Author(s): Rebecca W. Van Dyke, M.D., 2012

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M2 GI Sequence

Liver Tests: Use and Interpretation

Rebecca W. Van Dyke, MD

Winter 2012

Learning Objectives

• A. General: Understand the laboratory tests that are used in the clinical approach to liver disease and the pattern of abnormalities that occur in specific forms of liver injury.

– 1. When do we suspect a patient has liver disease? What tests can be used to accept or deny the presence of liver disease?

– 2. Can we define the type of liver disease the patient has by analyzing the results of the liver tests?– 3. How much functional liver tissue is present in a patient?

•  • B. Specific:

– 1. Be able to interpret panels of biochemical liver tests in terms of general type of liver disease, chronicity and severity.

– 2. Be able to construct a differential diagnosis for different patterns of liver test results.– 3. Be able to identify potential problems in interpreting liver tests.

Industry Relationship DisclosuresIndustry Supported Research and

Outside Relationships

• None

How Do We Tell Someone Has Liver Disease?

Clues that may lead to a suspicion of liver disease:

AnorexiaFatigue

Nonspecific NauseaVomitingMental confusion

Jaundice (“yellow eyes”) More specific Dark urine (“coca-cola” urine) (late findings) Abdominal swelling; ascites

Peripheral edema; leg swelling

Unfortunately none of these are specific markers of liver disease and for many patients these are very late findings.

Purpose of Liver Tests

1. Screen for clues to the presence of liver injury/diseaseliver cell injurybile flow/cholestasis

2. Quantitate degree of liver function/dysfunctionquantitative liver tests

3. Diagnose general type of liver diseasepattern of liver test abnormalities

4. Diagnosis of specific liver diseasedisease-specific tests such as serology forviral hepatitis

Tests of Liver Cell Injury/Death

Transaminases

Alanine amino transferase (ALT)

Aspartate amino transfersase (AST)

Transaminases are enzymes that catalyze the transfer of-amino groups from amino acids to -keto acids.These enzymes are important in gluconeogenesis.

ALT (alanine aminotransferase)

alanine pyruvic acid + +ketoglutarate glutamate

AST (aspartate aminotransferase)

aspartate oxaloacetic acid + +ketoglutarate glutamate

Transaminases:

AST(SGOT) ALT(SGPT)

Many tissues Liver only

Cytosol/mitochondria Cytosol

Normal blood levels: 20-70 IU/liter (depending on method)

Some AST/ALT release occurs normally

Require pyridoxal 5’-phosphate as an essential cofactor

Multi-channel Automated Analysis of Enzymes in Blood

Patient serum

Substrate

ALT +

substrate

Colored product

lamp

Photodetector

Absorbance converted to enzyme activity

ASTALT

Release of AST/ALT from Liver Cells during Acute Hepatocellular Injury

Transaminases

Why do we used these enzymes to indicate liver damage?

1. Convenient to measure

2. Present in liver cells in large amounts

3. Direct release of enzymes into blood throughfenestrated endothelium allows rapid“quantitative” assessment of ongoinghepatocyte necrosis

4. Blood level roughly proportional to the numberof hepatocytes that died recently (hours-days)

2000

100

200

500Serum Enzyme Level (IU/ml)

Cirrhosis (little ongoing

injury)

Mild chronic hepatitis C

(asymptomatic)

Acute viral hepatitis A

(clinically mild)

AST

ALT

Patterns: AST and ALT in Various Liver Diseases

Acute viral hepatitis A

(clinically severe)

3000

Ischemic infarction: how high would AST/ALT go?

Transaminases

Special considerations:

1. AST is also present in other tissues (muscle, brain, kidney, intestine). ALT is more specific for liver.

2. Even very mild liver abnormalities can cause slightly elevated AST/ALT

- for example, mild fatty liver.

Fatty Liver: Most Common Cause of Mildly Increased AST/ALT

(~1.5-3x upper limit of normal)

TransaminasesProblems with using transaminases to assess liver injury:

1. Only assess injury over the past 1-2 days as enzymes are cleared efficiently from blood by RES

2. May not accurately assess hepatocyte death from apoptosis

3. Magnitude of elevation does not necessarily correlate with extent of liver function or dysfunction at the present time or in the future.

