BILIRUBUN
METABOLISM
AN OVERVIEW
1
FATE OF RED BLOOD CELLS
Life
span in blood stream is 60-120 days
Senescent
RBCs are phagocytosed and/or
lysed
Normally, lysis
occurs extravascularly in the reticuloendothelial system
(mainly spleen) subsequent to RBC phagocytosis

Lysis can also occur intravascularly
(in blood stream)
2
NORMAL BILIRUBIN
METABOLISM
Unconjugated = Fat soluble
Conjugated = Water soluble
3
HYPERBILIRUBINEMIA
 Increased plasma concentrations of bilirubin (> 3 mg/dL) occurs
when there is an imbalance between its production and excretion
 Recognized clinically as jaundice
4
Prehepatic (hemolytic) jaundice


Results from excess production of bilirubin (beyond the
livers ability to conjugate it) following hemolysis
Excess RBC lysis is commonly the result of autoimmune
disease; hemolytic disease of the newborn (Rh- or ABOincompatibility); structurally abnormal RBCs (Sickle cell
disease); or breakdown of extravasated blood
High plasma concentrations of unconjugated* bilirubin
(normal concentration ~0.5 mg/dL)
*Fat soluble

5
Intrahepatic jaundice



Impaired uptake, conjugation, or secretion
of bilirubin
Reflects a generalized liver (hepatocyte)
dysfunction
In this case, hyperbilirubinemia is usually
accompanied by other abnormalities in
biochemical markers of liver function
6
Posthepatic jaundice




Caused by an obstruction of the biliary tree
Plasma bilirubin is conjugated, and other biliary
metabolites, such as bile acids accumulate in the
plasma
Characterized by pale colored stools (absence of
fecal bilirubin or urobilin), and dark urine
(increased conjugated bilirubin)
In a complete obstruction, urobilin is absent from
the urine
7
Diagnoses of Jaundice
AST (SGOT)/ ALT (SGPT) = Transaminases
ALP = Alkaline Phosphatse
8
Neonatal Jaundice
Common,
particularly in premature infants
Transient (resolves in the first 10 days)
Due to immaturity of the liver enzymes
High levels of unconjugated bilirubin are toxic to the
newborn – cause a type of mental retardation known as
kernicterus
Jaundice within the first 24 hrs of life or which takes longer
then 10 days to esolve is usually pathological and needs to
be further investigated
9
Regulation of iron metabolism
10
Overview

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Patterns of Liver damage
Review of individual tests
Appraise synthetic function
Non-hepatic causes of abnormal tests
Synthesize an approach to evaluation
11
What does the Liver do?

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

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A LOT!
 Detoxifies Poisons
Stores and Mobilizes Energy
 Externally-Derived Poisons
Controls Blood Sugar (Glucose)
Alcohol
 Regulates Glycogen
 Byproducts of Metabolism
Regulates Fat Storage
Bilirubin
Aids Digestion
 Breaks down Drugs
Produces Bile
 Produces Vitamins
Regulates Blood Clotting
 Vitamin D
Manufactures
 Stores Minerals
 Clotting factors
 Iron
 Other Blood Proteins
 Produces Essential Immune System
Produces Hormones
Factors
Manufactures Cholesterol
 Monitors, as Well as Manufactures,
Countless other Blood Proteins, to
Filters Blood
Maintain the Proper Levels of Numerous
Chemicals in the Body
12
Liver Function Key Points
Produces
bile
Produces proteins
Albumin
Clotting factors
Liver does TOO much for any single test or set of test to
determine
Focus on the basic test themselves and recognition of basic
patterns
13
Markers of Hepatocellular
Injury

Hepatocytes are damaged so they leak – so these enzymes
are HIGH
 Aspartate aminotransferase
 (AST/ SGOT)
 Alanine aminotransferase
 (ALT/ SGPT)
 Lactate dehydrogenase (LDH)
14
AST:ALT ratio
Alcoholic
hepatitis
Ratio is >1 90% of the time – often 2:1
Mechanism thought to be related to
B6 depletion in
alcoholics which leads to disrupted ALT synthesis and
therefore decreased levels.
This is NOT SPECIFIC!!
 Viral Hepatitis: Both ALT AND AST elevated
 Ratio < 1 70% of the time
Mechanism unclear
15
Causes of Hepatocellular
Damage
A
Autoimmune hepatitis – ANA, Anti-smooth muscle ab
(ASMA), Anti-Liver and Kidney Mitochondrial Ab. HEP A
B Hepatitis B
C Hepatitis C
D Drugs or toxins
E Ethanol / Hep E (Pregnancy)
F Fatty liver
G Growths (i.e., tumors)
H Hemodynamic disorder
(congestive heart failure or “shock liver”)
I Inborn errors - iron (hemochromatosis),
copper (Wilson's disease) or alpha1-antitrypsin deficiency
16
Level of Elevation
Giannini, E. G. et al. CMAJ 2005;172:367-379
17
Key points




