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Quick Compendium of CP: Chemistry

Quick Compendium of CP: Chemistry

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Quick Compendium of CP: Chemistry

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  1. Quick Compendium of CP: Chemistry Enzymes, Serum Proteins, Acid-Base and Electrolytes ZW 7/7/08

  2. Enzymes: Basics • Michaelis-Menton kinetics: • The rate of enzyme activity varies linearly with substrate concentration up to the point that the enzyme is fully saturated with substrate • At this point, enzyme working as fast as it can (Vmax) • Rate of reaction at this point varies only with enzyme concentration

  3. Michaelis-Menton Kinetics

  4. Enzymes • Can measure enzyme concentration by: • Using excess of substrate • Or, measure whether a reaction has taken place • Measure reaction products • NAD to NADH to NAD (NADH absorbs light at 340nm, NAD does not)

  5. Coupled Enzyme Assay (NAD/NADH) • Aspartate (Asp) + a-ketoglutarate  oxaloacetate (OAA) + glutamate • Does not utilize NADH • Aspartate (Asp) + a-ketoglutarate  oxaloacetate (OAA) + glutamate + NADH malate + NAD • Add excess NADH and aKG, along with catalysts AST and MD • The disappearance of NADH (absorbance at 340nm) can be used as a reflection of AST (enzyme at first arrow)

  6. Measurement of Enzyme Antigen • The quantity of enzyme determined by immunoassay corresponds to the enzyme ACTIVITY • Discordance between concentration and ACTIVITY usually takes the form of the immuno assay overestimating the activity • May be due to serum enzyme inhibitors • Deficiency in necessary cofactor • Defective enzyme • Proteolytically inactivated enzymes

  7. Cofactors, Coenzymes • Cofactors: • Substances that bind to an enzyme and enhance activity • Include inorgantic cofactors like zinc, calcium, magnesium, iron • Organic: also called coenzymes • Coenzymes: • Organic cofactors, include NAD, protein S, pyridoxine (vit B6)

  8. Macroenzymes • Ordinary enzymes bound to antibodies • Has 2 effects: • Makes it incapable of functioning • Prevents it from being cleared from blood

  9. Competitive INHIBITION

  10. Non-competitive Inhibition

  11. Uncompetitive Inhibition

  12. Enzyme Units • International Unit (IU): • The amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute • Katal • 1 katal= the amount of enzyme that catalyzes the conversion of 1 Mole of substrate per second • 1IU = 16.7 katals

  13. Hepatic Enzymes • Liver transaminases • AST and ALT • AST • Cardiac muscle, liver, skeletal muscle, kidney, brain, lung, pancreas (in descending order) • Found within cytoplasm (20%)and mitochondria (80%) • ALT • MORE SPECIFIC FOR LIVER, confined to liver and kidney • Found entirely within cytoplasm

  14. Hepatic Enzymes • In children • AST activity is slightly higher than ALT • Pattern reverses at age 20 • In adults, AST activity a little lower than ALT • May reverse with old age • Both AST/ALT activities higher in adult males over females, and in African-Americans • Hemolysis raises AST/ALT

  15. Hepatic Enzymes • Intra-individual variation more significant for ALT than AST • Marked diurnal variation (highest in afternoon) and day-to-day variation up to 30% • Both AST/ALT elevated in heparin therapy to around 3X baseline • In renal failure, both significantly lower than in healthy individuals

  16. Hepatic Enzymes • Lactate dehydrogenase (LDH): • Present in numerous tissues, traditionally separated into 5 isoenzymes by electrophoresis • Fastest moving are LD1 and LD2 • Found in heart, RBC, kidney • Slower moving are LD4 and LD5 • LIVER and skeletal muscle • LD3 in lung, spleen, lymphocytes, and pancreas • LD6- “sixth” LD is sometimes seen migrating cathodal to LD5 • PRESENCE THOUGHT TO BE A DIRE FINDING (hepatic insufficiency in setting of cardiovascular collapse)

  17. LDH • Concentrations: • LD2>LD1>LD3>LD4>LD5 • LD 1 elevation (with flipped LD ratio LD1>LD2): • Acute MI • Hemolysis • Renal infarction • Elevated LD4 and LD5: • LIVER DAMAGE or skeletal insult • Elevated LD1 and LD5: • Acute MI with liver congestion • Chronic alcoholism

  18. Alkaline phosphatase • Two types of phosphatases: • Alkaline (optimum pH is 9): • Bone, bile ducts, intestine, placenta • Separate reference ranges for women and children • Acid (optimum pH is 5): • Found in prostate, RBC, and bone • RBC acid phosphatase is susceptible to inhibition by 2% formaldehyde and resistance to inhibition by tartrate (this is also seen in hairy cell leukemia)

  19. Alkaline phosphatase • 4 isoenzymes by electrophoresis: • Each displays characteristic degrees of inactivation by heating, urea incubation, and l-phenylalanine • Heating produces significant inactivation of bone alk phos (bone burns), 50% inactivation of biliary alk phos, and NO inactivation of placental alk phos.

