measurement of ALP isoenzymes is useful for differentiating between bone and liver sources. 3) The highest elevations of ALP in patients with liver disease occur in patients with cholestasis or hepatic carcinoma.

SERUM ENZYME TESTS
γ -Glutamyl Transpeptidase ( γ -GT) Cholestasis Alcohol Drugs Acute hepatic damage Combining with alkaline phosphatase Male Female 11-50U/L 7~32U/L

SERUM ENZYME TESTS
1) γ -GT has been localized to the whole hepatobiliary treefrom hepatocytes to common bile duct in the liver, and also to pancreatic acini and ductules. 2) Serum γ -GT is elevated in association with a wide variety of pathologic states in addition to hepatobiliary disease, including chronic alcoholism, pancreatic disease, myocardial infarction, renal failure, chronic obstructive pulmonary disease and diabetes. 3) In liver disease, serum γ -GT activity correlates well with serum ALP, and is the most sensitive indicator of biliary tract disease.

SERUM ENZYME TESTS
5’-Nucleotidase (5’-NT) 1) 5’-NT is present in the intestines, brain, heart, blood vessels and endocrine pancreas in addition to the liver. 2) Elevation of 5’-NT in the serum are purported to be of hepatobiliary origin only despite the widespread localization of the enzyme in other body tissues. 3) In clinical hepatic disease, serum 5’-NT correlate closely with serum alkaline phosphatase. 4) Serum 5’-NT is particularly useful in diagnosing liver disease in childhood and in pregnancy.

SERUM ENZYME TESTS
monoamine oxidase, MAO  Β-Proline hydroxylase, PH Indices of hepatic fibrosis

Unlike some disorders such as acute pancreatitis (Amylase) and myocardial infarction (Myoglobin Troponin), for which there are enzyme markers that are primarily used for one disorder and have high diagnostic efficiencies, there are no enzyme markers that are specific for any single liver disease.

When evaluating these disorders, therefore, it is appropriate to consider a panel of markers, sometimes called live function tests (LFTs). usually includes bilirubin, AST, ALT, ALP, and sometimes GGT and 5'NT although these tests can reflect various disease processes in the liver, they do not reflect hepatic reserve for synthesis and metabolic functions

Other tests for Liver Excretion Function

Bile Acids

Bile Acids
The regulation of bile acid is a major function of the liver. Cholesterol homeostasis is in large part maintained by the conversation of cholesterol to bile acids and subsequent regulation of bile acid metabolism. Bile acid provides surface-active detergent molecules that facilitate both hepatic excretion of cholesterol to bile acids and solubilization of lipids for intestinal absorption.

The metabolism of bile acid

The clinical Significances of SERUM
BILE ACIDS
1. In the last 20 years, there have been major methodological advances in the measurement of serum bile acids. 2. Clinical significance: 1) Bile acids more specifically reflect excretory function. Increase of bile acids in serum suggest impaired hepatic uptake or secretion, or portalsystemic shunting 2) The high concentration of serum bile acids seem to occur in viral hepatitis and extrahepatic obstruction.

SERUM BILE ACIDS
3) Abnormal results of ALP, AST and rGT lacked the specificity of bile acids abnormalities. AST determinations are more sensitive than those for fasting serum bile acids in detecting mild liver diseases such as fatty liver or chronic persistent hepatitis, because mild or patchy hepatocellular injury does not impair hepatic function severely. 4) The ratio of cholic acid to chenodeoxycholic acid is between 0.5-1.0 in healthy subjects, but in extrahepatic obstruction it is increased 0.96-3.6.

SERUM BILE ACIDS

Reference value: Total Bile Acid : 0~6µ mol/L

Summary of Liver function tests
•A request for LFTs will usually generate results for bilirubin, the aminotransferases and alkaline phosphatase. • Raised activities of the aminotransferases(AST and ALT) indicate hepatocellular damage. •Increased bilirubin concentration and increased alkaline phosphatase activity indicate the presence of cholestasis, a blockage in bile flow. •Serial use of LFTs is of most value in following the progress or resolution of liver disease. •Measurement of γ GT can give an indication of hepatocellular enzyme induction due to drugs or

Tests of Viral Hepatitis
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Hepatitis A Hepatitis B Hepatitis C

Tests of Viral Hepatitis A

Hepatitis A(HAV) is a kind of RNA virus. It is transmitted by the fecaloral route. It is thus implicated in most instances of water-borne and food-transmitted infection and in epidemics of viral hepatitis.