AST and ALT elevations infer HEPATOCELLULAR
DAMAGE
NON-SPECIFIC TEST with other causes that can lead to
elevation
Levels in the 100’s ETOH, 1000’s viral, and 10,000’s toxin
related.
Ratio can SUGGEST but not diagnose alcoholic hepatitis
18
Markers of
Cholestasis/Obstruction

Cholestasis (lack of bile flow) results from the blockage
of bile ducts or from a disease that impairs bile
formation in the liver itself. Back leads to GRADUAL
increase in enzymes over the course of days.
 Alkaline phosphatase (ALP)
 Gamma-glutamyltransferase (GGT)
 Bilirubin
19
ALP



Originates primarily in Bone and Liver
 Other sources include intestine, kidney, placenta.
Isoenzymes can determine Bone vs Liver
May lag behind symptoms in rising.
20
GGTP


(gamma glutamyl transpeptidase):
elevated in bile duct disease and alcoholism.
21
Causes of Cholestasis
 Obstruction
 Intrahepatic
Primary Biliary Cirrhosis – young females. Test: Anti-Mitochondrial
Ab (AMA)
DRUGS – any number of medications, particularly antibiotics and
anti-seizure
Any Hepatocellular damage (CONFUSED?
The hepatocellular can cause cholestatis, but the necrosis is
greater)
Critical illness
 Extrahepatic
Common duct obstruction (stone/Tumor)
Primary Sclerosing Cholangitis – More often males with IBD
Pancreatic head obstruction (Stone/Tumor)
 Differentiate – use U/S to look at common bile duct dilation.
 If non-diagnostic do ERCP then liver bx.
22
Bilirubin Fractions Present in
Blood and Urine
Unconjugated
(90%)
Conjugated
In Serum
As:
Measured As:
Albumin-bound
Indirect-reacting
bilirubin
Unbound
Direct-reacting
bilirubin
Present
in Urine
Never
Yes, when serum
bilirubin exceeds
3-4 mg/dL
23
ACUTE ALCOHOLIC HEPATITIS
Pattern
of liver test abnormality is hepatocellular
AST level is higher than the ALT level but
rarely exceeds 400
IU/mL
AST is typically in the
 100 IU/mL to 200 IU/mL range,
 even in severe disease, and
The ALT may even be normal,
 even in severe cases
“In alcoholic hepatitis
AST:ALT ratio is 2:1”
24
Jaundice
25
Hyperbilirubinemia
Hyperbilirubinemia (Jaundice)
Prehepatic
(Hemolysis)
Hepatic
Genetic defects,
primary liver disease
Unconjugate
d Bilirubin
Mixed
Posthepatic
Bile Duct Obstruction
Pancreatic Head CA
Conjugated Bilirubin
26
Key Points



ALP and GGT combined are markers of cholestasis, but
other things can make them rise.
2 types of Bilirubin, only conjugated excreted in urine
Cholestasis can be extra or intrahepatic – remember
how to differentiate
27
LIVER FUNCTION TESTS
Protein production
Albumin
Clotting Factors
Total protein production
28
Albumin
 65% of serum protein
 ½ Life = 3 weeks
 Low levels can correlate with chronic liver dysfunction.
 Other reasons to be low?
Decrease production
Malnutrition
Chronic Inflammation
Increased Loss
Kidney – Nephrotic Syndrome
GI tract – Protein-losing enteropathy
Skin – Severe burn
29
A/G RATIO:


The value of the A/G ratio is not precise due to the
countless number of variables in the fractions (Total
Globulins and Albumin) associated with various
metabolic states.
Abnormal A/G ratios usually reflect a general index of
liver dysfunction.
30
Clotting Factors

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Most clotting factors are synthesized in the liver
½ shorter than Albumin
Prothrombin Time (PT) is a good functional test – but
usually use INR to correct for lab variability
PT/INR PROLONGED in liver disease
31
Clotting Factor


