Liver enzymology is used to evaluate liver injury and function. Elevations in "leakage enzymes" like ALT and AST indicate hepatocyte injury, while increases in "induction enzymes" like ALP and GGT suggest cholestasis or impaired bile flow. ALT is the most specific indicator of hepatocyte injury in dogs and cats. ALP and GGT are useful for detecting cholestasis, with GGT being more specific but less sensitive than ALP in dogs, and more sensitive but less specific in cats. Bilirubin levels rise with increased RBC breakdown, decreased hepatic uptake or conjugation, or disrupted bile flow. Together these tests provide insight into the type and severity of liver disease.
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Liver Enzymology
1. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry)
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VMP 978
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Disease vs. failure
o Failure usually results from some type of disease
o Recognized by failure to clear blood of substances normally eliminated, and failure
to synthesize substances normally produced
o 70-80% of functional hepatic mass must be lost before liver failure occurs
Tests
o Hepatocyte injury (“leakage”)
ALT, AST, SDH, GLDH
o Cholestasis (“induction”)
ALP, GGT
o Liver function
T. bili, bile acids, ammonia, BSP, ICG, ALB, GLOB, GLUC, BUN, CHOL,
coagulation factors
Leakage vs. induction
o Leakage
Enzymes is in cytosol and/or organelles
Damage to cell membrane/injury to organelles
Sublethal or lethal
No enzyme production needed = increases are seen in hours
o Induction
Enzyme is attached to cell membrane
Stimulus = increased enzyme release from cells = increased enzyme activity
in serum
Need for enzyme production = increases typically seen in days
o In diseases characterized primarily by hepatocyte injury, activities of leakage
enzymes tend to increased relatively more than those of induced enzymes
o In diseases characterized primarily by cholestasis, activities of induced enzymes
tend to be increased relatively more than those of leakage enzymes
o Many liver diseases, especially as they become more chronic, can result in both
hepatocyte injury and cholestasis differentiation of leakage vs. induction
enzymes and their relative increases does not always yield useful information
Hepatocyte injury (“leakage enzymes”)
o Alanine aminotransferase (ALT)
Also called serum glutamic pyruvic transaminase (SGPT)
Free in cytoplasm, highest concentrations in hepatocytes of dogs/cats
Very liver specific in these species, but can see increases with severe
muscle damage
Muscle activity: ~5% of liver activity in skeletal muscle, ~25% in cardiac
muscle
Despite decreased activity in muscle, because total muscle mass is
much greater than liver mass, this can be a significant potential
source of ALT leakage
Increases in ALT usually d/t hepatocyte death or sublethal injury,
but necrosis or sublethal damage to muscle cells should also be
considered look for other indicators of muscle damage =
creatinine kinase (CK)
Increased activity can be d/t hypoxia, metabolic alteration resulting in
hepatocyte lipid accumulation (hepatic lipidosis), bacterial toxins,
2. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry)
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inflammation (hepatitis), hepatic neoplasia (primary or metastatic), many
toxic chemicals and drugs
Also increased [blood glucocorticoid] from tx with GCs or
increased endogenous GC synthesis secondary to HAC
o Magnitude of increase associated with GCs is not necessarily
indicative of degree of hepatopathy
Anticonvulsants can also cause increased ALT related to either
hepatocyte injury and/or increased enzyme production
Acutely, serum activity of ALT is proportional to number of injured cells, but
magnitude of ALT activity is not indicative of cause of injury or type of
damage (sublethal vs. necrosis) to hepatocytes next slide
Serum ALT activity increases approximately 12 hours after an injury to
hepatocytes, peaks approximately 1-2 days after a single acute injury
Can be increased during recovery from an injury as a result of active
hepatocyte regeneration
In some cases of severe liver disease, markedly decreased hepatic mass =
may have too few remaining hepatocytes to result in a marked increase in
ALT, even if cells are injured and leaking ALT (see low numbers normally,
leakage may make higher, but remain WNL)
Chronic disease degree of active hepatocyte injury may be mild =
hepatocytes that remain don’t leak a large amount of ALT
Half-life: in dogs, is uncertain (ranges from a few to 60 hours); also
uncertain in cats, but thought to be shorter than that in dogs
Horses/ruminants: hepatocyte [ALT] is too low to be useful for detection of
liver disease
Moderate [ALT] in muscle = ALT increases in these species more
likely to be d/t muscle injury/disease
