CLINICAL CHEMISTRY CHAPTER 22

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CLINICAL CHEMISTRYCHAPTER 22

• LIVER FUNCTION

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• Introduction
• OK, so your beautiful new baby is a little
  yellow.
• And why did the doctor put your baby in a
  tanning booth?
• You told your family the baby was a little
  jaundiced. But what does that mean?
• Merriam-Webster Dictionary
• 2 exhibiting or influenced by envy, distaste,
  or hostility lt a jaundiced
  eye gt
• Does that mean your beautiful new baby is going
  to grow up to be evil?
• Good news!! Your baby is probably not the
  anti-Christ and he/she is also probably totally
  normal and healthy
• Say tuned to find out why!!!

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Key Terms

• Bile
• Bilirubin
• Total bilirubin
• Conjugated / Unconjugated Bilirubin
• Free bilirubin
• Kupffer cells
• Cirrhosis
• Jaundice
• Icteric
• Gallstones
• Urobilinogen
• UDPG
• Albumin
• Prehepatic, hepatic, post hepatic diseases

• Gilberts Syndrome
• Crigler Najjar Syndrome
• Dubin Johnson Syndrome
• Kernicterus
• HDNB
• Cholestasis
• Physiologic Jaundice of the newborn
• Diazo reagent
• Accelerators
• Ehrlich Reaction
• Evelyn Malloy Reaction
• Jendrassik Grof Reaction
• Direct Bilirubin Measurement
• Reyes Disease
• Reticuloendothelial ( RE ) System

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• Objectives
• List some of the general functions of the liver
  involving bile production and secretion,
  synthesis of other substances and detoxification
• Discuss the origin and formation of bilirubin
• Discuss the common methodologies and principles
  that are use to determine bilirubin
  concentrations
• Classify the three general types of jaundice and
  list causes for each
• Interpret laboratory bilirubin test results and
  identify possible disease conditions
• Discuss proper collection and processing of
  bilirubin specimens

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• Meet you liver !!!
• The liver is responsible for many important
  metabolic functions
• gt 100 lab tests associated with liver function
• Liver is connected to the circulatory system by
• Hepatic artery Provides its blood supply
• Portal vein Transports absorbed substances
  from the GI tract
• Lobules are the functional units, consisting of
  clusters of hepatocytes around a ventral vein
• Kupffer cells line vascular spaces called
  sinusoids. Blood from these spaces drain into
  the portal veins.

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• Other major hepatic functions
• Protein synthesis ( almost all proteins )
• Regulation of carbohydrate metabolism
• Regulation of lipoproteins
• Detoxification of drugs and chemicals
• Do not let Hannibal Lector eat your liver with
  fava beans and a fine chianti

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• Excretion of bile
• Liver produces up to 3 liters bile / day
• Bile excreted into bile canaliculi and stored in
  the gall bladder
• Bile is excreted into the GI tract to aid in
  digestion and absorption of lipids
• Bilirubin in the main bile pigment , derived from
  heme catabolism

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Formation of bilirubin

• RBCs are phagocytized in the spleen. Hemoglobin
  is catabolized into amino acids, iron and heme.
• Heme ring is broken open and converted to
  unconjugated ( indirect )
  bilirubin.
• RE cells in the spleen secrete unconjugated
  bilirubin into the plasma, where bilirubin is
  bound by albumin.
• Albumin bilirubin complex travels to the liver.
• Hepatocytes conjugates bilirubin with gluconic
  acid and UDPG enzyme.
• Conjugated bilirubin secreted into the bile ducts
  ( GI tract )
• GI bacterial normal flora convert conjugated
  bilirubin into urobilinogen.
• Urobilinogen may be excreted into the stool,
  reabsorbed into the plasma and excreted in the
  urine

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• Increased plasma bilirubin indicates
• Increased RBC catabolism
• Decreased hepatic conjugation and excretion of
  bilirubin
• Jaundice
• Yellowish discolorization of the skin and sclera
  from increased plasma bilirubin
• Icteric
• Plasma / serum with yellowish color from ?
  bilirubin
• Reference ranges
• Total Bilirubin ( conjugated unconjugated )
  0.2 - 1.0 mg / dl
• Conjugated bilirubin 0.0 - 0.2 mg / dl
• Fullterm newborns 2.0 6.0 mg / dl

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• General classifications of jaundice
• Prehepatic
• Excess RBC destruction ( Not impaired liver
  function )
• Increased unconjugated bilirubin
• Hepatic
• Defective liver function ( most common )
• Defective hepatocyte uptake conjugation
  secretion of bilirubin
• Cholestasis Impaired hepatic transport
• Posthepatic
• Impaired ability of liver to excrete bile into
  the GI tract ( gallstones, tumors )

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• Causes of increased bilirubin
• Excessive RBC catabolism
• Hemolytic anemias
• Hepatic inability to conjugate and excrete
  bilirubin
• TBIL usually lt 5.0 mg / dl
• Pre-hepatic jaundice
• ? Total Bilirubin ? Unconjugated Normal
  conjugated
• Negative Urine Bilirubin

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• Gilberts Syndrome
• Defective bilirubin transport into hepatocyte (
  TBIL lt 3.0 mg / dl )
• Crigler Najjar Syndrome
• UDPG deficiency
• Hepatocytes lack UDPG enzyme cannot conjugate
  bilirubin
• Dubib Johnson and Rotors Syndrome
• Defective secretion of conjugated bilirubin
• Physiological Jaundice of the newborn
• Immature liver at birth
• Temporary deficiency of UDPG
• Small / moderate elevated unconjugated bilirubin
  lasting a few days