Chronic cholestatic disease often have increased INR–
Why?
 Vit K def
 Vit K Fat soluable
 Cholestatic/obustructive so not enough bile secretion
so not enough Vit K absorption
How to differentiate Vit K def from Decreased synthesis?
 Give Vit K (take 24-48 hours to correct)
 Factor V NOT Vit K dependent so can be checked
directly
32
Key Points



The only liver FUNCTION test are test of PROTEIN
PRODUCTION
Low albumin due to liver dysfunction implies CHRONIC
(>3 week) liver damage
Always differentiate Vit K Def from Decreased liver
synthesis in pt with cholestatic disease
33
Further Testing



Ultrasound
 Good to visualize large bile ducts and large
masses. Cheap. Non-invasive
 Use: Obstruction
ERCP (Endoscopic Retrograde Cholangio
Pancreatography)
 Visualize smaller bile ducts, ampulla of Vater, and head
of pancreas. Provides t issue. Expensive. Highly
invasive
 Use: Obstruction
Liver Biopsy (rarely done)
 See hepatic pathology, particularly of the
hepatocytes. Gold Standard. Expensive.
Invasive
 Use: Hepatocelluar
34
Overview of Approach to Liver
Tests
Think
of NON-hepatic causes for abnormalities
Look at pattern – cholestatic (hepatobiliary) vs hepatocellular
Look at tests of FUNCTION for duration of dysfunction
What is your DDx?
What further testing is needed?
35
Caveats of Liver ‘Function’ Tests




There is NO PERFECT TEST
A group of tests is needed in order to ‘infer’ functional
liver status.
Mixed injury/obstruction patterns are common in REAL
LIFE !!
DO NOT assume that a NORMAL test result indicates
absence of liver disease. (example: AST & ALT can be
normal in End Stage Cirrhosis.)
36
Hepatitis A Serology
37
Anti-HBsAg
HBsAg
HBeAg
Anti-HBcAg
Anti-HBeAg
HBV DNA
38
Hepatitis B
39
USA: HCVGenotype
distribution
40
Why Genotype?
·
Genotypes 1, 2 and 3 =
North America and Western Europe
· Genotype 4 =
Africa, Egypt and the Middle East, but is increasingly seen in some parts of
Europe
·Genotype 5 = Africa and the Middle East
· Genotype 6 = Southeast Asia
·
Genotype 7 = Central Africa
41
Why Genotype?
1.
Those with genotypes 2 and 3:

Respond better (require only 24 week therapy)
2. Genotype 1 to respond poorly to therapy with
alpha interferon or the combination of alpha interferon and ribavirin.
3. A 48-week course of combination treatment is typically adequate for
those with genotype 1.
4. Data are mixed concerning genotype 4, though its response somewhere
in between the response of genotypes 2 and 3, and genotype 1.
5. Treating genotype 5 shows that its response to combination
treatment is similar to those with genotype 1.
6. genotype 6 lies at an intermediate level, between that
seen with
genotype 1 and genotypes 2
or 3.
7. Since it has just recently been discovered as having a distinct genetic
make-up, the response to standard combination therapy is not yet
established for genotype 7.
42
USA:
Prev:4 million
Incidence:35,000 to 185,000
Deaths: 10,000-20,000
43
Prevalence by Age & Ethnicity
USA
44
Hepatitis C
45
VIRAL LOAD TESTS
Measures
number copies made by a virus
Reported as ‘high’ or ‘low’
High: More than 2 million copies
Low: Less than 2 million copies
?Log drop: A 10 fold change- take
0ne zero off the end
of starting number for each ‘log drop’/’log kill’
46
RIBA
Recombinant
Immunoblot Assay
The EIA anti-HCV reactivity could represent a false-positive
reaction, recovery from hepatitis C, or continued virus infection
with levels of virus too low to be detected
If the immunoblot test for anti-HCV is positive, the patient has
most likely recovered from hepatitis C and has persistent antibody
without virus.
If the immunoblot test is negative, the EIA result was probably a
false positive.
47
PCR (Polymerase Chain Reaction)
Amplification
PCR
amplification can detect low levels of HCV RNA in
serum.
Testing for HCV RNA is a reliable way of demonstrating that
hepatitis C infection is present and is the most specific test
for infection.
48
Genotyping and Serotyping of
HCV
6
known genotypes and more than 50 subtypes of
hepatitis C
Helpful in epidemiology and deciding response to
therapy
Genotypes 2 and 3 are almost three times more likely to
respond to therapy with alpha interferon or the
combination of alpha interferon and ribavirin.
49
ELISA
Enzyme-Linked
ImmunoSorbent Assay, or ELISA, is a
biochemical technique used mainly in immunology to detect the
presence of an antibody or an antigen in a sample.
50
3 ways to test viral loads:
PCR
measure the amount of HCV RNA in the blood (can
measure very small loads as low as 50 IU/mL)
bDNA (Branched –chain DNA)- only measures medium
loads above 500 IU/mL
TMA (Transcription-mediated amplification) can measure
very small amounts (5-10 IU/mL)
51
Remember!