ALT rarely measured in these species = muscle-specific enzymes
(CK) used more often for detection of muscle injury/disease
o Aspartate aminotransferase (AST)
Previously called serum glutamic oxaloacetic transaminase (SGOT)
Highest concentrations in hepatocytes and both cardiac and skeletal muscle
cells of all species
NOT liver specific
Found in cytoplasm, but most is associated with mitochondrial
membranes
Increased serum activity related to hepatocyte death, sublethal
hepatocyte injury, + muscle cell death, sublethal muscle cell injury
ALT often only enzyme used to detect liver injury in dogs/cats d/t its
specificity, but AST can be increased for same reasons as ALT
Magnitude of increase usually less than ALT
Less specific than ALT, but more sensitive for certain types of
hepatocyte injury in dogs/cats
o Dogs: corticosteroids generally don’t result in increased
AST unless they result in steroid hepatophy
o Cats: AST often mildly increased with normal ALT
pyogranulomatous hepatitis secondary to FIP
3. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry)
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VMP 978
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As with ALT, because AST is present in liver and muscle, should use
muscle-specific enzymes (CK) to detect muscle injury vs. hepatocyte
injury
Horses/ruminants: AST not as liver specific as other enzymes (SDH, GLDH),
but more widely available for assay
Enzyme of choice for routine detection of hepatocyte injury in these
species
Increased can be seen for same reasons as increased ALT
Major problem is that increase can be seen with muscle injury as
well = check muscle-specific enzymes (CK) when assaying AST
o Increased AST with normal CK suggests that AST is coming
from liver and that hepatocyte injury has occurred
o Half-life of CK shorter than AST= both may have increased
because of muscle injury, but CK may have returned to
normal at time of assay, while AST remains elevated d/t
longer half-life
Can assay SDH, GLDH if truly suspicious of liver
disease in these species, but may have to send
out/may be more expensive
Activity may be normal/slightly increased with significant liver disease
(chronic/low-grade ± markedly decreased hepatic mass)
Half-life dog: 5 hours, cats: 1-2 hours, horses: 50 hours
o Sorbitol dehydrogenase (SDH)
High concentrations in hepatocytes of dogs, cats, horses, ruminants
Low concentration in other tissues = liver-specific
Free in cytoplasm
Increased activity suggests hepatocyte death or sublethal injury
Not superior to ALT in detecting hepatocyte injury in dogs/cats; not
commonly use in these species
Much more specific than AST for detecting hepatocyte injury in
horses/cattle
Half-life very short (<2 days) after acute injury serum activities may
return to normal within 4-5 days
Relatively stablein vitro (cattle/horses)
5 hours at room temp (in serum), up to 48 hours (72 hours in cattle)
when frozen
Keep this in mind when sending out assays ID a lab ahead of time
that can process sample before it becomes unstable
o Glutamate dehydrogenase (GLDH)
High concentration in livers of dogs, cats, horses, ruminants
Low concentrations in other tissues = liver-specific
Free in cytoplasm
Increase = hepatocyte death or sublethal hepatocyte injury
More stable in vitro than SDH, but still unstable compared to most other
diagnostic enzymes
Assay is difficult, not widely available
ALT superior to GLDH for detecting hepatocellular injury in
dogs/cats
4. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry)
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Horses/ruminants: useful because is more liver-specific than AST
and has better storage stability than SDH
Sensitive indicator of acute hepatocyte damage in ruminants, but is not very
sensitive for more chronic liver diseases
Cholestasis (“induction enzymes”)
o Alkaline phosphatase (ALP)
Synthesized by liver, osteoblasts, intestinal epithelium, renal epithelium, and
placenta most from liver
Half-life of intestinal, renal, and placental-origin ~6 minutes (dogs);
intestinal ~2 minutes (cats)
When increased, should consider increased osteoblastic activity,
cholestasis, drug induction (dogs), other chronic diseases (i.e.,
neoplasia)
Bone origin: increases are usually mild and in young, growing animals
Use age-specific intervals, if possible
In older animals, can see increases with osteosarcoma and other
bone neoplasms (primary and metastatic), but is inconsistent
Bone healing = localized increase in osteoblastic activity = mild,
sometimes no, detectable increase in ALP
Hyperparathyroidism = mild increase may be detected d/t increased
bone turnover
Liver origin
Marked cholestasis in dog, more varied in other species
o Increased intrabiliary pressure induces increased
hepatocellular (and possibly bile duct epithelial) ALP
production