Serologic markers of viral hepatitis A
Agent markersdefinition significances HAV AntiHAV IgM type IgG type Antibody Current or recent to HAV infection of HAV

Current or previous infection of HAV, confers immunity

Tests of Viral Hepatitis B

HBV is a kind of DNA virus consisted by core and surface components. HBV transmission occurs most commonly via blood and blood products, contaminated needles and intimate personal contact. HBV is present in all body fluids.

Serologic markers of viral hepatitis
Agent markers definition HBV HBsAG significances HBV surface Positive in acute or chronic antigen infection e antigen, a Transiently positive in acute component hepatitis B, reflects presence of viral replication and high infectivity

HBeAG

Anti-HBe Antibody to Transiently positive, may be e antigen persistently present in chronic cases, reflects low infectivity Anti-HBc IgG type Antibody to core antigen

Positive in all acute and chronic cases, reliable marker of infection past or current

Agent

markers Anti-HBs

definition Antibody to surface

significances confers immunity,

Tests of Viral Hepatitis C
• HCV is a kind of RNA virus and is similar to HBV, is largely parenterally transmitted. HCV is the main cause of posttransfusion hepatitis.

Serologic markers of viral hepatitis
Agent markers definition significances HCV AntiHCV Antibody Positive after clinical to HCV onset(15 weeks), Not protective, Persists in chronic infection

Liver Disease

Causes for liver Poisoning disease hepatitis (hepatitis Infection Viral
B) Inadequate perfusion Chronic excess alcohol ingestion Autoimmune disease Unusual cause of cirrhosis: α 1-antitrypsin deficient and Wilson’s disease

Liver disease
•Acute Hepatocellular Injury Viral Hepatitis Acute Liver failure •Cholestatic Liver Disease Intrahepatic Obstruction Extrahepatic Obstruction •Chronic Liver Disease Chronic Hepatitis Cirrhosis Liver Cancer •Alcoholic Liver Disease Fatty Liver Alcoholic Hepatitis

Acute Hepatocellular Injury
It may resolve OUTCOME It may progress to acute hepatic failure It may lead to chronic hepatic damage

Biochemic al findings in hepatic failure

Cholestasis

Cholestasis represents the demonstrable accumulation in the blood stream of substances normally excreted in bile (e.g., bilirubin, cholesterol, bile acids).

Cholestatic Liver Disease

Intrahepatic Obstruction Intrahepatic cholestasis often results from cirrhosis or hepatitis. Extrahepatic Obstruction Extrahepatic cholestasis is usually the result of mechanical obstruction of the common bile duct or hepatic duct.

Chronic Liver Disease

Chronic Hepatitis chronic active hepatitis chronic persistent hepatitis Cirrhosis Liver enzyme levels in cirrhosis are variably elevated and can be normal during the terminal stages of the disease. Primary biliary cirrhosis /Elevations in ALP and aminotransferases are expected along with high titers of antimitochondrial antibody. Liver Cancer The liver enzyme results tend to be more elevated in the active form; however, differentiation is best made by performing a liver biopsy.

Fibrosis of Liver tissues
Fibrosis is common to several chronic liver diseases and as such is the leading cause of morbidity and mortality from hepatic disease. Hepatic fibrosis may have biologic effects on cells as well as physical effects on blood flow and is a main cause of portal hypertension.

Cirrhosis
•The most common causes of cirrhosis are chronic excess alcohol ingestion, viral hepatitis and autoimmune diseases. •Cirrhosis is not reversible. There are no good biochemical indicators of cirrhosis in the early and stable period •Cirrhosis can develop in children as a result of a1-antitrypsin deficiency or Wilson’s disease and in adult due to haemochromatosis.

Terminal stage of cirrhosis
•Developing jaundice •Encephalopathy •Ascites •Bleeding tendenices •Terminal liver failure

Alcoholic Liver Disease
Fatty Liver The concentrations of traditional liver enzymes are typically within the normal range. Definitive diagnosis is made by a liver biopsy. The disease is considered benign and can be effectively reversed by immediate abstinence from ethanol intake. Alcoholic Hepatitis Liver enzyme concentrations are increased, notably AST and ALT

Normal Liver

Fatty Liver

Cirrhotic Liver

Case history

A 49-year-old woman attend her GP with an 8-day history of anorexia, nausea and flu-like symptom. She had noticed that her urine had been dark in colour over the past 2 days. Physical examination revealed tenderness in the right upper quadrant of the abdomen. LFTS were as follows: Bilirubin AST ALT ALP γ -GT TP Alb mol/L U/L (g/L) 63 936 2700 410 312 68 42 Comment on these results. What is the differential diagnosis?