Antibodies (Ab) suggests immune response
IgM-Ab means acute infections

IgG-Ab means NO active infection

52
Immunoassay





A test that measures the concentration of a substance in a
biological liquid:
typically serum or urine,
uses the reaction of an
Antibody (Ab) or antibodies
to its antigen (Ag)
53

HBsAg+;
X Anti-HBs –ve;


Anti HBc Ag +ve;
Anti-HBc IGM Ab +ve
Suggests:




A.
B
C.
D.
Acute hepatitis B
Chronic hepatitis B-Low infectivity
Chronic hepatitis B-High infectivity
Immunized against HBV infection
54
HBsAg detected;
X Anti-HBs -ve;
 Anti HBc Ag +ve;
X Anti-HBc IGM Ab –ve;
 Anti- HBe Ab +ve;

Suggests:




A.
B
C.
D.
Acute hepatitis B
Chronic hepatitis B-Low infectivity
Chronic hepatitis B-High infectivity
Immunized against HBV infection
55
X HBsAg not detected;

Anti-HBs +ve;
X Anti-HBc IGM Ab –ve;
Suggests:




A.
B
C.
D.
Acute hepatitis B
Chronic hepatitis B-Low infectivity
Chronic hepatitis B-High infectivity
Immunized against HBV infection
56

HBsAg detected;
X Anti-HBs not detected;
X Anti-HBc IGM Ab not detected;
X Anti-HBe Ag not detected
Suggests:
 A. Acute hepatitis B
 B Chronic hepatitis B-Low infectivity
 C.Chronic hepatitis B-High infectivity
 D.Immunized against HBV infection
57



HBsAg not detected;
Anti-HBs not detected;
Anti-HBc IGM Ab not detected;
Suggests:





A.
B
C.
D.
E.
Acute hepatitis B
Chronic hepatitis B-Low infectivity
Chronic hepatitis B-High infectivity
Immunized against HBV infection
Susceptible to HBV infection
58
PANCREATIC DATA
59
PANCREATIC CANCER
ENDOCRINE-
INSULINOMA
EXOCRINE- HEAD/BODY/
TAIL/AMPULLA
OBSTRUCTIVE “PAINLESS” JAUNDICE/ WEIGHT LOSS
Fourth leading cause of cancer death
DIAGNOSIS BY IMAGING
60
USEFUL BIOCHEMISTRY


CMP
CA-19-9/ CEA 19 Useful in follow up after therapy but not
for diagnosis
61
PANCREATIC DISEASES


Endocrine -- which produce the hormones insulin and
glucagon
Exocrine -- which make powerful enzymes to digest fats,
proteins, and carbohydrates
62
PANCREATITIS




ALCOHOL
T2 DIABETES
CYSTIC FIBROSIS
PANCREATIC INSUFFICIENCY
63
? PANCREATIC INSUFFICIENCY






Lack of digestive enzymes:
presents with symptoms of malabsorption,
malnutrition,
vitamin deficiencies,
weight loss, and is often associated with
steatorrhea (loose, fatty, foul-smelling stools).
Diabetes may also be present in adults with pancreatic
insufficiency.
64
?Tests




Fecal Fat- look for fat globules
Trypsin-stool trypsin levels
Trypsinogen (serum)
Imaging: MRI Studies/ ERCP
65
Pancreatitis

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

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
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


Amylase- increase after 2-12 hrs peaks at
12-72 hrs
Lipase- increase after 4-8 hrs peaks at 24 hrs
Trypsin/Trypsinogen
Complete Blood Count (including white blood cell count)
Comprehensive Metabolic Panel (Bilirubin, liver function tests)
Glucose
Calcium
Magnesium
C-Reactive Protein
66