AST and ALT = rate of destruction of hepatocytes

Liver function = number of functional hepatocytes left

Not all liver abnormalities cause liver cell death: simple liver cyst with

normal liver tests

Transaminases and Alcoholic Liver Disease: A Twist

Pyridoxal 5’-phosphate (P5P) deficiency:

AST and ALT require P5P (vit. B-6) as an enzymatic cofactor

Alcoholics are often deficient in P5P as their major calorie source is alcohol

P5P deficiency results in lower synthesis of AST/ALT (less in hepatocytes) and very low enzymatic activity (ALT worse than AST)

Less AST/ALT released into blood and it isn’t measured by lab assays

Transaminases and Alcoholic Liver Disease

Further: Mitochondrial AST and alcohol Alcohol shifts mAST from mitochondria to plasma

membrane where it readily enters blood – thus AST easier to remove from hepatocytes.

Therefore: AST>>ALT is released into bloodfrom damaged hepatocytes

AND

both AST/ALT enzymatic activities in bloodare lower than expected from the extent of liverdamage/dysfunction.

AST/ALT and Pyridoxal 5’ Phosphate

P5’P

Numerous active enzymes with P5’P Few inactive enzymes without P5’PPoorly measured by lab assays

AST is Affected Less than ALT so AST>ALT

Release of AST/ALT from Liver Cells After Alcohol Exposure

ASTALT

Alcohol increases mitochondrial AST on liver cell plasma membrane where it readily enters blood. Thus AST>>ALT in blood.

1000

100

200

650Serum Enzyme Level (IU/ml)

Alcoholic hepatitis

Mild chronic hepatitis C

Acute viral hepatitis A

AST

ALT

Patterns: AST versus ALT

Ratio 2.4 Ratio 0.8Ratio 0.65

Tests of Cholestasis/Reduced Bile Flow

Enzymes released as a Accumulation in liver/blood consequence of decreased of substances normally bile flow excreted in bile

Alkaline phosphatase Bilirubinor

5’-nucleotidase Bile saltsLeucine aminopeptidase-glutamyl transpeptidase

Alkaline Phosphatase: Location at Canalicular (Apical) Membrane

Alkaline phosphatase Origin of enzyme and mechanism of increase in cholestatic liver disease:

1. Apical membrane of hepatocyte and bile duct cells 2. Very sensitive to any changes in bile flow, obstruction of large or small bile ducts. 3. Amplified by bile acid retention 4. Easily released into blood as it is a GPI-anchored protein solubilized from membrane by detergents (bile acids) Easily measured spectrophotometrically Purpose: ? Detoxifies lipopolysaccharide (LPS) from bacteria

Bile Acids (Bile Salts) such asTaurocholate

stimulate productionof alkaline phosphatasemolecules.

Normal (A) and(B) BlebbedHepatocytes

Bile acid-induced injury

Alkaline Phosphatase 5’ Nucleotidase GGTP

GPI-Anchored Proteins

Release of GPI-Anchored Proteins From Liver During Cholestasis

Hepatocyte

Blood

Bile canaliculus

1000

100

200

500Serum Enzyme Level (IU/ml)

Hepato- cellular disease

Mild early partial

bile duct obstruction

Acute bile duct

obstruction

Alkaline Phosphatase in Various Liver Diseases

Long-standing bile duct

obstruction

1500

Degree of elevation of AP is highly variable depending on duration and extent of cholestasis and other unknown factors.

Alkaline Phosphatase

Interpretation of elevated levels:1. Cholestasis (especially in extrahepatic

obstruction2. Infiltrative diseases (granulomas)3. Neoplastic disease infiltrating liver

Sensitive test as will go up if only some small ducts are obstructed and/or if there is only partial obstruction of major ducts.

Disadvantages:Not completely specific because of isoenzymes

in other organs (bone, intestine, placenta)Ex: bone disease, intestinal obstruction, pregnancy.

Serum Bilirubin (Bile Acids)

Rationale:Liver is virtually the only mechanism for excretionCholestasis from any cause results in “back-up”

of these compounds in blood

Interpretation:Cholestasis: extrahepatic or intrahepatic

Disadvantages:Does not distinguish hepatocellular disease,

in which hepatocytes don’t make bile,from bile duct obstruction

Bilirubin

An organic anion

The byproduct of heme breakdown

In mammals bilirubin must be conjugated toglucuronic acid and excreted in bile

Blood levels go up if any steps in production or hepatocyte excretion are altered. However obstruction at the level of bile ducts must be complete or virtually complete for bilirubin levels in blood to change