Markers For Liver Function And Disease

Markers For Liver Function And Disease
Disease or function Markers Function evaluation Normal synthesis capacity Albumin, prealbumin prothrombin time, Excretory function Bilirubin, bile acids , globulins Metabolic function Ammonia, amino acids, lipids, serum protein electrophoresis Pathological evaluation Hepatocellular injury Obstruction AST, ALT Bilirubin, alkaline phosphatase, γGT , 5 ’- nucleotidose bile acids, serologies CEA, α-fetoprotein

Infections Viral and autoimmune Malignancies

ALT and AST in acute liver injury
disease acute hepatitis
(viral or toxic)

enzyme marker ALT and AST elevated>1000U/L ALT elevated in 3 to 4 weeks after infection ALT return to normal within 8 to 12 weeks

HAV HBV and HCV chronic active hepatitis end-stage liver disease

preclinical incubation phase is longer and ALT and AST may remain normal for 2 to 6months ALT and AST return to normal within 2 to 3months ALT and AST elevated 5 to10 fold ALT and AST return to normal or subnormal

Relationship of AST and ALT to ALP and GGT in Hepatitis

Relationship of AST and ALT to ALP and GGT in Cholestasis

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The best markers for intrahepatic and extrahepatic cholestasis are ALP, GGT, and 5'NT AST and ALT are generally only slightly elevated in cholestasis, rarely more than 500 U/L. The largest elevations (four- to 10-fold) of ALP are typically seen in obstruction owing to gallstones or malignancy and in biliary cirrhosis. Measurement of total and direct bilirubin are also important in making the diagnosis of obstructive jaundice.

Ratio (AST/ALT)

The Ratio of (AST/ALT)

Further differentiation of specific liver diseases is aided by calculating the ratio of AST to ALT levels. — acute or chronic ? — intra- or extrahepatic ?

recommended by the International Federation of Clinical Chemistry (IFCC) The ratio is normally approximately 1.15

ALT versus AST levels in various liver diseases

AST/ALT
Disease AST/ALT Acute disorders of the liver <1.0 acute hepatitis Chronic disorders of the liver >1.0 alcoholic liver disease chronic active hepatitis Chronic persistent hepatitis normal Extrahepatic obstruction acute passage of a stone < 1.5 intrahepatic cholestasis biliary cirrhosis and malignancy ≥1.5

Relationship of AST and ALT to ALP and GGT in Malignancy

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Relationship of AST and ALT to ALP and GGT in Alcoholic Live Disease

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Abnormal Liver Function Tests Hepatocellular Normal Disease
Albumin

Cholestatic Disease
Decrease d Albumin Normal Albumi n Decreased Albumin

Acute Chronic Hepatitis Hepatitis

Acute Cholestasi s Ultrasound Detection

Chronic Cholestasis

Intrahepatic Cholestasis

Extrahepatic Cholestasis

Case history
A 60-years-old female with a history of breast carcinoma treated by mastectomy three years previously is now complaining of general malaise and bone pain. Biochemistry showed that fluid and electrolyte, total protein, albumin and calcium values were all normal. LFTs were as follows: Bilirubin GT µ mol/l 7 32 33 38 AST ALT Alkaline phosphatase U/l 890 γ

Summary

Biochemical monitoring of liver disease is by sequential measurements of the aminotransferase, bilirubin and alkaline phosphatase. In acute liver damage there is usually intrahepatic obstruction as well as hepatocellular damage. Severe cases of acute liver damage may progress to hepatocellalur failure. Cirrhosis is the end point of both acute and chronic liver damage, as well as being caused by a number of metabolic and autoimmune diseases. Biochemical tests may be of little value in making a specific diagnosis. A liver biopsy is frequently more helpful.

The clinical significances of each liver function test? The differentiation diagnosis of jaundice. The evaluations of biochemical tests of liver functions in different liver diseases.

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