Liver Function Test

Chief metabolic organ in the body

Liver

Liver

• receives _ mL of blood per minute

15 mL

Liver

• composed of 2 cell types:

• hepatocytes

• kupffer cells

Liver

• cells are arranged into the _ > the anatomic unit of the liover

lobule

Liver

• has a unique capacity to generate by _

cell division

Liver

• _ % of the liver must be destroyed to abolish its functions

• 80%

Major Metabolic Functions of the Liver (5)

• Synthetic Function

• Conjugation Function

• Detoxification and Drug Metabolism

• Excretory and Secretory Function

• Storage Function

Major Metabolic Functions of the Liver

• Synthetic Function

• Plasma proteins > 12 g albumin, globulins

• Carbohydrates

• Cholesterol

• Triglycerides and Lipoproteins

• Ketone bodies

• Enzymes

Major Metabolic Functions of the Liver

• Conjugation Function

-

involves bilirubin metabolism > 200 to 300 mg produced daily

Major Metabolic Functions of the Liver

• Detoxification and Drug Metabolism

• protects the body from potentially injurious substances absorbed fro the intestinal tract and toxic by-products of metabolism

• ammonia →urea

Major Metabolic Functions of the Liver

• Excretory and Secretory Function

• Bile excretion

• Bile acids are conjugated with amino acids glycine and taurine to form bile salts

Major Metabolic Functions of the Liver

• Storage Function

• Vitamins A, D, E, K and B12

• Glycogen

Some Examples of Liver Dysfunction (8)

• Hepatocellular disease

• Cholestasis > obstruction of bile flow

• Cirrhosis

• Hepatitis

• Jaundice

• Liver cancer

• Steatosis > fatty liver

• Genetic disorders > hemochromatosis, iron storage

Liver Function Tests (4)

• Noninvasive methods for screening liver dysfunction

• Help in identifying general types of disorder

• Assess severity and allow prediction of outcome

• Disease and treatment follow-up

Liver Function Tests

• Broadly classified as: (2)

• Tests to detect hepatic injury

• Tests to assess hepatic function

Liver Function Tests

• Tests to detect hepatic injury (2)

• Mild or severe; acute or chronic

• Nature of liver injury > hepatocellular or cholestasis

Classification of Liver Function Tests

• 3 GROUPS

• Group I: Markers of Liver Dysfunction or Synthetic Function Tests

• Group II: Markers of Hepatocellular Injury

• Group III: Markers of Cholestasis

Classification of Liver Function Tests

• Group I: Markers of Liver Dysfunction or Synthetic Function Tests (4)

• Serum Bilirubin > total and conjugated

• Urine > bile salts and urobilinogen

• Total protein, serum albumin, and albumin/globulin ratio

• Prothrombin time

Classification of Liver Function Tests

• Group II: Markers of Hepatocellular Injury (2)

• Alanine aminotransferase (ALT)

• Aspartate aminotransferase (AST)

Classification of Liver Function Tests

• Group III: Markers of Cholestasis (2)

• Alkaline phosphatase (ALP)

• g-glutamyltransferase (GGT)

Limitations of Liver Function Tests (2)

• Normal LFT values do not always indicate absence of liver disease > liver has very large reserve capacity

• Asymptomatic people may have abnormal Liver Function Tests results > diagnosis should be based on clinical examination

Common Serum Liver Chemistry Test (8)

• Alanine aminotransferase

• Aspartate aminotransferase

• Bilirubin

• Alkaline phosphatase

• Prothrombin time

• Albumin

• g-glutamyltransferase

• Bile acids

Common Serum Liver Chemistry Test

• Alanine aminotransferase

• hepatocellular damage

Common Serum Liver Chemistry Test

• Aspartate aminotransferase

• Hepatocellular damage

Common Serum Liver Chemistry Test

• Bilirubin

• Cholestasis

• Impaired conjugation

• Biliary obstruction

Common Serum Liver Chemistry Test

• Alkaline phosphatase

• cholestasis

• infiltrative disease

• biliary obstruction

Common Serum Liver Chemistry Test

• Prothrombin time

synthetic function

Common Serum Liver Chemistry Test

• Albumin

Synthetic function

Common Serum Liver Chemistry Test

• g-glutamyltransferase

• cholestasis

• biliary obstruction

Common Serum Liver Chemistry Test

• Bile acids

• cholestasis

• biliary obstruction

Common Serum Liver Chemistry Tests

• A by-product of RBC breakdown

• Major metabolite of heme

Bilirubin

Bilirubin

• approximately - to - mg is produced in healthy adults daily

• - % is derived from turnover of senescent RBCs

• approximately 250 to 350 mg is produced in healthy adults daily

• 85 % is derived from turnover of senescent RBCs

Bilirubin

• yellowish pigment observed in -

jaundice

Bilirubin

• high bilirubin levels are observed in (3)