Bile Canaliculus

Hepatocyte

MRP-2: Multispecific organic anion transporter

ATP

Conjugated bilirubin Glutathione S-conjugates other organic anions

Unconj Bilirubin

SER

Unconj BR

Conj BR

Conj BR

RBC breakdown in RES

Hepatic Bilirubin Transport

Blood

UDP-glucuronide

Unconj BR+

Conj BR

Bile Canaliculus

Hepatocyte

Multispecific organic anion transporter

ATP

Conjugated bilirubin Glutathione S-conjugates other organic anions

Unconj Bilirubin

SER

Unconj BR

Conj BR

Conj BR

Gilbert's syndrome (mild) Crigler-Najjar syndrome (severe)

Dubin-Johnson syndrome Rotor's syndrome ?estrogen/cyclosporin

Hemolysis

Hepatic Bilirubin Transport and Mechanisms of Hyperbilirubinemia

Blood

Unconj Bilirubin

SER

Unconj BR

Conj BR

Mechanism of Hyperbilirubinemia in Liver Disease

UDP-glucuronide

Unconj BR+

Conj BR

Overall Rate-Limiting Step

Conj BR

Albumin

Interpretation of Elevated Serum Bilirubin

Conjugated hyperbilirubinemia:BR reached liver and was conjugated but not excreted in bile

1. Cholestasis/biliary obstruction (must be essentially complete)2. Hepatocellular damage (collateral damage to all liver functions)

bile formation impaired >> conjugation impaired3. Rare disorders of canalicular secretion of conjugated bilirubin

Unconjugated hyperbilirubinemia:BR didn't reach liver efficiently or wasn't conjugated

1. Massive overproduction - acute hemolysis2. Impaired conjugation

common: Gilbert's syndrome (mild)rare: Crigler-Najjar syndrome (severe)

BRAlb

Alb BR

ER

BR

BR-Glu Bile

Hepatocyte

Blood

BR-Glu

BR-Glu

BR-Glu

Alb

Bili-albumin conjugate or "delta bilirubin"

Formation of Bilirubin-Albumin Conjugates

Further: Bilirubin Undergoes Non-Enzymatic Reaction with Albumin

Why is Bili-Albumin of Clinical Interest?

• Not of interest during liver disease as this form of bilirubin is measured as conjugated bilirubin.

• However, after resolution of cholestasis/liver disease, bili-albumin is cleared like albumin– Albumin half-life: several weeks– Conj. bilirubin half-life: hours to days–

• Thus resolution of jaundice is often SLOW compared to improvement of other liver functions.

Purpose of Liver Tests

1. Screen for clues to the presence of liver injury/diseaseliver cell injurybile flow/cholestasis

2. Quantitate degree of liver function/dysfunctionquantitative liver tests

3. Diagnose general type of liver diseasepattern of liver test abnormalities

4. Diagnosis of specific liver diseasedisease-specific tests such as serology forviral hepatitis

TESTS OF LIVER FUNCTION

AlbuminClotting factors

BilirubinBile Acids

14C-Aminopyrine

Blood Level

Production

Elimination

Metabolism

Blood Level

Metabolites 14CO2

Albumin

• Rationale– Liver is the sole source

• Interpretation of Decreased Level– Decreased liver production

– Increased renal/GI loss (nephrotic syndrome; protein losing enteropathy in inflammatory bowel disease

– Protein malnutrition

• Disadvantages– Prolonged half-life

– No unique interpretation

Prothrombin Time

• Rationale– Liver is sole source of vitamin K-dependent clotting

factors, including those critical for PT– Factor VII has very short half-life (hours)

• Interpretation of Increased PT– Hepatocyte protein synthesis impaired– Vitamin K deficiency/Coumadin therapy– Disseminated intravascular coagulopathy

• Advantages– Rapidly reflects changes in liver function

Interpretation of Abnormal Albumin/Prothrombin Time

Liver markedly diseased - reserve function gone

Albumin: monitors slow changes in liver function(months to years)

reflects long-term liver dysfunction

Protime: monitors rapid changes in liver function(hours to days/weeks)

reflects either short or long-term liverdysfunction

Other Clues to Globally Impaired Liver Function

Bilirubin: goes up with any disease that globally impairsliver function (OR blocks bile flow)

Glucose: hypoglycemia (late finding; indicates very poor liver function)

BUN: low BUN is a late and poorly specific finding in liver dysfunction due to poor urea synthesis