o Sequestration of bile in biliary system causes solubilization
of ALP molecules attached to cell membranes increased
release of these into blood
o Half-life of cholestasis-induced ALP (“liver ALP”, LALP) ~72
hours in dogs, ~6 hours in cats
o May also see concurrent increase in bilirubin, bile acids
ALP often increases before bilirubin; can also see increased
urinary bilirubin
Causes of cholestasis (and therefore increased ALP) include lesions
involving the intra-/extra-hepatic biliary system (most common),
but also any hepatic disease resulting in significant hepatocellular
swelling can obstruct small bile canaliculi and therefore induce
increased ALP production and release
o Lipidosis, inflammation of hepatic parenchyma
Drug-induced
Best documented in dogs (glucocorticoids exogenous and
endogenous CiALP (corticosteroid induced ALP))
o Can be distinguished from ALP of liver origin, but
significance of this is uncertain, as GCs can cause an increase
in LALP as well as CiALP
5. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry)
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o Chronic diseases (including chronic hepatobiliary disease)
cause long-term stress = increases in LALP as a result of
disease, but also CiALP as a result of stress
o Perform other tests (bilirubin, bile acids) to detect
hepatobiliary origin vs. corticosteroid-induced
Concurrent presence of hyperbilirubinemia is
strongly suggestive of cholestatic cause of increased
ALP
Secondary to neoplasms
Bone origin = d/t osteoblastic activity
Primary/metastatic liver or biliary tree neoplasia = d/t cholestasis
Pituitary/adrenal glands = d/t increased glucocorticoid production
Mechanisms uncertain for others, but have been associated with
increased ALP mammary adenocarcinoma, squamous cell
carcinoma, hemangiosarcoma
Neoplasia + subclinical liver disease should always be considered as
causes of increased ALP in older animals with an unexplained
increase in ALP
Half-life is short in cats (6 hours) = mild increases in ALP are more
significant in cats than other species
GGT recommended for evaluation of cholestatic disease in cats
May see moderate increase in ALP with hyperthyroidism (both bone
and liver isoenzyme) cause is unclear; may be d/t effects of
thyroxine on liver and bone
In horses: increases not well-documented, but most that have been detected
have been associated with cholestasis or osteoblastic activity
Wide reference intervals = reduced sensitivity for detection of liver
disease in horses
In ruminants: increases most commonly from cholestasis or increased
osteoblastic activity (i.e., young, growing animals or nutritional secondary
hyperparathyroidism)
Wide reference intervals = reduced sensitivity for detection of liver
disease
o γ-Glutamyltransferase (GGT)
Induced enzyme, but increases can be seen with acute hepatic injury
(possibly d/t release of membrane fragments to which GGT is attached)
Synthesized by most body tissues, highest concentration in
pancreas/kidneys; lower in hepatocytes, bile duct epithelium, intestinal
mucosa and high concentrations in mammary glands of cattle, sheep, and
dogs
Most of the serum GGT originates from the liver release from renal
epithelial cells = increased urinary GGT activity (not serum); when released
from pancreatic cells, passed out with pancreatic secretions rather than into
the bloodstream
Increases in dogs associated with cholestasis and glucocorticoids
Cholestasis = increased production, solubilization of GGT attached to
cell membranes (as a result of detergent action of bile acids that are
not passing to intestines at a normal rate)
6. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry)
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o Increases at approximately the same rate as ALP
Detection of liver disease
More specific, less sensitive than ALP in dogs
More sensitive, less specific than ALP in cats
Advised to measure both GGT and ALP at the same time to detect
hepatobiliary disease
Dogs: GC-induced appears to be associated with increased enzyme
production by the liver; parallels increase in ALP
Can see mild increases in animals that are being treated with
anticonvulsants marked increases in these animals may be
indicative of idiosyncratic reaction resulting in cholestatic liver
disease with life-threatening implications
Horses/ruminants: narrower reference interval = superior to ALP for
detection of cholestasis
High serum GGT activity in colostrum of cattle and sheep can see
extremely high activities in young calves/lambs that have consumed
colostrum (can be more than 200-fold the upper limit of adult
reference interval during first 3 days of life)
Tests of liver function
o Bilirubin
Is derived from porphyrin-containing compounds, mainly RBCs released
from macrophages, attaches to protein, and is transported to the liver
Passage through hepatocyte membrane facilitated by carrier
