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Liver Enzymology


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Notes to accompany liver enzymology presentation for senior clinical pathology rotation (VMP 978)

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Liver Enzymology

  1. 1. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry) Omega Cantrell VMP 978 1 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. 2. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry) Omega Cantrell VMP 978 2 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. 3. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry) Omega Cantrell VMP 978 3 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. 4. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry) Omega Cantrell VMP 978 4 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. 5. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry) Omega Cantrell VMP 978 5 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. 6. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry) Omega Cantrell VMP 978 6 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. 7. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry) Omega Cantrell VMP 978 7 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. 8. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry) Omega Cantrell VMP 978 8 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. 9. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry) Omega Cantrell VMP 978 9  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. 10. LIVER ENZYMOLOGY (from Chapter 23 of Thrall’s Veterinary Hematology & Clinical Chemistry) Omega Cantrell VMP 978 10  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