1

BILIRUBUN METABOLISM

AN AN OVERVIEWOVERVIEW

2

FATE OF RED BLOOD CELLSLife 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)

3

NORMAL BILIRUBIN METABOLISMUnconjugated = Fat solubleConjugated = Water soluble

4

HYPERBILIRUBINEMIA Increased plasma concentrations of bilirubin (> 3 mg/dL) Increased plasma concentrations of bilirubin (> 3 mg/dL)

occurs occurs when there is an imbalance between its production and when there is an imbalance between its production and

excretion excretion Recognized clinically as jaundiceRecognized clinically as jaundice

5

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 ABO- incompatibility); 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

6

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

7

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

8

Diagnoses of Jaundice

AST (SGOT)/ ALT (SGPT) = TransaminasesAST (SGOT)/ ALT (SGPT) = Transaminases

ALP = Alkaline PhosphatseALP = Alkaline Phosphatse

9

Neonatal Jaundice

Common, particularly in premature infantsTransient (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 kernicterusJaundice 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

10

Regulation of iron metabolism

11

Overview

Patterns of Liver damage Review of individual tests Appraise synthetic function Non-hepatic causes of abnormal tests Synthesize an approach to evaluation

12

What does the Liver do? A LOT! Stores and Mobilizes Energy Stores and Mobilizes Energy Controls Controls Blood Sugar (Glucose)Blood Sugar (Glucose)

Regulates Glycogen Regulates Glycogen Regulates Fat Storage Regulates Fat Storage Aids Digestion Aids Digestion Produces Bile Produces Bile Regulates Blood Clotting Regulates Blood Clotting Manufactures Manufactures

Clotting factorsClotting factors Other Blood Proteins Other Blood Proteins

Produces Hormones Produces Hormones Manufactures Cholesterol Manufactures Cholesterol Filters BloodFilters Blood

Detoxifies Poisons Externally-Derived Poisons

Alcohol Byproducts of Metabolism

Bilirubin Breaks down Drugs Produces Vitamins Vitamin D Stores Minerals Iron Produces Essential Immune System

Factors Monitors, as Well as Manufactures,

Countless other Blood Proteins, to Maintain the Proper Levels of Numerous Chemicals in the Body

13

Liver Function Key Points

Produces bileProduces proteinsAlbuminClotting factors

Liver does TOO much for any single test or set of test to determineFocus on the basic test themselves and recognition of basic patterns

14

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)

15

AST:ALT ratio

Alcoholic hepatitisRatio is >1 90% of the time – often 2:1Mechanism 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 timeMechanism unclear

16

Causes of Hepatocellular DamageA Autoimmune hepatitis – ANA, Anti-smooth muscle ab (ASMA), Anti-Liver and Kidney Mitochondrial Ab. HEP AB Hepatitis B C Hepatitis C D Drugs or toxins E Ethanol / Hep E (Pregnancy)F Fatty liverG 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

17

Level of Elevation

Giannini, E. G. et al. CMAJ 2005;172:367-379

18

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

19

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

20

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.

21

GGTP

(gamma glutamyl transpeptidase): elevated in bile duct disease and alcoholism.

22

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.

23

Bilirubin Fractions Present in Blood and Urine

In Serum

As:Measured As:

Presentin Urine

Unconjugated

(90%) Albumin-bound Indirect-reacting

bilirubin Never

Conjugated UnboundDirect-reacting

bilirubin

Yes, when serum bilirubin exceeds 3-4 mg/dL

24

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”

25

Jaundice

26

Hyperbilirubinemia

Hyperbilirubinemia (Jaundice)

Prehepatic(Hemolysis)

HepaticGenetic defects,

primary liver disease

PosthepaticBile Duct ObstructionPancreatic Head CA

Unconjugated Bilirubin

Mixed Conjugated Bilirubin

27

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

28

LIVER FUNCTION TESTS

Protein production Albumin Clotting Factors Total protein production

29

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

30

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.