Purpose of Liver Tests

1. Screen for clues to the presence of liver injury/diseaseliver cell injurybile flow/cholestasis

2. Quantitate degree of liver function/dysfunctionquantitative liver tests

3. Diagnose general type of liver diseasepattern of liver test abnormalities

4. Diagnosis of specific liver diseasedisease-specific tests such as serology forviral hepatitis

Interpretation of Liver Tests

A. Consider nonhepatic causes of abnormal liver tests

B. Examine the pattern of liver test abnormalities to categorize liver disease:

1. cholestatic versus hepatocellular

2. acute versus chronic

3. decompensated versus mild functional function impairment

Patterns of Abnormal Liver Tests

Hepatocellular Cholestasis

Major: AST/ALT Alkaline Phosphatase

Minor: Bilirubin BilirubinPT/albumin AST/ALT

PT (Vit K deficiency)

20

1

2

10

Fold Increase

AST or ALT PTBilirubinAlkaline Phosphatase

Hepatocellular disease

Cholestatic disease

Patterns: Acute Liver Disease

Clues to Acute vs Chronic Liver Disease

Abnormalities of PT versus albumin

Known duration of abnormal liver tests

History of exposure to potential causative agents

Clinical signs of consequences of long-standing liver disease

Tempo of subsequent changes in AST/ALT, bilirubin, PTchronic tends to change slowlyacute tends to change quickly

20

1

2

10

Fold Increase

AST or ALT PTBilirubinAlkaline Phosphatase

Patterns: Chronic Liver Disease with Impaired Liver Function

Albumin

-2

Clues to Severity of Liver Dysfunction

Prothrombin time

Albumin

(Bilirubin, glucose)

(Clinical signs of consequences of severe liver dysfunction such as hepatic encephalopathy)

Clues to severityof liver damageand, potentially, toseverity of liverdysfunction may come from thetemporal sequenceof changes in readily available livertests.

For example, rapidfall in AST/ALTin severe hepatitismay not be a goodsign.

Purpose of Liver Tests

1. Screen for clues to the presence of liver injury/diseaseliver cell injurybile flow/cholestasis

2. Quantitate degree of liver function/dysfunctionquantitative liver tests

3. Diagnose general type of liver diseasepattern of liver test abnormalities

4. Diagnosis of specific liver diseasedisease-specific tests such as serology forviral hepatitis

These are discussed in future lectures

Summary

• Use biochemical tests to assess– Presence of liver disease– General type of liver disease– Sense of severity of liver dysfunction– Sense of acute versus chronic disease

• Use liver tests with other data to work through differential diagnosis– See algorithms in syllabus and textbook

Approach to the Patient with Jaundice (Bilirubin)

History, PE Lab tests: AP, AST/ALT, Alb. PT

Abnormal findings suggesting liver disease

Suspect intrahepatic cholestasis or hepatocellular disease

Suspect extrahepatic cholestasis

Noninvasive imaging of the biliary tree: ultrasound or CT (may go to direct duct visualization in some cases)

Specific diagnostic tests hepatitis screen AMA, ANA, SMA Ceruloplasmin Fe/TIBC, ferritin Stop drugs Consider liver biopsy Consider CT to r/o structural disease Medical management/ observation

Normal ducts

Dilated ducts

Normal ducts, still suspect extrahepatic cholestasis

Relief of biliary obstruction: surgical endoscopic percutaneous

Direct duct visualization (ERCP or PTC)

Obstruction visualized

No obstruction visualized

Normal liver tests

Fractionate bilirubin

Unconjugated hyperbilirubinemia

Conjugated hyperbilirubinemia

Hemolysis Gilbert’s syndrome Crigler-Najjar syndrome

Dubin-Johnson syndrome Rotor syndrome

Approach to Patient with Increased Alkaline Phosphatase

Exclude pregnancy, physiologic causes

Obtain additional biochemical markers of cholestasis (GGTP, 5'-NT, or AP isoenzymes, serum bilirubin)

Nonhepatic cause of AP Hepatic cause of AP

Consider: bone disease (eg, Paget's disease, hyperparathyroidism, bone metastasis) ectopic AP secretion

Large (> 3-fold) elevation of AP

Modest (< 3-fold) elevation of AP

Consider: hepatocellular injury (eg, viral and alcoholic hepatitis)

Abdominal ultrasound or CT scan

Dilated ducts Non-dilated ducts

Consider: choledocholithiasis, cancer of the pancreas, cholangiocarcinoma, biliary stricture, pancreatitis

Focal hepatic defectsNormal or diffusely abnormal liver

Consider: primary or metastatic cancer of the liver, pyogenic/amebic abscess

Liver biopsy and consider: primary biliary cirrhosis, granulomas

Modified from: Kelley Textbook of Internal Medicine, 3rd edition, 1997, pp 663.

Alkaline phosphatase (AP)

A variety of cases are provided in your syllabus to allow practice in analyzing liver tests.