saturation of this mechanism does not occur under normal
conditions
Attaches to binding protein (ligandin) to prevent excretion back to
bloodstream once in hepatocyte, then conjugated
Most conjugated bilirubin is secreted into bile canaliculi and
excreted in bile
o This form is not protein-bound and is more soluble than
protein-bound unconjugated form
o Small amount of conjugated bilirubin passes back into blood
from hepatocytes, and if remains unbound to protein, is
excreted through glomerular filtration
o Conjugated secreted into bile canaliculi, passes with bile into
SI and is converted to urobilinogen (90% excreted as
stertobilinogen in feces, other 10% is reabsorbed and either
re-enters hepatocytes or is excreted in urine)
Increased bilirubin can result from increased Hb production (increased RBC
destruction), decreased uptake/conjugation by hepatocytes, or disruption of
bile flow
Increased Hb production usually from RBC destruction
extravascular hemolysis
o Bilirbuin overwhelms carrier mechanism, cannot enter
hepatocytes and “backs up” to result in increased serum
bilirubin
Decreased uptake/conjugation result of decreased delivery of
bilirubin to hepatocytes secondary to decreased hepatic blood flow,
7. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry)
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marked decrease in hepatocyte numbers because of acute/chronic
hepatocyte destruction, or defects in either bilirubin uptake or
conjugation by hepatocytes
Disruption of bile flow usually from blockage (partial or complete) in
biliary system
o Cholestasis, accumulation of bile in biliary system (biliary
inspissation)
o Cholestasis most often associated with inflammation or
neoplasia in biliary system, but can also be secondary to
calculi in biliary system
o Can also be caused by diseases that affect parenchyma rather
than biliary system (lipidosis, parenchymal inflammation)
hepatocyte swelling blocks small bile canaliculi in liver,
prevents normal flow of bile, or secondary to blockage of
upper small intestine
If obstruction is the cause, ALP/GGT are more
sensitive indicators than bilirubin, as they will
increase more quickly
Urinary concentration of bilirubin may also be more
sensitive (cholestasis, bile leakage), especially in
species with a low renal threshold
Species differences
Fasting hyperbilirubinemia d/t decreased food intake (anorexia,
starvation) can be seen and is most marked in horses
o Mechanisms include competition with FFAs for ligandin-
binding sites = more unconjugated bilirubin in serum
o Other mechanisms include decreased hepatic blood flow,
decreased affinity of hepatocyte membrane carriers for
bilirubin molecules, competition for hepatocyte bilirubin
uptake by substances other than FFAs that accumulate
during fasting
Dogs: low renal threshold for bilirubin = trace bilirubin normal in dog urine
Ruminants: concentrations not consistently increased in animals with liver
disease significant hyperbilirubinemia most often results from hemolysis
o Bile acids
Synthesized in hepatocytes from cholesterol, then conjugated to amino acids
(increases water solubility), then secreted into biliary system
In animals with gall bladders, are stored and concentrated there
secreted into SI at the time of a meal
In those without, are continuously secreted into intestinal tract
Emulsification of fat promote digestion and absorption of fat and fat-
soluble vitamins (ADEK)
Most BAs reabsorbed into blood from ileum, and cleared via portal
circulation (most on first-pass) = should normally see only a slight
postprandial increase
Those cleared by hepatocytes are secreted into biliary system and
recirculate this occurs several times after a meal
Causes of increased [BA]
8. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry)
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VMP 978
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Deviation of portal circulation (PSS, severe cirrhosis): blood shunted
from hepatocytes = less/no first-pass clearing of BAs
Decreased intrinsic hepatocyte uptake (hepatitis, necrosis, GC
hepatopathy); in some diseases, relates to decreased functional
hepatic mass
Decreased BA excretion via biliary system: subsequent regurgitation
into systemic circulation most often from cholestasis (cholangitis,
bile duct blockage, intestinal obstruction, neoplasia), but can also be
from leakage from bile duct or gall bladder
BAs stable at room temperature for several days, and assays are widely
available NOTE: hemolysis can result in falsely decreased [BA], lipemia
can result in falsely elevated [BA]
When testing, fast for 12 hours, take sample, then feed a fatty diet to
stimulate contraction of gall bladder; postprandial sample is taken 2 hours
after meal
Fasting >20umol/L and postprandial >25umol/L are very specific
for liver disease in dogs and cats
