Proteins.docx

Digestion Diet →Stomach: partial digestion (HCl & pepsin) →Duodenum: short peptide, dipeptide, aa (trypsin, chymotrypsin, carboxypeptidase (pancreas) → Ileum & jejunum: aa absorption → immediately synthesized and stored into Protein. ** Excess aa Degraded in the liver Stored as “FAT.” [aa...

Digestion Diet →Stomach: partial digestion (HCl & pepsin) →Duodenum: short peptide, dipeptide, aa (trypsin, chymotrypsin, carboxypeptidase (pancreas) → Ileum & jejunum: aa absorption → immediately synthesized and stored into Protein. ** Excess aa Degraded in the liver Stored as “FAT.” [aa]blood: 35-65 mg/dl Protein turnover rate: 125-220g/day Kidney Filters & reabsorption ~7g/dl (150-300mg/24h) excreted. 1/3 of normally excreted protein is “Tamms Horsfall Protein” “Uromodulin.” Two pathways for protein catabolism Lysosomal pathway: Intra & extracellular proteins Cytosolic pathway: Intracellular proteins Central Catabolic Reaction: Transamination “Transaminase” Except: Lysine, threonine, proline, hydroxyproline Serum Protein Total Protein: Approx 6.5-8.3 g/dl Total Albumin: Approx 3.5-5.5 g/dl Total Protein - Albumin = Globulin A/G ratio 1:1 ~ 1.5:1; 1.5:1 ~ 2.5:1 Total Protein = Albumin + Globulin ↑ Total protein = ↑ Globulin ↓ Total protein = ↓ Albumin Acute Phase Reaction The response to the acute inflammation! Protein that DECREASES in the Acute Phase: Negative Acute Phase Reactant Negative Phase Reactant Protein that INCREASES in the Acute Phase: Acute Phase Reactant Acute Phase Protein Acute Phase Reactant: Involves defense or protective function CRP Amyloid P component (Serum) Mannose binding lectin (MBL) Complements Fibrinogen Prothrombin Factor VIII von Willebrand factor Plasminogen activator inhibitor (PAI-1) α2 macroglobulin Ferritin Ceruloplasmin Haptoglobin α1-antitrypsin (down regulating inflammation) α1-antichymotrypsin Negative Phase Reactant: Due to the aa pools are being shunted to other protein needs (required in the current inflammatory process) Albumin Transferrin Transthyretin (thyroid hormone carrier, prealbumin) Retinol binding protein (retinol (Vit A) carrier protein) Antithrombin Transcortin (also called corticosteroid binding globulin, or serpin 6) Hemoconcentration & Hemodilution Mainly due to the plasma volume change Relative protein concentration changes A/G ratio: no change Low Body H2O → “Hemoconcentration” Excess Body H2O→ “Hemodilution” Total protein abnormality Hypoproteinemia Excretion in urine in renal disease ✴Nephrotic syndrome or Nephrosis Leakage into the GI tract ✴Inflammatory conditions of digestive system (spruce) Loss of blood ✴Open wounds or internal bleeding Decreased Intake ✴Malnutrition/ malabsorption Decreased synthesis ✴Liver disease, Inherited immunodeficiency [defective plasma cells] Increased protein catabolism ✴Burn, Trauma, increased energy demand [pregnancy] Hyperproteinemia NOT common as hypoproteinemia Causes: Dehydration (most common) Increased γ-globulin (myeloma, Waldenstrom’s macroglobulinemia, Chronic infection) Serum Protein Electrophoresis Pre-albumin fraction Prealbumin [transthyretin (TTR) / thyroxine-binding prealbumin (TBPA)] Serves as a transport protein for a small fraction of thyroid hormones, esp. thyroxine Also binds with retinol-binding protein to transport retinol (Vitamin A) ~ 180-450 mg/L (0.1-0.4 g/L) Not seen on typical SPE Clinical Significance: A very sensitive marker of poor protein NUTRITIONAL STATUS. Decreased in hepatic damage, acute phase inflammatory response, & tissue necrosis. Increased in patients receiving steroids, & chronic renal failure Albumin fraction NR: 3.5-5.5 g/dl; MW: 66 kD 50-60% of total protein Extremely Negative Charged In an alkaline buffer, moves rapidly toward Anode. Functions: Maintenance of colloid osmotic pressure (oncotic pressure) of the intravascular fluid Binding of various substances in the blood “negativity” i.e., Unconjugated bilirubin, salicylic acid, FA, Ca2+, Cortisol, & drugs Serves as a nutritional source of amino acids when necessary. Negative Acute Phase Reactant Buffering capacity Pathology Hypoalbuminemia Decreased Production Malnutrition (inadequate source of aa) Liver disease Increased