Liver basics

Dina N Greene, PhD, DABCC

Assistant Professor
Associate Director, Chemistry
Department of Laboratory Medicine
dngreene@uw.edu
 Learning Objectives

!  Describe the basic anatomy and physiology

of the liver
!  Compare the analytical assays used to
evaluate liver function
!  Describe the pathology associated with
various liver function test results
!  Evaluate a case of elevated AST
Questions to consider
!  What is the function of the liver?
!  What role does the liver play in digestion?

!  What are the biomarkers we use to determine

if the liver is in acute or chronic distress?
www.medivisuals.com
www.medivisuals.com
 Primary Liver Functions

!  Metabolism/nutrient !  Detoxification
storage !  Microorganisms
!  Carbohydrate !  Drugs
!  Protein !  Toxins
!  Fat
!  Excretion
!  Bile
Liver-synthesized proteins
1.  Albumin
2.  Alpha and beta globulins
3.  Factor II, VII, IX, and X
Liver Anatomy
 Liver Circulatory System
Blood from the
Heart
Blood Sources Combine In
the Sinusoid

Blood from the GI,

Pancreas, & Spleen

http://physics4ct.blogspot.com/2009/03/liver-circulatory-system.html
Hepatic Architecture
Hepatic Micro-Architecture

http://www.accessscience.com/
Functional Unit of the Liver
Portal Tract

Portal Trac

Central Vein

Lobule

Portal Tract
 Central Vein

Bile Flow Blood Flow

Portal Tract
 Portal Tract

Bile Duct

Portal venula

Hepatic Arteriola
 Hepatocytes

Sinusoids
Thanks to Dr Greg Cheeney for the AP slides!
Hepatocyte
 Primary Liver Functions

!  Metabolism/nutrient !  Detoxification
storage !  Microorganisms
!  Carbohydrate !  Drugs
!  Protein !  Toxins
!  Fat
!  Excretion
!  Bile
The Case
!  66 y/o female
!  Retired school teacher

!  Presents with chest discomfort and dyspnea

Krishnamurthy et al, Clin Chem 2009

Patient Summary
!  No PMH of liver disease
!  No obvious abnormalities

!  Takes no meds

!  Sclerae anicteric

!  Affirmed alcohol use @ <2 oz spirits daily

2 oz
Initial Laboratory Tests
!  Increased aspartate amino transferase
544 U/L (ref int 11-47 U/L)
 Hepatocyte Damage Releases
Intracellular Enzymes

Damaged
AST
ALT

Robbins Pathology 8th Ed

Copyright © 2010 by Saunders
 Aspartate Aminotransferase
!  Found in many tissues
!  liver > kidney > heart > skeletal muscle
!  Cytoplasmic and mitochondrial forms
!  Activity in males > females
!  Marker for hepatitis, damaged/necrosing hepatic cells
!  Can also be elevated in:
!  Hemochromatosis
!  non-alcholic fatty liver disease
!  Wilson’s disease

!  Non-liver reasons for elevation include

!  Hemolysis
!  Damage to cardiac muscle
!  Damage to skeletal muscle
!  Renal failure
 Quantifying AST

AST*
L-Aspartate + α-Oxoglutarate L-Glutamate + Oxalacetate

MDH
Oxalacetate Malate

NADH + H+ NAD+

*Requieres P5P as a cofactor

 AST = 544 U/L
~12x higher than the upper limit of
the reference interval…
But, if this were liver disease we
would expect additional markers
to be elevated
 Alanine Aminotransferase

!  Considered the most important test for

recognition of acute and chronic liver injury
!  Like AST, present in other tissues, but activity is
relatively higher in liver (vs. AST)
!  Exclusively cytoplasmic and has a half life of ~47
hrs (~3x longer than AST)
!  Thus, ALT is usually >AST

Patient’s ALT = 23 U/L (ref int 7-53 U/L)

 Quantifying ALT

ALT*
L-Alanine + α-Oxoglutarate L-Glutamate + Pyruvate

LDH
Pyruvate Lactate

NADH + H+ NAD+

*Requieres P5P as a cofactor

What are some mechanisms for erroneous AST/ALT results?

Factors affecting AST and ALT
(besides liver injury)

Dufour et al, Clin Chem 2000

 Alkaline Phosphatase

!  Found in liver, bone, intestine, and placenta

!  Elevations are not totally specific to liver
!  Involved in metabolite transport across cell
membranes
!  If liver isoenzyme is elevated it’s usually a
result of cholestatic liver injury
!  GPI anchored to membrane - bile salts facilitate
release
Cholestatic Liver Injury
 Quantifying Alk Phos

ALP
pNPP + AMP pNP + AMP-PO4
Mg+2

pNPP = p-Nitrophenyl Phosphate

AMP = 2-Amino-2Methyl-1-propanol
 γ-Glutamyltransferase
!  Facilitates transport of amino acids across cell
membranes
!  Like Alk Phos, GPI anchor solublized by bile,
leading to increased concentration in the serum
during cholestatic injury
!  Helps discriminate elevated Alk Phos
!  Elevated by chronic alcohol use
!  Is this accounted for when making the reference
interval?
 Quantifying GGT