31

Clotting Factors

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

32

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

33

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

34

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

35

Overview of Approach to Liver Tests

Think of NON-hepatic causes for abnormalitiesLook at pattern – cholestatic (hepatobiliary) vs hepatocellularLook at tests of FUNCTION for duration of dysfunctionWhat is your DDx?What further testing is needed?

36

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.)

37

Hepatitis A Serology

38

Anti-HBAnti-HBssAgAg

HBV HBV DNADNA

HBeAgHBHBssAgAg

Anti-HBAnti-HBccAgAg

Anti-HBAnti-HBeeAgAg

39

Hepatitis B

40

USA: HCVGenotype distribution

41

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

42

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.

43

USA:Prev:4 million Incidence:35,000 to 185,000Deaths: 10,000-20,000

44

Prevalence by Age & Ethnicity USA

45

Hepatitis C

46

VIRAL LOAD TESTS

Measures number copies made by a virusReported as ‘high’ or ‘low’High: More than 2 million copiesLow: 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’

47

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.

48

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.

49

Genotyping and Serotyping of HCV

6 known genotypes and more than 50 subtypes of hepatitis C Helpful in epidemiology and deciding response to therapyGenotypes 2 and 3 are almost three times more likely to respond to therapy with alpha interferon or the combination of alpha interferon and ribavirin.

50

ELISAEnzyme-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.

51

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/mLTMA (Transcription-mediated amplification) can measure very small amounts (5-10 IU/mL)

52

Remember!

Antibodies (Ab) suggests immune response IgM-Ab means acute infections

IgG-Ab means NO active infection

53

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)

54

HBsAg+;

X Anti-HBs –ve; Anti HBc Ag +ve; Anti-HBc IGM Ab +ve

Suggests:

A. Acute hepatitis B B Chronic hepatitis B-Low infectivity C. Chronic hepatitis B-High infectivity D. Immunized against HBV infection

55

HBsAg detected;X Anti-HBs -ve; Anti HBc Ag +ve;X Anti-HBc IGM Ab –ve; Anti- HBe Ab +ve;

Suggests:

A. Acute hepatitis B B Chronic hepatitis B-Low infectivity C. Chronic hepatitis B-High infectivity D. Immunized against HBV infection

56

X HBsAg not detected; Anti-HBs +ve;

X Anti-HBc IGM Ab –ve;

Suggests:

A. Acute hepatitis B B Chronic hepatitis B-Low infectivity C. Chronic hepatitis B-High infectivity D. Immunized against HBV infection

57

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

58

HBsAg not detected; Anti-HBs not detected; Anti-HBc IGM Ab not detected;

Suggests:

A. Acute hepatitis B B Chronic hepatitis B-Low infectivity C. Chronic hepatitis B-High infectivity D. Immunized against HBV infection E. Susceptible to HBV infection

59

PANCREATIC DATA

60

PANCREATIC CANCER

ENDOCRINE- INSULINOMAEXOCRINE- HEAD/BODY/

TAIL/AMPULLAOBSTRUCTIVE “PAINLESS” JAUNDICE/ WEIGHT LOSSFourth leading cause of cancer death DIAGNOSIS BY IMAGING

61

USEFUL BIOCHEMISTRY

CMP CA-19-9/ CEA 19 Useful in follow up after therapy but not

for diagnosis

62

PANCREATIC DISEASES Endocrine -- which produce the hormones insulin and

glucagon Exocrine -- which make powerful enzymes to digest fats,

proteins, and carbohydrates

63

PANCREATITIS

ALCOHOL T2 DIABETES CYSTIC FIBROSIS PANCREATIC INSUFFICIENCY

64

? 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.

65

?Tests

Fecal Fat- look for fat globules Trypsin-stool trypsin levels Trypsinogen (serum) Imaging: MRI Studies/ ERCP

66

Pancreatitis

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