Loss/Use after synthesis GI tract loss via intestinal leakage Loss in renal disease Burns (leakage) Ascites Inflammation/neoplasm Pregnancy (used by baby) Can cause edema (↓ Oncotic pressure → water leakage into tissue) Hyperalbuminemia Relative increase Usually due to dehydration Rarely seen due to other reasons Genetic Origin: Bisalbuminemia Unusual molecular characteristics. Does not appear to be harmful to individual. Analbuminemia Extremely low or absent albumin Measurement(s): Chemical, Electrophoresis, Dye binding α1- Globulin Fraction α1-antitrypsin (APR) ~90% of α1 fraction 0.2-0.4 g/dl **Immunoassay FUNCTION: neutralizes trypsin & trypsin-like enzymes Protease Inhibitor “Neutrophil elastase” Deficiency: severe, degenerative, EMPHYSEMATOUS PULMONARY DISEASE & juvenile hepatic cirrhosis Acquired Deficiency: Chronic Liver Disease Severe protein deficiency conditions Increase: Acute Phase Reaction, pregnancy, oral contraceptive use α1 ACID GLYCOPROTEIN (OROSOMUCOID)APR Functions: maintaining mucus membrane integrity cell membrane formation & fibers in association with collagen Increase: Acute Phase Reaction, pregnancy, cancer, pneumonia, RA, conditions related to the cell proliferation Decreased: Inborn errors of metabolism Measurement(s): cf. AAG: has high carb content, “NOT well stained in SPE” Immunonephelometry, Immunofixation, Immunoassay(s) α1-FETOPROTEIN (AFP) Synthesized by FETAL yolk sac & parenchymal cells of the FETAL liver Gradually decreased after birth Function not fully established in adult Detectable in maternal blood during pregnancy up to 7-8 months Used as screening test for several fetal conditions between 12 & 20 weeks (gestational age) Elevated AFP: Anencephaly Spina bifida neural tube defects atresia fetal distress ataxia-telangiectasia (Louis-Barr Syndrome) Also increased in Twins Low level of AFP: 3-4x increased risk for Down’s syndrome Tumor marker in adult High density Lipoprotein (HDL) α2-Globulin Fraction α2 Macroglobulin Largest major nonimmunologic circulating protein (mw 725 - 820 kD) Function: Neutralize enzymes, [thrombin, trypsin, pepsin, plasmin] Carrier for zinc Role in the innate or nonspecific immune response **Increase in nephrotic syndrome. Measurement: Nephelometry, Immunoassay Prothrombin Coagulation factor II; converted to thrombin in process of coagulation; thrombin acts on fibrinogen to convert it to fibrin Decreased in liver disease, Vitamin K deficiency Measurement: prothrombin time Thyroid Binding Globulin Function: transport protein for T3 & T4 Measurement: Immunoassay Indirect measurement is by T3 Uptake (currently decreasing in use) (TBG Binding Capacity) Haptoglobin (APR) Function: bind free Hb to prevent loss of Hb via urine, and to transport it to the liver Increases: burns, nephrotic syndrome, rheumatic disease, stress, infection, acute infection, tissue necrosis Decreases: IV hemolysis Measurement: Immunonephelometry haptoglobin electrophoresis immunoassay Ceruloplasmin (APR) Transport protein for copper Enzymatic activities: Cu oxidase, histaminase, ferroxidase Increases: inflammatory processes, pregnancy, malignancies, oral estrogen therapy. Decreases: Wilson’s Disease, Menkes’ kinky-hair syndrome Measurement: Immunonephelometry Immunoassay Erythropoietin Protein hormone produced in tubules of kidney Functions Stimulates erythropoiesis ➡ Acts on erythroid cell precursors in bone marrow An potent cytoprotective (anti-apoptotic) hormone Angiotensinogen Precursor to angiotensin I After conversion to angiotensin II, regulates, or increases blood pressure via vasoconstriction α2 Lipoproteins (VLDL) Preβ β-Globulin Fraction β1 Lipoproteins (LDL) Fibrinogen Coagulation Factor I Converted to fibrin by thrombin Acute phase reactant Increased: inflammatory conditions, pregnancy, oral contraceptives Measurement: coagulation Forms a distinct band between the β and γ areas when plasma is electrophoresed Plasminogen Fibrinolytic protein activated to enzymatic status (plasmin) in coagulation process Plasmin slowly lyses fibrin clots Complement Fractions except C1 & C2 Enhances nonspecific cellular immune response, such as phagocytosis, anaphylaxis, lysis Decreased: malnutrition, DIC, SLE, RA, recurrent infection Measurement: immunonephelometry immunoassay CH50 Total Hemolytic Complement Transferrin (Dierophilin) C-1 Esterase Inhibitor Inhibits C-1 esterase (activated C-1) Deficiency results in angioneurotic edema Hemopexin Function is to bind free heme Increased in acute phase reaction Decreases: IV hemolysis & hemolytic anemia Measurement: Nephelometry Immunoassay β2 Microglobulin Light chain of HLA antigen Found on surface of most nucleated cells, esp. high on lymphocytes MW ~11 kD Filtered by renal glomerulus, but 99% is reabsorbed and catabolized in the proximal tubules Increases: tubular damage (poor clearance by kidney) Overproduction of certain cell types, such as occurs in certain inflammatory diseases (RA, SLE) In HIV patients, a high β2 level in the absence of renal failure indicates a large lymphocyte turnover, suggesting the viral killing of lymphocytes Measurement: immunoassay γ-Globulin Fraction C1 & C2 IgG Predominant immunoglobulin of the secondary or anamnestic response Crosses the placental barrier Associated with long-term immunity Highest concentration in blood of all immunoglobulins Usually reacts best at 37 degrees IgA Found both in blood and on mucous membranes in secretions Exists as monomer and dimer, possibly trimer Associated with protection of respiratory and gastrointestinal disease IgM First immunoglobulin to be produced in immune response Dominant immunoglobulin in primary immune response Pentamer structure Largest immunoglobulin Isohemagglutinin (Blood group antibodies) IgD Function in serum unknown Found on surface of mature B lymphocytes as part of antigen recognition system of B-cells Very low concentration in serum IgE Associated with Type I hypersensitivities Bound to mast cells in tissues and basophils in blood Low concentration in serum Liver Function and Bilirubin Metabolism General liver function Largest organ in the body and is responsible for: METABOLISM AND SYNTHESIS: carbohydrates, proteins, lipids, porphyrins, and bile acids. Synthesizes most plasma proteins except immunoglobulins & hemoglobin STORAGE: Iron, glycogen, metabolic end products and xenobiotics to less/non-toxic form. Increases water solubility for excretion 1400-1600 in normal adults Divided into 2 primary lobes that are abundantly vascularized (~15 ml/min) from hepatic artery to hepatic vein Lobule is structural unit of the liver: cords of liver cells (hepatocytes radiate from central vein. Each lobule contains a branch of hepatic artery, portal vein, and bile duct. Boundary formed by portal tract made of connective tissue. Between cords are vascular spaces (sinusoids) lined by endothelial and Kupffer’s cell. Hepatocytes- (parenchymal) cells are responsible for metabolic functions Kupffer’s cells- phagocytic macrophages Bile canaliculi are channels located between hepatocytes that interconnect and eventually drain into larger bile ducts. Stimulated state: Transformed into COLLAGEN producing cells →Responsible for Fibrosis & eventually cirrhosis Stellate cells: B/t endothelial cells and sinusoid Normal (Quiescent) State: Vit A storage, NO synthesis Liver Function Carbohydrate Metabolism Glycogenesis (storage of glucose in form of glycogen) Glycogenolysis (release of glucose from glycogen) Gluconeogenesis (synthesis of glucose from amino acid or lactic acid precursors). Glycogen: Primary source for the blood glucose [Glycogen]liver fluctuates depending on usage Synthesis & release: controlled by Insulin, glucagon, glucocorticoids, T3/T4 (Thyroid hormone) Normal blood glucose: 70-110 mg/dl, (regardless of last meal time) ** Required for normal brain and tissue function ATP and NADH are produced from oxidation of glucose and fatty acids. After meal liver has 14 hrs of glycogen reserve. ** Depletion of reserve leads to increased lipolysis (increasing the FA) and gluconeogenesis. Process endogenous and exogenous compounds by BIOTRANSFORMATION (a series of chemical alteration by “ENZYME ACTIVITY” Protein metabolism All plasma proteins (except Igs & Hb) are synthesized in the liver. Neonates retain some hemoglobin synthesis Metabolic pools of amino acids are maintained in the liver. Amino