Liver Enzymology
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Presentation for senior clinical pathology rotation (VMP 978)
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Liver Enzymology
- 2. Disease vs. Failure Failure usually results from some type of disease Recognized by failure to clear blood of substances normally eliminated, and failure to synthesize substances normally produced 70-80% of functional hepatic mass must be lost before liver failure occurs
- 3. Tests Hepatocyte injury (“leakage”) ◦ ALT, AST, SDH, GLDH Cholestasis (“induction”) ◦ ALP, GGT Liver function ◦ T. bili, bile acids, ammonia, BSP, ICG, ALB, GLOB, GLUC, BUN, CHOL, coagulation factors
- 4. Leakage vs. Induction Leakage ◦ In cytosol and/or organelles ◦ Damage to cell membrane/injury to organelles Sublethal or lethal ◦ No enzyme production needed = increases are seen in hours Induction ◦ Attached to cell membrane ◦ Stimulus = increased enzyme release from cells = increased enzyme activity in serum ◦ Need for enzyme production = increases typically seen in days
- 5. HEPATOCYTE INJURY (“LEAKAGE”) Alanine aminotransferase (ALT) Aspartate aminotransferase (AST) Sorbitol dehydrogenase (SDH) Glutamate dehydrogenase (GLDH)
- 6. Alanine Aminotransferase (ALT) Highest concentrations in dog/cat hepatocytes ◦ Very liver specific, but may see increases with severe muscle damage Increased activity from any disease that causes hepatocyte injury ◦ Membrane injury cell death ◦ Many potential causes Degree of elevation ◦ Acute vs. chronic injury
- 7. Lassen, ED. (2006). Laboratory Evaluation of the Liver. In: Thrall, MA, et al. Veterinary Hematology and Clinical Chemistry. Ames, IA: Blackwell. 372.
- 8. ALT (cont’d) Activity vs. time ◦ 12 hours 1-2 days ◦ Recovery Severe liver disease ◦ Decreased hepatic mass ◦ Chronic disease Half-life ◦ Dogs vs. cats Horses/ruminants ◦ Hepatic vs. muscle injury
- 9. Aspartate Aminotransferase (AST) Hepatocytes and myocytes all species Less liver specific, but can be more sensitive Horses/ruminants Half-life
- 10. Sorbitol Dehydrogenase (SDH) Mainly in hepatocytes liver-specific! Not superior to ALT in dogs/cats Horses/cattle Half-life Stability (in vitro) less than other enzymes
- 11. Glutamate Dehydrogenase (GLDH) Mainly in hepatocytes liver-specific! ◦ Dogs, cats, horses, ruminants More stable than SDH Assay is difficult, not widely available
- 13. Alkaline Phosphatase (ALP) Many sources, but some have very short half-life Bone origin Liver origin Induced by drugs Secondary to some neoplasms
- 14. γ-Glutamyltransferase (GGT) Highest concentrations in pancreas/kidneys, but most serum GGT from liver More specific, less sensitive than ALP (dogs) More sensitive, less specific than ALP (cats) Horses/ruminants: GGT superior to ALP
- 15. TESTS OF LIVER FUNCTION Substances normally eliminated by the liver Bilirubin, bile acids, CHOL, ammonia Substances normally synthesized by the liver ALB, GLOB, BUN, CHOL, coagulation factors
- 16. Bilirubin Breakdown product of Hb, other porphyrin-containing compounds Metabolized in the liver to conjugated form Causes of increased bilirubin ◦ Increased Hb (increased RBC destruction), decreased uptake/conjugation by hepatocytes, disruption of bile flow Species differences
- 17. Bile Acids Synthesized in hepatocytes from cholesterol, then secreted into biliary system ◦ Recirculates (via liver reuptake and secretion) Causes of increased concentration ◦ Deviation of portal circulation, decreased hepatocyte uptake, decreased excretion via biliary system Measured pre- and post-prandially Only one sample collected in horses, ruminants, and llamas
- 18. Ammonia Produced in digestive tract, absorbed from intestine into blood, removed by liver Increased concentration typically in animals with PSS, but not sensitive for diagnosis ◦ Can also see with loss of >60% loss of functional hepatic mass Tolerance vs. concentration
- 19. Bromosulfophthalein Excretion (BSP) Dye, given IV, removed by hepatocytes, conjugated, excreted in bile Test of hepatic blood flow, ability of hepatocytes to remove/conjugate/excrete, patency/int egrity of biliary system No longer commercially available
- 20. Indocyanine Green Excretion (ICG) Dye, given IV, removed by hepatocytes, excreted in bile (unconjugated) Test assess hepatic blood flow, ability of hepatocytes to remove ICG from blood, patency/integrity of biliary system Commercially available, but complicated
- 21. 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
- 22. Globulins (GLOB) Most synthesized in lymphoid tissue, but some synthesis is performed by liver Decrease not usually as marked as albumin
- 23. Glucose (GLUC) After SI absorption, transported to liver and converted to glycogen Synthesize via gluconeogenesis Release stores via glycogenolysis Concentrations vary in animals with hepatic failure
- 24. Urea (BUN) Synthesized from ammonia ◦ Decreased conversion in liver disease/failure ◦ Causes increased [blood ammonia], decreased [BUN]
- 25. Cholesterol (CHOL) Liver is a major site of synthesis Excreted via bile
- 26. Coagulation Factors Most are synthesized by the liver ◦ I, II, V, IX, X Need for bile ◦ Vitamin K II, VII, IX, X PT/APTT
- 27. Lassen, ED. (2006). Laboratory Evaluation of the Liver. In: Thrall, MA, et al. Veterinary Hematology and Clinical Chemistry. Ames, IA:
- 28. References Lassen, ED. (2006). Laboratory Evaluation of the Liver. In: Thrall, MA, et al. Veterinary Hematology and Clinical Chemistry. Ames, IA: Blackwell. 355-375.