Single sample taken for horses, ruminants, and llamas
Tend to have a wider reference interval
Increased [BA] is suggestive of hepatic disease, but results should be
correlated with other laboratory findings and clinical signs
o Ammonia
Produced in the digestive tract, absorbed from intestine into blood, carried
by portal circulation to liver, then removed
Alterations in hepatic blood flow or markedly decreased numbers of
functional hepatocytes result in increased [blood ammonia]
Measured using plasma, but is very unstable after collection
Measurement is useful, but [BA] is more sensitive and easier to
perform
Increased [plasma ammonia] most commonly seen in animals with PSS
(congenital or secondary to severe cirrhosis), but results are not sensitive
for diagnosis of these disorders
Can also see increases with loss of 60% or more of hepatic functional
mass
Tolerance is only performed in animals suspected of PSS but high baseline
concentration not present (if performed in animals with high baseline
concentration, could result in markedly increased [blood ammonia] =
adverse clinical effects)
o Bromosulfophthalein excretion (BSP)
Dye is administered IV, circulates bound to protein (primarily ALB), and is
removed from blood by hepatocytes
In hepatocytes, is conjugated and then excreted in bile
Useful for assaying liver functions in animals, but has caused occasional
anaphylactic reactions in humans = no longer commercially available
[BA] is more specific and sensitive, and easier to perform
o Indocyanine Green Excretion (ICG)
Dye is administered IV, circulates bound to protein (ALB, B-lipoprotein),
removed from blood by hepatocytes, excreted unconjugated in bile
9. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry)
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Commercially available, but requires several timed blood collections after
injection = labor-intensive, more complicated compared to [BA]
No significant advantages compared to [BA]
o Albumin (ALB)
Synthesized by liver
Increases are always affected by extrahepatic factors (mainly dehydration)
Decreases due to hepatic factors not seen until 60-80% hepatic function is
lost
Species differences in hypoalbuminemia accompanying liver disease
Very common in dogs with CLD (>60% have hypoalbuminemia), but
not as common in horses with CLD (~20% have hypoalbuminemia)
o Globulins (GLOB)
Most globulins functioning in the immune system are synthesized in
lymphoid tissue, but several other types are synthesized in the liver
Hepatic failure can result in decreased synthesis = decreased serum
concentration
Usually does not decrease as much as albumin
A:G commonly decreases because of hepatic failure
Concentration may increase with chronic liver disease (suspected to be d/t
decreased clearance of foreign proteins by Kupffer cells = come into contact
with immune system in other parts of the body = immune response =
hyperglobulinemia)
Frequently, beta and gamma globulin concentrations increase, and
may see bridging between these two fractions on an
electrophoretogram
o Especially well-documented in horses (more than 50% of
those with CLD also have increased globulin concentrations)
o Glucose (GLUC)
Glucose that has been absorbed by SI in transported to liver via portal
circulation, enters hepatocytes, converted to glycogen
Synthesized via gluconeogenesis, stored glucose released via glycogenolysis
Concentration varies in animals with hepatic failure
Increased because of decreased hepatic glucose uptake = prolonged
postprandial hyperglycemia
Decreased because of reduced hepatocytic gluconeogenesis or
glycogenolysis
o Urea (BUN)
Synthesized in hepatocytes from ammonia
In liver failure, decreased hepatocyte numbers = decreased conversion from
ammonia to urea = increased [blood ammonia], decreased [BUN]
o Cholesterol (CHOL)
Bile is a major route of cholesterol excretion from the body
Interference with bile flow (cholestasis) can result in increased
[serum cholesterol] = hypercholesterolemia
Liver is a major site of synthesis
Decreased synthesis can lead to hypocholesterolemia
Levels vary with type of liver disease decreased synthesis vs. cholestasis
o Coagulation Factors
10. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry)
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Liver synthesizes most of the coagulation factors (1, 2, 5, 9, 10)
Blockage of bile flow can result in decreased absorption of fat-soluble
vitamins (i.e., K), and decreased production of vitamin K-dependent
coagulation factors (2, 7, 9, 10)
Liver failure: reduced synthesis of these factors can prolong the one-stage
prothrombin time and activated partial thromboplastin time
Prolonged if concentration of any factor involved in the test
decreases to less than 30% of normal
Coagulation disorders are common in dogs with liver failure, but rare in
large animals with liver failure