• gallstones

• acute hepatitis

• chronic hepatitis

Breakdown of RBCs

Jaundice

• comes from the French “_” = _

• yellow discoloration of skin, mucous, membrane and sclera of the eyes

• jaune = yellow

Jaundice

• visible indication of _

hyperbilirubinemia

Jaundice

• yellow discoloration of serum

icterus

Jaundice

• evident when bilirubin levels exceed _

2 to 3 mg/dL

Principle of Bilirubin Assays

• diazotization of bilirubin to produce azobilirubin

Van Den Bergh Reaction

Principle of Bilirubin Assays

• Van Den Bergh Reaction (3)

• Evelyn and Malloy Method

• Jendrassik and Grof Method

• Modified Jendrassik and Grof Method

Principle of Bilirubin Assays

• Van Den Bergh Reaction

→Evelyn and Malloy Method

Coupling accelerator:

Diazo Reagents:

Final Reaction:

Coupling accelerator:

• 50% methanol

Diazo Reagents:

• A - 0.1% sulfanilic acid + HCl

• B - 0.5% sodium nitrite

• BLANK - 1.5% HCl

Final Reaction:

• Positive > pink to purple azobilirubin (560 nm)

Principle of Bilirubin Assays

• Van Den Bergh Reaction

→most sensitive and most widely used

→more sensitive than Evelyn Malloy

Jendrassik and Grof Method

Principle of Bilirubin Assays

• Van Den Bergh Reaction

→Jendrassik and Grof Method

1. Popular technique for _

2. Not falsely elevated by _

3. Coupling accelerator:

4. Buffer:

5. Final reaction:

1. Popular technique for DISCRETE ANALYZERS

2. Not falsely elevated by HEMOLYSIS

3. Coupling accelerator: CAFFEINE SODIUM BENZOATE

4. Buffer: SODIUM ACETATE > less sensitive to pH change

5. Final reaction: POSITIVE > pink to blue azobilirubin (600 nm)

Principle of Bilirubin Assays

• Van Den Bergh Reaction

→candidate reference method for total bilirubin

Modified Jendrassik and Grof Method

Principle of Bilirubin Assays

• Van Den Bergh Reaction

→Modified Jendrassik and Grof Method

Coupling accelerator:

Coupling accelerator: caffeine-benzoate

Serum Bilirubin Levels - Normal Values

Class of Jaundice (3)

• Pre-hepatic or Hemolytic (Unconjugated)

• Hepatic or Hepatocellular

• Post-hepatic

Class of Jaundice

• Pre-hepatic or hemolytic (Unconjugated)

→LAB FINDINGS (5)

• increased amount of bilirubin being presented to the liver

• B1 markedly increased, B2 normal

• negative urine bilirubin

• increased urobilinogen

• dark stool

Class of Jaundice

• Pre-hepatic or hemolytic (Unconjugated)

→CAUSES (7)

• abnormal red cells

• antibodies

• drugs and toxins

• thalassemia

• hemoglobinopathies

• Gilbert's

• Crigler-Najjar syndrome

Class of Jaundice

• Hepatic or Hepatocellular

→LAB FINDINGS (4)

• intrinsic liver defect

• increased B1 and B2

• increased urobilinogen

• positive urine bilirubin

Class of Jaundice

• Hepatic or Hepatocellular

→Causes (3)

• viral hepatitis

• toxic hepatitis

• intrahepatic cholestasis

Class of Jaundice

• Post-hepatic

→LAB FINDINGS (6)

• Obstruction in the bile duct

• B2 markedly increased

• B1 normal

• positive urine bilirubin

• decreased urobilinogen

• pale-colored stool

Class of Jaundice

• Post-hepatic

→Causes (4)

• Extrahepatic cholestasis

• Gallstones

• Tumors of the bile duct

• Carcinoma of pancreas

Congenital Hyperbilirubinemia (5)