5-Amino-2-
L-γ-Glutamyl-3- + Glyclyglycine GGT
nitrobenzoate
carboxy-4-nitroanilide

+ L-γ-Glutamylglycylglycine
Alk Phos & GGT results

!  Alk Phos = 95 IU/L (38-126 IU/L)

!  GGT = 25 IU/L (11-50 IU/L)

Bilirubin

http://diaglab.vet.cornell.edu/clinpath/modules/chem/images/bilirubin%20metabolism.jpg
Bilirubin Quantification
Bilirubin Quantification

No color change

alcohol
Bilirubin Quantification
 Pathologies associated with
bilirubin elevation
!  Unconjugated
!  Intravascular hemolysis
!  Liver failure
!  Liver hypertension
!  Genetic
!  Conjugated
!  Genetic
!  Acute hepatitis
!  Biliary tract obstruction
 Patient’s Bilirubin Results

!  Total
!  0.5 mg/dL (0.0-1.3 mg/dL)
!  Direct
!  0.2 mg/dL (≤0.3 mg/dL)
 Albumin
!  Evaluation of protein synthetic ability
!  Decreased due to extensive liver damage
or restriction of blood flow through the
portal vein
!  Half life 21 days
!  Not liver specific, but if liver is the cause of
low albumin, it indicates extensive damage

Patient’s Albumin = 3.8 g/dL (ref int 3.6-5.0 g/dL)

 Albumin Quantification

Bromocresol + Albumin Green Complex

 Lactate Dehydrogenase
!  Highly non-specific test
!  Found in cytoplasm of all cells and tissues
!  Most tissues have LDH activity 500-1000x > than serum
activity
!  Small amount of breakdown leads to significant elevation
!  Tends to be slightly higher in person’s >65 y/o
!  For liver most important when there is also a significant
in Alk Phos w/ normal AST, ALT, and other liver markers
!  Indicated space occupying lesion of the liver

Patient’s LDH = 373 U/L (ref int 100-250 U/L)

LDH Quantification

LDH
Lactate Pyruvate

NAD+ NADH + H+
 And after all of those labs
didn’t help diagnosis, they ran
a few more…
More Lab Results
hemoglobin 139 g/L (121-151g/L)

reticulocytes 0.008 (0.005-0.015)

haptoglobin 0.97 g/L (0.27-2.20 g/L)

TSH 3.60 mIU/L (0.35-5.50 mIU/L)

ANA 1:80 (negative)

Anti Sm Muscle Ab 1:80 (<1:20)

Anti Mitochondrial Ab negative

More Lab Results

α1 antitrypsin 167 mg/L (100-200 mg/L)

ferritin 202 mg/L (10-291 mg/L)

ceruloplasmin 380 mg/L (180-460 mg/L)

Hepatitis antigens nonreactive

Aldolase 4.5 U/L (<8 U/L)

CK 116 U/L (38-234 U/L)

Radiographic studies showed that abdomen and chest

were unremarkable
Any tests that you are surprised
they didn’t order?

•  Troponin
•  PTT
“…without signs or symptoms
of liver disease, the patient was
advised to discontinue alcohol
consumption, and the clinical
laboratory was contacted for
additional studies.”
The clinical lab decided to look
for the presence of a
macroenzyme
 Macroenzymes

http://www.path.cam.ac.uk/~mrc7/igs/migg2a.html http://www.uhnresearch.ca/labs/chirgadze/research.html

!  High molecular mass forms of serum enzymes

!  Can be Ig or non Ig bound
!  Macroamylase first described 45 years ago
!  Frequently interfere with serum enzyme results
!  False positive results
!  False negative results (hook effect or polymers that block one of
the antibodies from binding, substrate depletion)
What could you do to test for
macroAST in this patient?
Krishnamurthy et al, Clin Chem 2009
 This patient is positive for
macroAST…but, what exactly
does that mean for her?
Why do antibodies to enzymes occur?
Do macroenzymes cause disease?

Can macroenzymes be used as

diagnostic markers?
Why do antibodies to enzymes occur?
Theory 1: Antigen Driven Theory

Non-self antigen or
self antigen
modified self antigen

Theory 2: Dysregulation of Immune Tolerance

All self antigens

Do macroenzymes cause disease?

!  No convincing evidence exists

What next?
!  This patient can not use AST as a marker of
liver function
!  Inform and educate patient about this result

!  Probably no correlation to disease or risk, but

that’s not really known
 Conclusions

!  Liver function tests include the quantification

of a variety of enzymes and proteins
!  Interpretation of liver pathology should
involve the combined assessment of these
markers
!  Interferences should be investigated if a
falsely elevated or decreased result is
suspected