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• Hemolytic Disease of the Newborn ( HDNB )
• Mother Newborn Blood Group incompatibility (
  ABO, Rh )
• Maternal IgG antibodies cross the placenta and
  attack fetal RBC
• Increased hemolysis of newborns RBC
• Newborns immature liver cannot conjugate and
  excrete bilirubin
• Plasma albumin is saturated with unconjugated
  biliribin
• Excess free bilirubin ( unbound ) penetrates
  blood brain barrier
• Kernicterus - Perminant brain damage to the
  newborn
• Kernicteris usually occurs when TBIL gt 20.0 mg /
  dl
• Treatment of HDNB
• UV light
• Exchange Transfusions

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• Cirrhosis
• Irreversible structural damage ( scaring ) to the
  liver
• Common causes
• Alcohol abuse
• Viral hepatitis
• Hemochromatosis
• Obstructions of hepatic circulation
• Autoimmune diseases
• Reyes Syndrome

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Classic Bilirubin Techniques
EHRLICH REACTION Bilirubin Diazotized
Sulfanilic ( Diazo reagent )
Red-blue chromogen EVELYN-MALLOY
REACTION Bilirubin Diazo 50 Methanol
Red-blue
chromogen JENDRASSIK-GROF REACTION Bilirubin
Diazo CaffeineA Benzoate-Acetate
Red-blue chromogen
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• The variety of these early techniques led to the
  discovery that there were two different form of
  bilirubin
• Conjugated Bilirubin
• Always reacts with diazo reagent
• Direct, esterfied
• Water soluble
• Unconjugated bilirubin
• Will not react with diazo reagent unless there
  are accelerators ( alcohol , caffeine-benzoate-a
  cetate ) added
• Indirect , non-esterfied
• Water insoluble

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Conjugated Bilirubin Methodology
Bilirubin Diiazo ( No
Accelerators )

Ascorbic Acid Tartrate
Azobilirubin
Absorbs light _at_ 600 nm
Only conjugated
bilirubin reacts Note Normal plasma has
little to no direct bilirubin . The 0.0
0.2 mg / dl reference represents a small false
positive. But why?
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Total Bilirubin methodology BILIRUBIN
SODIUM ACETATE ( BUFFER ) CAFFEINE (
ACCELERTOR ) SODIUM BENZOATE ( ACCELERATOR )
DIAZOTIZED SULFANILIC ACID
Ascorbic acid destroys excess diazo Alkaline
tartrate promotes color formation
Azobilirubin
( Absorbs light _at_ 600 nm ) The
addition of accelerators allows conjugated and
unconjugated bilirubin to react
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Classic Bilirubin Methodology ( Diazo Technique
)
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• Review

CONJUGATED BILIRUBIN
UNCONJUGATED BILIRUBIN DIRECT INDIRECT WATER
SOLUBLE WATER INSOLUBLE ESTERFIED NON-ESTERF
IED FOUND IN URINE NOT FOUND IN URINE DOESNT
NEED ACCELERATOR NEEDS ACCELERATOR TOTAL
BILIRUBIN CONJUGATED UNCONJUGATED
BILIRUBIN LABORATORIES ROUTINLY MEASURE TOTAL
BILIRIBIN ( TBIL ) AND CONJUGATED BILIRUBIN (
DBIL ) . UNCONJUGATED BILIRUBIN IS CALCULATED
BY TBIL DBIL UNCONJUGATED BILIRUBIN
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• Direct measurement of bilirubin
• Because bilirubin has a distinctive color, it is
  possible to measure bilirubin directly
• No chemical reaction is necessary !
• Unfortunately, this procedure has limited value
  because of other colored plasma substances that
  interfere
• Only newborns lack these interfering substances.

A455 nm - A 575 nm Absorbance of Total
Bilirubin ( A 575 nm Corrects for absorbance
from hemolysis )
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• Bilirubin specimen requirements
• Serum / plasma
• Protect from light - Bilirubin is light
  sensitive
• Hemolysis causes false increased bilirubin

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• Miscellaneous other tests related to hepatic
  disease
• Elevated Enzyme activity
• ( AST , ALT, GGT, ALK, LDH )
• Plasma proteins
• ? albumin
• ? Protime ( PT )
• Non protein nitrogens
• ? Ammonia
• Viral infections
• HAV, HBV, HCV, HDV, CMV

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REVIEW OF CONJUGATED / UNCONJUGATED BILIRUBIN
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Bilirubin Reference Ranges

• Total Bilirubin 0.2 - 1.0 mg / dl
• Conjugated bilirubin 0.0 - 0.2 mg / dl
• Fullterm newborns 2.0 6.0 mg /dl TBil
• Visible jaundice around 3.0 mg/dl TBil
• Exchange Transfusions considered around 15.0
  20.0 mg/dl TBil

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Liver / Bilirubin Links
http//www.drhull.com/EncyMaster/B/bilirubin.html
http//www.nlm.nih.gov/medlineplus/ency/article/0
03479.htm http//www.pediatrics.wisc.edu/children
shosp/parents_of_preemies/jaundice.html http//ww
w.drgreene.com/21_633.html