Hinweis der Redaktion
- In diseases characterized primarily by hepatocyte injury, activities of leakage enzymes tend to increased relatively more than those of induced enzymesIn diseases characterized primarily by cholestasis, activities of induced enzymes tend to be increased relatively more than those of leakage enzymesMany 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
- Also called serum glutamic pyruvic transaminase (SGPT)Free in cytoplasm, highest concentrations in hepatocytes of dogs/catsVery liver specific in these species, but can see increases with severe muscle damageMuscle activity: ~5% of liver activity in skeletal muscle, ~25% in cardiac muscleDespite decreased activity in muscle, because total muscle mass is much greater than liver mass, this can be a significant potential source of ALT leakageIncreases 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, inflammation (hepatitis), hepatic neoplasia (primary or metastatic), many toxic chemicals and drugsAlso increased [blood glucocorticoid] from tx with GCs or increased endogenous GC synthesis secondary to HACMagnitude of increase associated with GCs is not necessarily indicative of degree of hepatopathyAnticonvulsants can also cause increased ALT related to either hepatocyte injury and/or increased enzyme productionAcutely, 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 injuryCan be increased during recovery from an injury as a result of active hepatocyte regenerationIn 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 ALTHalf-life: in dogs, is uncertain (ranges from a few to 60 hours); also uncertain in cats, but thought to be shorter than that in dogsHorses/ruminants: hepatocyte [ALT] is too low to be useful for detection of liver diseaseModerate [ALT] in muscle = ALT increases in these species more likely to be d/t muscle injury/diseaseALT rarely measured in these species = muscle-specific enzymes (CK) used more often for detection of muscle injury/disease
- Previously called serum glutamic oxaloacetic transaminase (SGOT)Highest concentrations in hepatocytes and both cardiac and skeletal muscle cells of all speciesNOT liver specificFound in cytoplasm, but most is associated with mitochondrial membranesIncreased serum activity related to hepatocyte death, sublethal hepatocyte injury, + muscle cell death, sublethal muscle cell injuryALT often only enzyme used to detect liver injury in dogs/cats d/t its specificity, but AST can be increased for same reasons as ALTMagnitude of increase usually less than ALTLess specific than ALT, but more sensitive for certain types of hepatocyte injury in dogs/catsDogs: corticosteroids generally don’t result in increased AST unless they result in steroid hepatophyCats: AST often mildly increased with normal ALT granulomatous hepatitis secondary to FIPAs with ALT, because AST is present in liver and muscle, should use muscle-specific enzymes (CK) to detect muscle injury vs. hepatocyte injuryHorses/ruminants: AST not as liver specific as other enzymes (SDH, GLDH), but more widely available for assayEnzyme of choice for routine detection of hepatocyte injury in these speciesIncreased can be seen for same reasons as increased ALTMajor problem is that increase can be seen with muscle injury as well = check muscle-specific enzymes (CK) when assaying ASTIncreased AST with normal CK suggests that AST is coming from liver and that hepatocyte injury has occurredHalf-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-lifeCan assay SDH, GLDH if truly suspicious of liver disease in these species, but may have to send out/may be more expensiveActivity 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
- High concentrations in hepatocytes of dogs, cats, horses, ruminantsLow concentration in other tissues = liver-specificFree in cytoplasmIncreased activity suggests hepatocyte death or sublethal injuryNot superior to ALT in detecting hepatocyte injury in dogs/cats; not commonly use in these speciesMuch more specific than AST for detecting hepatocyte injury in horses/cattleHalf-life very short (<2 days) after acute injury serum activities may return to normal within 4-5 daysRelatively stable in vitro (cattle/horses)5 hours at room temp (in serum), up to 48 hours (72 hours in cattle) when frozenKeep this in mind when sending out assays ID a lab ahead of time that can process sample before it becomes unstable
- High concentration in livers of dogs, cats, horses, ruminantsLow concentrations in other tissues = liver-specificFree in cytoplasmIncrease = hepatocyte death or sublethal hepatocyte injuryMore stable in vitro than SDH, but still unstable compared to most other diagnostic enzymesAssay is difficult, not widely availableALT superior to GLDH for detecting hepatocellular injury in dogs/catsHorses/ruminants: useful because is more liver-specific than AST and has better storage stability than SDHSensitive indicator of acute hepatocyte damage in ruminants, but is not very sensitive for more chronic liver diseases