  20. Alkaline phosphatase • Biliary alk phos: • Most sensitive marker of hepatic metastases • Bone alk phos: • Produced by osteoBLASTS and reflects bone reforming activity • Highest levels seen in Paget’s disease of bone • A specific immunoassay for bone alk phos available

  21. Regan Isoenzyme • Observed in about 5% of individuals with carcinoma • Appears identical to placental alkaline phosphatase

  22. Intestinal Alk Phos • Elevation • Can be factitious in non-fasting individuals, particularly in Lewis positive type B or O pts • Ingesting a meal can elevate alk phos by 30% in 2-12 hours • Repeat fasting alk phos

  23. Alk Phos • Minor elevations are a common clinical problem • Usually higher in men than women • Higher in African-Americans • Threshold of 1.5 times normal limit for further investigation (repeat in 6 months if borderline) • Causes: • Pregnancy, CHF, hyperthyroidism, drugs

  24. Alkaline phosphatase • Sensitive indicator for hepatic metastases • In women, investigation should include assay for anti-mitochondrial antibodies

  25. Gamma-glutamyl transferase (GGT) • GGT: • best test to confirm if elevate alk phos if of biliary tree origin • Found in biliary epithelial cell, particularly those of the small interlobular bile ducts and ductules • Exquisitely sensitive to biliary injury • Also elevated in: • Steatosis, diabetes, hyperthyroidism, RA, acute MI, COPD

  26. GGT • Present within the smooth endoplasmic reticulum of hepatocytes • Whenever there is induction due to excess toxin, GGT levels increase • This includes warfarin, barbiturates, dilantin, valproic acid, methotrexate, EtOH • 2-3X normal limit in heavy drinkers • Returns to normal after 3 weeks abstinence and can be followed as marker for alcohol consumption

  27. 5’ Nucleotidase • Main source is biliary epithelium • Levels highest in cholestatic conditions • Another test to confirm if elevated alk phos is due to hepatobiliary disease • Low sensitivity, best as confirmatory test, utility less than GGT

  28. Ammonia • Hyperammonemia nearly always due to liver failure • In children, it should raise the suspicion for an INBORN ERROR IN METABOLISM • Sources of ammonia: • Skeletal muscle and gut • Bacteria in GI tract produce ammonia • Normally functioning liver removes this ammonia and discards it in the form of urea which is excreted in urine

  29. Ammonia • Blood ammonia can become disastrously high when: • Too much collateral circulation • Excess protein in gut (excess hemoglobin from variceal bleed) • SIGNIFICANT HEPATOCYTE DISFUNCTION • In cirrhotic patients, these conditions are often met, and neurotoxicity can result

  30. AMMONIA • Measurement requires a FRESH specimen which has been CHILLED during transport and has undergone NO hemolysis • Smoking patients must abstain for several hours before draw • Anyone care to comment on the current state of the ammonia level as it can or cannot be ordered?

  31. Bilirubin • Unconjugated (indirect) bilirubin: • Water-insoluble form produced by breakdown of heme • Taken to liver tightly bound to albumin where it under goes glucuronidation to produce water-soluble (as in bile) conjugated (direct) bilirubin • Conjugated bili excreted in bile where intestinal bacteria convert to urobilinogen

  32. Bilirubin • Urobilinogen ends up in feces, some of which is reabsorbed and excreted in urine • Some urobilinogen is converted by colonic bacteria into brown pigments (complete biliary obstruction leads to yellow-white stool- the Silver Stool of Thompson)

  33. Bilirubin • Unconjugated bilirubin, even when it is quite high, does NOT appear in urine • Thus, bilirubinuria indicates CONJUGATED hyperbilirubinemia • 2 test methods • Diazo-colorimetric methods: • Rely on formation of colored dye through reaction of bili with diazo compound • Without the addition of an accelerator (alcohol), only conjugated bilirubin is measured • Addition of accelerators measures combined unconj and conjugated (total) bilirubin • Direct spectrophotometry • Bilirubin concentration measured by absorbance (455nm)

  34. Causes of Hyperbilirubinemia • Unconjugated: • Hemolysis (extravascular) • Blood shunting (cirrhosis) • Right heart failure • Gilbert syndrome • Drugs: rifampin • Crigler-Najjar syndrome • Hypothyroidism

  35. Causes of Hyperbilirubinemia • Conjugated: • Dubin-Johnson syndrome • Hepatitis • Endotoxin (sepsis) • Pregnancy (estrogen) • Drugs: estrogen, cyclosporine • Mechanical obstruction: • PBC, PSC, tumor, stricture, stone

  36. Additional Hepatic Function Tests • PT: • Factor VII has half life of 12 hours (ON RISE EXAM) • Sensitive marker for impaired hepatic synthetic function • Impaired bile secretion can lead to Vit K deficiency (bile salts required for absorption) • How do you distinguish between a prolonged PT because of cholestasis/impaired Vit K absorption and hepatocyte injury? • Add parental vit K • Gammaglobulins • Serum gammaglobulins elevated in liver injury, especially autoimmune