• Gilbert Syndrome

• Crigler-Najjar Syndrome

• Dubin-Johnson Syndrome

• Rotor Syndrome

• Lucey-Driscoll Syndrome

Congenital Hyperbilirubinemia

• bilirubin transport deficit

Gilbert Syndrome

Congenital Hyperbilirubinemia

• bilirubin conjugation deficit

Crigler-Najjar Syndrome

Congenital Hyperbilirubinemia

• 2 types of Crigler-Najjar Syndrome

• Type 1: Complete deficiency of enzyme UDPGT

• Type 2: Partial deficiency of enzyme UDPGT

Congenital Hyperbilirubinemia

• bilirubin excretion deficit

Dubin-Johnson Syndrome

Congenital Hyperbilirubinemia

• defective excretion of bilirubin WITHOUT liver pigmentation

Rotor Syndrome

Congenital Hyperbilirubinemia

• caused by a circulating inhibitor to bilirubin conjugation

Lucey-Driscoll Syndrome

• severe unconjugated hyperbilirubinemia characterized by deposition of bilirubin in the brain, particularly affecting the basal ganglia, mainly the lenticular nucleus

• causes motor dysfunction, retardation, death

Kernicterus

Kernicterus

• the danger of kernicterus is a certainty at levels _

exceeding 20 mg/dL

• yellowish discoloration of the skin but not the sclera due to increases ingestion of carotenoids

Carotenemia

Liver Diseases (5)

• Biliary Obstruction

• Cirrhosis

• Tumors

• Reye's Syndrome

• Drug- and Alcohol-Related Disorders

Liver Diseases

• Biliary Obstruction

→In adults, _ or presence of bile stones is the most common cause of hyperbilirubinemia

→_ is bile stones in the common bile duct

→cholelithiasis

→choledocholithiasis

Liver Diseases

• Cirrhosis

→_ replaces normal healthy liver

→most common cause:

→signs and symptoms:

→SCAR TISSUE replaces normal healthy liver

→most common cause: CHRONIC ALCOHOLISM

→signs and symptoms: OCCUR IN LATE STAGES

Liver Diseases

• Tumors

→Primary:

→Metastatic/Secondary:

→Tumors of the liver may also be classified as benign or malignant:

→Primary: BEGINS IN THE LIVER CELLS

→Metastatic/Secondary: Tumors from other parts of the body spread to the liver, accounts for 90% to 95% of all hepatic malignancies

→Tumors of the liver may also be classified as benign or malignant:

1. BENIGN: hepatocellular adenoma

2. MALIGNANT: hepatocellular carcinoma, bile duct carcinoma

Liver Diseases

• Reye's Syndrome

→precise cause is unknown but studies found strong epidemiological association between _ and subsequent development of Reye's syndrome

→an acute illness, characterized by - and - of the liver

→precise cause is unknown but studies found strong epidemiological association between INGESTION OF ASPIRIN DURING A VIRAL SYNDROME and subsequent development of Reye's syndrome

→an acute illness, characterized by NON-INFLAMMATORY ENCEPHALOPATHY and FATTY DEGENERATION of the liver

Liver Diseases

• Drug- and Alcohol-Related Disorders

→Drug-induced liver disease accounts for _ to _ of all reported cases of acute liver failures

→most important drug-associated with hepatoxicity

→one of the most common drugs associated with serious hepatic injury

→Drug-induced liver disease accounts for 1/3 to ½ of all reported cases of acute liver failures

→most important drug-associated with hepatoxicity = ETHANOL

→one of the most common drugs associated with serious hepatic injury = ACETAMINOPHEN / PARACETAMOL

• colorless end-product of bilirubin metabolism that is oxidized by intestinal bacteria

Urobilinogen

Urobilinogen

• is oxidized into (3)

• urobilin

• stercobilin

• mesobilin

Urobilinogen

• After formation in the intestines: (2)

• most urobilinogen are reabsorbed and re-excreted by the liver; a minor fraction may be excreted in the urine

• some urobilinogen in the stool are converted into stercobilin

Urobilinogen

• absence in the urine or stool denotes _

complete biliary obstruction

Urobilinogen

• Specimen for testing:

• _ sample is preferred because of alkaline tide

• avoid exposure to _

• Specimen for testing: 2-HOUR URINE SAMPLE

• 2 to 4 PM sample is preferred because of alkaline tide

• avoid exposure to LIGHT

Urobilinogen

• Method for testing:

• Principle:

• _ to form RED color which is read spectrophotometrically

• Method for testing: EHRLICH'S REACTION

• Principle: REACTION OF EHRLICH'S REAGENT

• PDAB or PARA-DIMETHYL-AMINO-BENZALDEHYDE to form RED color which is read spectrophotometrically

Urobilinogen

• Reference Range:

→Urine

→Feces

→Urine > 0.1 to 1.0 Ehrlich unit/ 2 hours

→Feces > 75 to 275 Ehrlich units/ 100 g

Urobilinogen

• Clinical Significance

→In post-hepatic obstruction, urobilinogen formation is - because of impaired bilirubin excretion into the intestine