- Synthesized by liver, osteoblasts, intestinal epithelium, renal epithelium, and placenta most from liverHalf-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 animalsUse age-specific intervals, if possibleIn older animals, can see increases with osteosarcoma and other bone neoplasms (primary and metastatic), but is inconsistentBone healing = localized increase in osteoblastic activity = mild, sometimes no, detectable increase in ALPHyperparathyroidism = mild increase may be detected d/t increased bone turnoverLiver originMarked cholestasis in dog, more varied in other speciesIncreased intrabiliary pressure induces increased hepatocellular (and possibly bile duct epithelial) ALP productionSequestration of bile in biliary system causes solubilization of ALP molecules attached to cell membranes increased release of these into bloodHalf-life of cholestasis-induced ALP (“liver ALP”, LALP) ~72 hours in dogs, ~6 hours in catsMay also see concurrent increase in bilirubin, bile acids ALP often increases before bilirubin; can also see increased urinary bilirubinCauses 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 releaseLipidosis, inflammation of hepatic parenchymaDrug-inducedBest documented in dogs (glucocorticoids exogenous and endogenous CiALP (corticosteroid induced ALP))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 CiALPChronic diseases (including chronic hepatobiliary disease) cause long-term stress = increases in LALP as a result of disease, but also CiALP as a result of stressPerform other tests (bilirubin, bile acids) to detect hepatobiliary origin vs. corticosteroid-inducedConcurrent presence of hyperbilirubinemia is strongly suggestive of cholestatic cause of increased ALPSecondary to neoplasmsBone origin = d/t osteoblastic activityPrimary/metastatic liver or biliary tree neoplasia = d/t cholestasis Pituitary/adrenal glands = d/t increased glucocorticoid productionMechanisms uncertain for others, but have been associated with increased ALP mammary adenocarcinoma, squamous cell carcinoma, hemangiosarcomaNeoplasia + subclinical liver disease should always be considered as causes of increased ALP in older animals with an unexplained increase in ALPHalf-life is short in cats (6 hours) = mild increases in ALP are more significant in cats than other speciesGGT recommended for evaluation of cholestatic disease in catsMay 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 boneIn horses: increases not well-documented, but most that have been detected have been associated with cholestasis or osteoblastic activityWide reference intervals = reduced sensitivity for detection of liver disease in horsesIn 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
- 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 dogsMost 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 bloodstreamIncreases 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)Increases at approximately the same rate as ALPDetection of liver diseaseMore specific, less sensitive than ALP in dogsMore sensitive, less specific than ALP in catsAdvised to measure both GGT and ALP at the same time to detect hepatobiliary diseaseDogs: GC-induced appears to be associated with increased enzyme production by the liver; parallels increase in ALPCan 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 implicationsHorses/ruminants: narrower reference interval = superior to ALP for detection of cholestasisHigh 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)
- Is derived from porphyrin-containing compounds, mainly RBCs released from macrophages, attaches to protein, and is transported to the liverPassage through hepatocyte membrane facilitated by carrier saturation of this mechanism does not occur under normal conditionsAttaches to binding protein (ligandin) to prevent excretion back to bloodstream once in hepatocyte, then conjugatedMost conjugated bilirubin is secreted into bile canaliculi and excreted in bileThis form is not protein-bound and is more soluble than protein-bound unconjugated formSmall amount of conjugated bilirubin passes back into blood from hepatocytes, and if remains unbound to protein, is excreted through glomerular filtrationConjugated 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 flowIncreased Hb production usually from RBC destruction extravascular hemolysisBilirbuin overwhelms carrier mechanism, cannot enter hepatocytes and “backs up” to result in increased serum bilirubinDecreased uptake/conjugation result of decreased delivery of bilirubin to hepatocytes secondary to decreased hepatic blood flow, marked decrease in hepatocyte numbers because of acute/chronic hepatocyte destruction, or defects in either bilirubin uptake or conjugation by hepatocytesDisruption of bile flow usually from blockage (partial or complete) in biliary systemCholestasis, accumulation of bile in biliary system (biliary inspissation)Cholestasis most often associated with inflammation or neoplasia in biliary system, but can also be secondary to calculi in biliary systemCan 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 intestineIf obstruction is the cause, ALP/GGT are more sensitive indicators than bilirubin, as they will increase more quicklyUrinary concentration of bilirubin may also be more sensitive (cholestasis, bile leakage), especially in species with a low renal thresholdSpecies differencesFasting hyperbilirubinemia d/t decreased food intake (anorexia, starvation) can be seen and is most marked in horsesMechanisms include competition with FFAs for ligandin-binding sites = more unconjugated bilirubin in serumOther 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 fastingDogs: low renal threshold for bilirubin = trace bilirubin normal in dog urineRuminants: concentrations not consistently increased in animals with liver disease significant hyperbilirubinemia most often results from hemolysis