  37. Neonatal Jaundice • Most cases of neonatal jaundice are entirely benign (“physiologic jaundice”) • Hepatic enzymes not yet at full capacity leading to build-up of unconjugated bilirubin • Usually noted between days 2-3 of neonatal life • Usually peaks at 4-5 days; rarely exceeds 5-6 mg/dL

  38. Severe hyperbilirubinemia in neonates • Most common causes: • Hemolytic disease of the newborn (HDN) • Sepsis • Poorly developed blood-brain barrier causes unconjugated bili to pass to CNS and cause damage (kernicterus)

  39. When to worry about neonatal jaundice? • Appearance in first 24 hours of life • Rising bili beyond 1 week • Persistance of jaundice past 10 days • Total bili that exceeds 12 mg/dL • Single-day increase of >5 mg/dL • Conjugated bili (direct) that exceeds 2 mg/dL

  40. Therapy for neonatal jaundice • Phototherapy: • Consider when bili exceeds 10 mg/dL before 12 hours of age; 12 mg/dL before 18 hours of age; 14 mg/dL before 24 hours of age • Phototherapy converts unconj bili to a molecule that can be excreted WITHOUT conj • Not useful for conj hyperbili • Exchange transfusion: • When bili exceeds 20 mg/dL

  41. DDX of neonate hyperbili • Jaundice in 1st 24 hours: • Erythroblastosis fetalis • Concealed hemorrhage • Sepsis • TORCH infection • Jaundice between 3rd and 7th day: • Bacterial sepsis (usually UTI origin) • Arising after 1st week: • Breast milk jaundice, sepsis, extrahepatic biliary atresia, cystic fibrosis, congenital paucity of bile ducts (Alagille syndrome), neonatal hepatitis, glactosemia, inherited hemolytic anemia (PK def, hered. Spherocytosis, G6PD def)

  42. Lab Eval of Acute Liver Injury • May be symptomatic, Jaundice, Elevated transaminases • May be due to viral hepatitis (HAV, HBV, HCV), autoimmune hep, toxin, drug, ischemia, or Wilson disease • Labs: • Hepatitis serologies, ANA, ceruloplasmin, clinical history (new drugs usually cause damage within 4 months of starting)

  43. Acute liver injury labs • Acute viral hepatitis due to HAV, HBV most often leads to complete recovery • Acute HCV goes to chronic HCV in >80% of cases • Serologic testing for HAV, HBV are very dependable for diagnosing acute infx (IgM anti-HAV, IgM anti-HBc, HBsAg) • Anti-HCV test only about 60% sensitive for acute infx • HCV RNA testing 90-95% sensitive

  44. Transaminases • Acute hepatic injury due to ischemic or toxic injury produce PROFOUND elevations in transaminases- often >100X upper limit of normal (RARE in acute hepatitis) • AST > 3,000 U/L = toxin in 90% of cases • AST 10X upper limit of normal in acute viral hepatitis, but reaches this level RARELY in alcoholic hepatitis • AST:ALT ratio is over 2 in 80% of pts with toxic, ischemic, and EtOH hepatitis (<1 in viral hep) • Amount of transam elevation poorly correlates with LEVEL of injury

  45. PT/BILIRUBIN • PT (protime)- • Probably the best indicator of prognosis in acute hepatic injury • >4.0 secs indicates severe injury/unfav prog • BILI • Jaundice in 70% of pts with EtOH, HAV • Jaundice in <20% of pts with HBV, HCV • Jaundice rare in kids with acute viral hep, rare in toxic or ischemic injury • >15 mg/dL indicates severe liver injury, bad prognosis

  46. Pancreatic Enzymes • AMYLASE: • Serum amylase = salivary and pancreatic isoenzymes • On electrophoresis 6 bands result • 1st three are salivary • Slower 3 are pancreatic • Can be separated by inhibition as well • Salivary amylase sensitive to inhibition by wheat germ lectin (treticum vulgaris) • Assays based on monoclonal Abs directed against specific isoenzymes are very accurate

  47. Serum Amylase • Rises within 2-24 hours of onset of acute pancreatitis • Returns to normal in 2-3 days • Higher levels don’t correlate with severity • Higher levels are more specific for acute pancreatitis • Persistance in elevation suggests complication like pseudocyst

  48. Urine amylase • Nearly all pts have concomitant increase in urine amylase • Amylase primarily cleared by glomeruli • Renal insufficiency = spurious amylase elevation • Fractional excretion of compound (x) = FEx

  49. Amylase • Sensitivity of serum amylase for acute pancreatitis is 90-98% • Specificity is only around 70-75% • Specificity of urine amylase and FEamylase is higher

  50. Additional causes of increased amylase • Diabetic ketoacidosis, peptic ulcer dz, acute cholecystitis, ectopic pregnancy, salpingitis, bowel ischemia, intestinal obstruction, macroamylasemia, and renal insufficiency, opioid analgesics (contraction of sphincter of oddi)