→This is evidenced by a (2)

→increased urine urobilinogen is associated with (2)

→In post-hepatic obstruction, urobilinogen formation is DECREASED because of impaired bilirubin excretion into the intestine

→This is evidenced by:

1. CLAY-COLORED > partial biliary obstruction

2. CHALKY WHITE STOOL > complete biliary obstruction

→increased urine urobilinogen is associated with (2)

1. hemolytic disease

2. hepatocellular disease like hepatitis

Total Protein

• important in assessing: (2)

• nutritional status

• severe diseases involving liver, kidney, and bone marrow

Total Protein

• about _% higher in ambulatory persons

10%

Total Protein

• Plasma total protein is _ higher than serum total protein (fibrinogen)

• 0.2 to 0.4 g/dL

Total Protein

• Transudates have a TP of _

• Exudates have a TP of _

• Transudates have a TP of <3.0 g/dL

• Exudates have a TP of >3.0 g/dL

Total Protein

• Reference Value:

6 to 8 g/dL

Total Protein

• INCREASED (3)

• malignancy

• multiple myeloma

• Waldenstrom's macroglobulinemia

Total Protein

• DECREASED (4)

• hepatic cirrhosis

• glomerulonephritis

• nephrotic syndrome

• starvation

Total Protein

• Specimen Requirements and Result Variations

→ Use _ rather than plasma

→_ is not needed

→ _ may affect results

→_ falsely elevates total protein

→as person ages, there is a slight decrease in _

→lower total protein are also seen in _

→ Use SERUM rather than plasma

→FASTING is not needed

→ LIPEMIA may affect results

→HEMOLYSIS falsely elevates total protein

→as person ages, there is a slight decrease in ALBUMIN

→lower total protein are also seen in PREGNANCY

Total Protein

• Laboratory Methods (8)

• Kjeldahl Method (Indirect Method)

• Biuret Method (Direct Method)

• Folin-Ciocalteau (Lowry) Method

• Ultraviolet Absorption Method

• Salt Fractionation

• Refractometry

• Turbidimetric and Nephelometric Methods

• Serum Protein

• Others (2)

1. Coomassie Brilliant Blue Dye

2. Ninhydrin

Total Protein

• Laboratory Methods

→standard reference method

→quantifies protein by nitrogen content

→uses serum with tungstic acid, forming protein free-filtrate

Kjeldahl Method (Indirect Method)

Total Protein

• Laboratory Methods

→Kjeldahl Method (Indirect Method)

REAGENT:

END-PRODUCT:

REAGENT: sulfuric acid > digesting agent

END-PRODUCT: ammonia

Total Protein

• Laboratory Methods

→most widely used method

→requires 2 peptide bonds and alkaline medium

Biuret Method (Direct Method)

Total Protein

• Laboratory Methods

→Biuret Method (Direct Method)

PRINCIPLE:

PRINCIPLE: cupric ions react with peptide bonds in alkaline medium producing violet colored complex at 545 nm

Total Protein

• Laboratory Methods

→Reagents (4)

• Copper sulfate > main reagent

• Potassium sodium tartrate > keeps copper in solution

• Potassium iodide > stabilizer

• Sodium hydroxide < alkalinizes solution

Total Protein

• Laboratory Methods

→highest analytical sensitivity

Folin-Ciocalteau (Lowry) Method

Total Protein

• Laboratory Methods

→Folin-Ciocalteau (Lowry) Method

PRINCIPLE:

REAGENT:

COLOR ENHANCER:

PRINCIPLE: oxidation of tyrosine, tryptophan, and histidine to give a deep blue color

REAGENT: phenol/ phosphotungstic-molybdic acid

COLOR ENHANCER: Biuret reagent

Total Protein

• Laboratory Methods

→proteins absorb light at 210 nm due to peptide bonds

Ultraviolet Absorption Method

Total Protein

• Laboratory Methods

→globulins can be separated from albumin by salting out procedures using sodium salts

Sal Fractionation

Total Protein

• Laboratory Methods

→Salt Fractionation

SALT USED FOR ANALYSIS

Sodium sulfate

Total Protein

• Laboratory Methods

-.based on the measuremnt of refractive index due to solutes in serum

Refractometry

Total Protein

• Laboratory Methods

→Turbidimetric and Nephelometric Methods

Reagents:

Scatter incident light:

Block light:

Reagents:

• sulfosalicylic acid (SSA),

• trichloroacetic acid (TCA

Scatter incident light:

• Nephelometry

Block light:

• Turbidimetry