acids are protein building blocks: Under normal conditions rate of protein synthesis = rate of protein degradation. Excessive amino acids are converted to NH3 & urea for excretion Degradation of protein restores amino acid pools. Amino acids can also be used for gluconeogenesis, transamination, or deamination reactions Transamination reactions are catalyzed by alanine aminotransferase (ALT) & aspartate aminotransferase (AST) Useful in diagnosis of liver disease Damaged hepatobiliary cells: AST and ALT Damaged canalicular membrane and biliary obstruction: Alkaline phosphatase (ALP) & 5’-nucleotidase (NT) Hepatocellular and obstructive disorders: γ-glutamyl transferase (GGT)**GSH/GSSG, Glutathione Acute Hepatic Diseases: less change in plasma protein concentration Severe Liver diseases: Short lived hepatic proteins [Transthyretin (T4 carrier), Prothrombin] decreases to the abnormal value. → ½ life of transthyretin: 24 -48 hrs, prothrombin: about 60 hrs In advanced Cirrhosis: all liver-synthesized plasma proteins decrease vs. Increased Igs Proteins synthesized in liver serve many functions: Nutrition Lipid Metabolism ▪ Liver synthesizes cholesterol (free & esters), free fatty acids, triglycerides, sphingolipids, and phospholipids Increased synthesis with dietary intake: lipids is repackaged by liver with specific proteins and transported as lipoproteins (VLDL, LDL) Increased carbohydrate intake → Acetyl CoA → FFA →TG 70% of synthesized cholesterol is esterified with FA from phosphatidyl choline by LCAT (lecithin cholesterol acyltransferase) Cholesterol synthesis necessary for Cell membrane fluidity Bile acid formation (helps emulsify digested lipids for absorption) BILE ACIDS Final excretory metabolite of cholesterol 80% of cholesterol is converted into 4 major bile acids: cholic acid, chemodeoxycholate, deoxycholic acid, and lithocholic acid Bile acids are conjugated with amino acids (glycine & taurine) forming conjugated bile salts. Bile salts flow from bile ducts to gall bladder. Released into intestinal lumen to emulsify and absorb TG and cholesterol from diet Liver breaks down FAs (lipolysis) for energy (occurs when glycogen is depleted) FFA→ AcetylCoA →CO2 +NADH Excess acetyl-CoA formation (as might occur in Fasting, DM or Alcohol intoxication) and limited amounts of NAD+/NADP+ results in hepatic synthesis of ketone bodies. Storage Iron, glycogen, amino acids, vitamins (A & B12), Lipid (transient). Iron is stored as ferritin (and some hemosiderin) Only the liver & kidney contains **glucose-6-phosphatase which converts glucose-6-phosphate to glucose. Detoxification Excretory function of the liver Liver is primarily responsible for detoxification of poisons, drugs and toxic metabolic end products (NH3) Bilirubin← biliverdin reductase← Biliverdin← Heme oxygenase← Heme Detoxification by: Inactivation of agent by binding with serum protein (albumin binds bilirubin) Chemical modification of agent in liver to increase likelihood of excretion (NH3 converted to urea, or bilirubin is conjugated with glucuronic acid). Modifications can also occur via cytochrome P-450 system located in microsomes Modification includes hydroxylation, sulfation or conjugation with a carbohydrate or amino acid. Porphyrin Synthesis Porphyrins are chemical intermediates in the synthesis of hemoglobin, myoglobin, and Respiratory cytochromes. (Within the liver), heme synthesis is controlled by aminolaevulinic acid (ALA) Synthase. Jaundice Conjugated bilirubin: less than 0.3 mg/dL (5.1 µmol/L) Total bilirubin: 0.1 to 1.2 mg/dL (1.71 to 20.5 µmol/L) Caused by hyperbilirubinemia (25-50 mg/L).: Interference of bilirubin metabolism Yellow discoloration of skin, mucous membrane, and sclera. CANNOT be present in many individuals with liver diseases such as chronic liver disease Conditions that interfere with bilirubin metabolism: Prehepatic (overproduction Over production: **Excessive hemolysis (e.g. chronic hemolytic anemia, pernicious anemia) exceeds liver’s capacity to clear bilirubin Serum bilirubin rarely exceeds 50 mg/L [almost entirely “Unconjugated”] Chronic bilirubin production may lead to formation of bilirubin containing gallstones. Hepatic (impaired uptake by hepatocytes, conjugation defects, reduced excretion into bile) Impaired hepatocyte uptake caused by: drugs (competition for ligand binding) a hereditary disorder (**Gilbert’s syndrome: GS) Degree of hyperbilirubinemia is variable but is made worse with fasting. No other apparent symptoms. Lack/defect of conjugating enzyme (glucuronosyltransferase): Found in Crigler-Najjar Syndrome (type I / II) & Neonates. Type I: inherited, complete lack of enzyme! leads to infant mortality by 1 year Type II: inherited, partial deficiency: bilirubin levels not as high as with type I Neonatal physiologic jaundice caused by immaturity of glucuronosyltransferase enzyme (one of the last to be developed in the liver) Decreased albumin and incomplete blood-brain barrier (BBB) place infant at great risk of encephalopathy. Increased deposition of bilirubin in the Brain can result in kernicterus. Neonatal jaundice can be treated with UV radiation or exchange transfusion. → Conditions above result in increased unconjugated bilirubin. Defective excretion to the bile Excretion of bile is rate-limiting step of metabolism Dubin-johnson & Rotor syndromes are rare hereditary disorders of excretion. Dubin-Johnson syndrome is distinguished by dark pigment in hepatocytes. **Serum conjugated bilirubin levels are increased. Hepatocellular damage & necrosis Can affect uptake, conjugation, and excretion into bile. Most associated with Hepatitis and Cirrhosis. Post-hepatic (obstruction of bile flow) Obstruction of bile flow by gallstones or tumors which increase conjugated serum bilirubin. Diagnostic Enzymology Enzymes are mainly proteins that facilitate biochemical reactions. (Ribozyme: RNA splicing) Enzymes are biological catalysts. Hundreds of different types of enzymes are present in the body, on or within cells. Systemic Enzyme (tissue/ cell) specific enzyme ex: Liver, heart, pancreas Tissues/cells injury→ Start to leak out into the blood Can measure the ACTIVITY of these enzymes in the blood to ascertain whether these organs have been or are being damaged. Abnormal serum enzyme levels are found in various diseases and inflammation Cofactor/coenzyme: Protein or non-protein Permanent or temporary Essential factor for the enzymes which require cofactor Inhibition of Enzyme Activity: Competitive inhibition Allosteric inhibition Proenzyme (Zymogen): Inactive or less active precursor of enzyme Proteolytic modification required to be activated Example: Angiotensinogen, trypsinogen, pepsinogen, chymotrysionogen, prolipase Enzyme Kinetics Enzymes speed up reactions by: Lowering activations time Increasing “Rate Constant” Increasing “substrate specificity” (or substrate concentration) Enzyme concentration ↑[Enzyme], the faster the product formation b/c more enzymes = more enzyme/substrate complexes Substrate Concentration Substrate readily binds to the enzyme at low concentrations. ↑ [substrate], the rate of reaction increases. But [substrate] too high, enzyme Saturation. Enzymes work by converting a substrate into a product via an enzyme-substrate complex: Factors Influencing the rate of reaction “The shape of the protein affects its activity” Anything that alters the conformation of the protein/enzyme will have an impact on its activity. Enzyme concentration Substrate Concentration pH Fluctuation of pH: alter “ionizable group(s)” on the enzyme affect the enzymes shape. **significant change in pH can cause the enzyme to denature. Change equilibrium position [H+ involved reaction] Enzymes require a pH: 7 – 8 Exceptions: alkaline phosphatase, acid phosphatase, pepsin Temperature Temperature increases increase in molecular collisions increase in the rate of reaction. **Too high temperature causes the protein denature decreasing the rate of reaction. Optimal temperature is close to physiological temperature (37ºC) Presence of Inhibitor Enzyme activity evaluation Single (end) point Assays Incubate sample with substrate for a period Measure the end absorbance (O.D.) Calculate enzyme level by comparing to the [STD] Kinetic Assays Incubate sample with substrate Measure the absorbance over time at certain increments An average change in absorbance (product formation) is used to calculate “enzyme activity” Calculating Enzyme activity IU or U = The amount of enzyme that produces 1μMof product per minute under standard conditions Using data: Calculate change in absorbance per minute Correct for dilution factor and serum volume Use molar absorptivity to convert absorbance to μM Δ absorbance / molar absorptivity x dilution factor (total volume / sample volume) Muscle Enzyme: CK & LDH Creatine Kinase (CK/CPK) 82kD dimeric enzyme [CKBB, CKMB, CKMM] Catalyzes reversible phosphorylation of Creatine Mg2+ is an important cofactor [too much Mg2+ inhibit the CK] Active site of enzyme: R-SH which is easily oxidized in serum; therefore, it is unstable in serum CK can be partially restored by treating with antioxidant such as GSH & NAC Other inhibitors: Mn2+, Ca2+, Cu2+, iodoacetate, other SH binding proteins Highest activity in the MUSCLE: skeletal (2500 U/g tissue) & cardiac (550 U/g tissue) cf. Also in the brain, kidneys, liver & GI tract. Three CK isomers: Subunits: M & B (40 kD each) Combination of subunits: Three CK isomers CK-MM (skeletal & cardiac) CK-MB (Cardiac) CK-BB (Brain) Both Subunits (M&B) Have Lys residue @ C-terminus Lysine in M subunits can be hydrolyzed by “carboxypeptidase” → 2 CK MM isoforms: 2 Lys removed → CK MM1 1 Lys removed → CK MM2 CK MB could undergo same modification: CK MB1 / CKMB2 Other CK isomers: CK-mt: CK in between inner and outer mitochondrial membrane CK in macroform: macro CK Up to 6% of hospitalized patients’ sera Type I (CK (CK-BB) + Immunoglobulin (IgG); Type II oligomeric CK-mt Clinical Significance • Muscle Injury, Inflammation, Myocardial infarction, Necrosis of Skeletal muscle and Cardiac muscle ALL Type of Muscular Dystrophy Progressive Muscular dystrophy➡ highest in infancy and childhood **Duchenne Sex-Linked MD (x chromosome) 50-100 x URL Asymptomatic Female Carrier; 3-6x increased CK Severe Physical Crush Injury 200x URL 3 days after injury: CK value less than 5000U/L➡ Less probability of developing Acute renal failure Drugs (pharmacological dose) that could increase serum CK activity Statins, Fibrates, Antiretroviral, Ang II Receptor antagonist Measurement of CK Sources of error: Hemolysis, ↑ 10 U/L per Hb (1g/L), due to RBC AK Exercise Reference Range: Female: 15-171 IU/L Male: 46-180 IU/L CK-MB: less than 6% of total CK Lactate Dehydrogenase (LD/ LDH) Reversible reaction Reaction equilibrium favors the reduction of pyruvate to lactate @ physiological pH Increased pH will favor the oxidation of lactate to pyruvate Too much pyruvate or lactate inhibits LD activity EDTA inhibits LDH activity by binding Zn2+ MW: 134 kD Composed of 4 peptide chains of two type: M (or A) & H ( or B) LD1 (HHHH; H4) LD2 (HHHM; H3M1) LD3 (HHMM; H2M2) LD4 (HMMM; H1M3) LD5 (MMMM; M4) Other isoforms: LD-X (LD-C): four X (or C) subunits LD-6: from severely ill patient Clinical Significance Invariably found only in the cytoplasm of the cell Hepatic, Cardiac, Skeletal muscle, Hematological Disorders➡ Significantly increased “Tissue specific” LDH isomers LD1 & LD2: Heart, Kidney, RBC LD3: Lung, Spleen, Lymph node, WBC, PLT LD4 & LD5: Skeletal muscle, Liver Measurement of LD The reaction lactate to pyruvate is recommended Quantification of total LD activity: Monitor NADH at 340 nm Serum is preferred sample [should be stored at “room temperature”] cf. Plasma sample: more chance for PLT contamination; LD4 & 5 are labile at cold temperature Hemolytic sample (X) cf. 4000 times more LD in RBC than serum EP separation for separation of LD isoenzymes Reference range: 125 – 220 IU/L Cardiac Markers AMI - Myoglobin, Troponin I &T, CK-MB CHF- BNP Diagnostic Enzymology Alterations in LIVER enzyme activities Hepatocellular damage: aminotransferase activity Cholestasis (suppression of the normal flow of bile) : activities of alkaline phosphatase, 5’-nucleotidase, γ -glutamyl transferase Aminotransferase coenzymes: Pyrudoxal-5’-phosphate (P-5’-P) Prydoxamine-5’-phosphate Liver Specific enzymes Aspartate aminotransferase (AST) NR: 5-30 IU/L Liver, Kidney, Striated Muscle, RBCs (source of error: hemolysis) Cytoplasm & Mitochondrial forms ↑↑ Acute hepatocellular disorders ↑ Myocardial muscle or other conditions AST < ALT Alanine Aminotransferase (ALT) NR: 6-37 IU/L Predominantly in the liver (& kidney) lesser amount in Striated Muscle Exclusively in the cytoplasm Liver diseases (Acute): ALT>AST and remains elevated longer Ref: alcoholic hepatitis, hepatic cirrhosis, liver neoplastic Clinical significance of Aminotransferase LIVER disease: might 100 times the URL. (Usually 10-40-fold increase) Hepatic necrosis: prior to symptom, both ENZYMES activity increase Threshold for Diagnosing Acute Liver Injury: 7 times URL Peak activity of ALT & AST: 7th and 12th day Chronic hepatitis: ALT (> 6 months) [Periodic measurement] Toxic hepatitis v. Infections hepatitis Acetaminophen-induced hepatic injury: The peak activity: 85 x URL (about 90% of case) Infectious hepatitis: rarely seen this level of elevation Medication–induced elevation of ALT & AST activity NSAID, antibiotics, antiepileptic drug, statin, etc. Non-Alcohol Fatty Liver Disease: h ALT, AST Metabolic syndrome Higher body mass index increased waist circumstance ↑serum TG ↑ fasting insulin, LDL/HDL cholesterol Primary & metastatic malignancy of liver: AST>ALT; both enzymes 2-5 x h activity Cf. Early stage: activity increases within normal range AST/ALT ratio (AAR) greater than 1: Presence of advanced fibrosis in patients with chronic liver diseases (≈ 90% positive predictive value) Liver specific enzymes continues… Alkaline Phosphatase (ALP) Small intestine (mucosa), kidney (PCT), Osteoblast, Liver (Most of ALP activity in healthy adult sera), Placenta Lipid transport in the intestine and with the calcification process in bone Activators: CO2+, Mg2+, Mn2+ Inhibitors: Phosphate, borate, oxalate, cyanide ion Hepatobiliary Diseases: Serum enzyme activity could h up to 12 x depend on the degree of obstruction Biliary tree obstruction →hepatocytes→ synthesis of ALP Extra hepatic obstruction 3 x elevation of ALP activity vs. intra hepatic. Primary liver cancer patients or widespread secondary hepatic metastasis Infectious hepatitis: mild increase or normal ALP activity. Drug therapy could increase ALP activity. ** narcotic pain medicines, NSAIDs, propranolol, tranquilizers, tricyclic antidepressants Bone Diseases Hypophosphatasia: severe bone disease and impaired bone growth Paget disease (osteitis deformans) enlarged & deformed bones Vit D deficiency Osteomalacia Ricket 2-4x URL Osteoporosis Bone cancer (Primary / secondary) hyperparathyroidism Slight to moderate ↑ ALP activity is temporarily increased during healing from the fracture. Analysis 4-NPP or PNPP is the most popular chromogenic substrates for ALP O.D. of p-nitrophenol at 405 nm alkaline pH (~10) Serum/plasma w/o hemolysis at room temp within 4 hrs after collection EDTA & citrate CANNOT be used Be preferentially measured in fasting sera NR: 30 – 90 U/L Gamma Glutamyltransferase Transfer γ-glutamyl group from donor to an acceptor Substrate: 1) the γ-glutamyl acceptor, 2) AA or peptide, 3) water. Liver (hepatobiliary system), Kidney (PCT), Pancreas, & Intestine. Cytoplasmic protein [Membrane Bound] Clinical significance: Hepatobiliary disease [critical marker] Intra- & post-hepatic biliary obstruction: 5-30 x URL Acute and chronic pancreatitis Pancreatic malignancies (With biliary obstruction) Primary and metastatic liver neoplasm Alcoholic hepatitis Anticonvulsant drugs Analysis: NR (URL): Adult: Male - 70 U/L ; Female – 40 U/L 2-fold higher in people of African ancestry At birth: 6-7 times of adult reference interval γ-glutamyl-p-nitroanilide: less water soluble, It is difficult to reach the saturating concentration of substrate. derivatives of GGPNA in which various groups have been introduced into the benzene ring to ↑water solubility GGT activity is stable for 1 month @ 4C/ 1 year @ -20 C Non hemolyzed serum (preferred)/ EDTA-plasma Heparin [Turbidity of sample]; Citrate, oxalate, Fluoride [↓ GGT activity 10-15%] Pancreas specific enzyme Amylase (p type) & Lipase NR (URL): 30–100 IU/L (serum), 1–7 U/L (urine) Amylase 4-nitrophenyl (4-NP)-glycoside [Substrate] Free NP product detected by its OD at 405 nm Serum reference interval: 31-107 U/L All anticoagulants inhibit AMY activity (except