- Synthesized in hepatocytes from cholesterol, then conjugated to amino acids (increases water solubility), then secreted into biliary systemIn animals with gall bladders, are stored and concentrated there secreted into SI at the time of a mealIn those without, are continuously secreted into intestinal tractEmulsification 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 increaseThose cleared by hepatocytes are secreted into biliary system and recirculate this occurs several times after a mealCauses of increased [BA]Deviation of portal circulation (PSS, severe cirrhosis): blood shunted from hepatocytes = less/no first-pass clearing of BAsDecreased intrinsic hepatocyte uptake (hepatitis, necrosis, GC hepatopathy); in some diseases, relates to decreased functional hepatic massDecreased 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 bladderBAs 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 mealFasting >20umol/L and postprandial >25umol/L are very specific for liver disease in dogs and catsSingle sample taken for horses, ruminants, and llamasTend to have a wider reference intervalIncreased [BA] is suggestive of hepatic disease, but results should be correlated with other laboratory findings and clinical signs
- Produced in the digestive tract, absorbed from intestine into blood, carried by portal circulation to liver, then removedAlterations in hepatic blood flow or markedly decreased numbers of functional hepatocytes result in increased [blood ammonia]Measured using plasma, but is very unstable after collectionMeasurement is useful, but [BA] is more sensitive and easier to performIncreased [plasma ammonia] most commonly seen in animals with PSS (congenital or secondary to severe cirrhosis), but results are not sensitive for diagnosis of these disordersCan also see increases with loss of 60% or more of hepatic functional massTolerance 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)
- Dye is administered IV, circulates bound to protein (primarily ALB), and is removed from blood by hepatocytesIn hepatocytes, is conjugated and then excreted in bileUseful 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
- Dye is administered IV, circulates bound to protein (ALB, B-lipoprotein), removed from blood by hepatocytes, excreted unconjugated in bileCommercially available, but requires several timed blood collections after injection = labor-intensive, more complicated compared to [BA]No significant advantages compared to [BA]
- Hypoalbuminemia due to liver disease is not noted until 60-80% of functional hepatic mass has been lostSpecies differences: very common in dogs with CLD (>60% have hypoalbuminemia), but not as common in horses with CLD (~20% have hypoalbuminemia)
- Most globulins functioning in the immune system are synthesized in lymphoid tissue, but several other types are synthesized in the liverHepatic failure can result in decreased synthesis = decreased serum concentrationUsually does not decrease as much as albuminA:G commonly decreases because of hepatic failureConcentration 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 electrophoretogramEspecially well-documented in horses (more than 50% of those with CLD also have increased globulin concentrations)
- Glucose that has been absorbed by SI in transported to liver via portal circulation, enters hepatocytes, converted to glycogenSynthesized via gluconeogenesis, stored glucose released via glycogenolysisConcentration varies in animals with hepatic failureIncreased because of decreased hepatic glucose uptake = prolonged postprandial hyperglycemiaDecreased because of reduced hepatocytic gluconeogenesis or glycogenolysis
- Synthesized in hepatocytes from ammoniaIn liver failure, decreased hepatocyte numbers = decreased conversion from ammonia to urea = increased [blood ammonia], decreased [BUN]
- Bile is a major route of cholesterol excretion from the bodyInterference with bile flow (cholestasis) can result in increased [serum cholesterol] = hypercholesterolemiaLiver is a major site of synthesisDecreased synthesis can lead to hypocholesterolemiaLevels vary with type of liver disease decreased synthesis vs. cholestasis
- 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 timeProlonged if concentration of any factor involved in the test decreases to less than 30% of normalCoagulation disorders are common in dogs with liver failure, but rare in large animals with liver failure