Heparin) Serum or heparinized plasma Stable 4 days at room temp, 2 weeks at -4 °C, 1 year at -25 °C, 5 years at -80 °C P-AMY determination: selective S-AMY inhibition with monoclonal Ab Lipase Cofactors: bile salts & colipase Pancreas, gastric and intestinal mucosa. (5000 x greater than other tissues) Single chain glycoprotein: 48 kD 100% filtered & 100% reabsorption DOES NOT PHYSIOLOGICALLY DETECTED IN URINE CLINICAL SIGNIFICANCE NR: 10 – 160 U/L ↑LPS in Serum: acute pancreatitis Similar symptoms: Perforated gastric, or duodenal ulcer, GI obstruction, Mesenteric Vascular obstruction ↑serum LPS activity to greater than 3 x the URL (w/o renal failure) More sensitive than increase in AMY activity LPS remains elevated longer than AMY does With reduced GFR, serum LPS activity is increased ** Cystic fibrosis, Crohn’s disease, Celiac disease patients: may NOT produce enough Analysis Use TG or non-TG substrate Titrimetric, turbidimetric, spectrophotometry, fluorometric, immunologic techniques Methylresorufin can be detected at 580 nm bluish purple chromophore No sex and age-related differences LPS activity: stable for 1 week at room temp; 3 weeks in 4 °C; several years at -20 °C Miscellaneous Enzymes Acid Phosphatase (ACP) All phosphatase with optimal activity under acidic pH (< pH 7.0) Lysosomal and extra-lysosomal enzyme Greatest concentrations of ACP: Prostate Bone Spleen Platelet RBC Low ACP activity of serum: tartrate-resistant type (TR-ACP, TRAP or TRAPase) Activities of this fraction (bone) are increased in growing children conditions of increased osteolysis & bone remodeling ALP? Paget, hyperparathyroidism, malignant invasion in bone Can be used in the detection of PROSTATE CANCER. Measured the same way as ALP, but under ACIDIC conditions (pH = ~5) Reference range: 0 – 3.5 ng/ml Cholinesterase (CHE) Hydrolyzes esters of choline, e.g. acetylcholine Metabolize/clearance of drugs cf. deactivation of octanoyl ghrelin Found in the brain, serum, liver, and RBCs Two main types: Acetylcholinesterase (true cholinesterase, RBC Che, cholinesterase I) RBC, Lung, Spleen, Nerve endings, gray matter of the brain. Pseudocholinesterase (Acetylcholine acylhydrolase, Serum(/plasma) cholinesterase (CHE, SchE), cholinesterase II), current Butyylcholinesterase(BChe) Liver, Pancreas, Heart, White matter of brain, Serum Clinical Significance Liver function test ↑in CHE activity: impaired synthesis of the enzyme by liver used for monitoring of liver function after “liver transplantation” Indicator of possible insecticide poisoning Insecticides: CHE inhibitors (both cholinesterase) CHE falls more rapidly ↓ in RBC enzyme is used as a measure of “chronic exposure” For the detection of patients with atypical forms of the enzyme Muscle relaxants: Succinylcholine, mivacurium Hydrolyzed by CHE Analysis Substrate: acyl thiocholine ester (ATCl) Chromogenic disulfide agents: DTNB (Elliman’s reagent) Measure spectrophotometrically 5-thio-2-nitro-banzoic acid @ 410 nm Serum: activity stable for several weeks @ 4 °C; several years @-20 °C Glucose-6-Phosphate Dehydrogenase Enzyme is the first step in the pentose-phosphate shunt. Leads to the production of NADPH Very important enzyme in the RBCs. Functions to maintain NADPH levels. NADPH (glutathione) helps protect cell/hemoglobin from oxidation. A deficiency in G6PDH results in an inadequate supply of NADPH and inability to protect cell. Hemolysis can occur leading to hemolytic anemia Analysis Glucose-6-phosphate + NADP+ → 6-phosphogluconate + NADPH NADPH is measured spectrophotometrically (340 nm). “Erythrocyte hemosylate” is assayed for enzyme deficiency Diagnosis of Acute Myocardial Infarction Elevated LDH LD not specific to cardiac tissue – found in various other tissues as well. Increase in serum level from ~12 - 24 h post infarc, peaks after ~ 48-72 h, gradually returns to normal by ~ 7 – 14 days Elevated Cardiac Enzymes (CK) CK-MB is heart specific Rises about 4-8 h after infarct, peaks at 12-24 h, and returns to normal in 2-3 days CKMB index = CKMB activity/Total CK activity x 100 CKMB: <6% total CK Troponins Troponins are proteins involved in muscle contraction. TnI, TnT, TnC: found in skeletal/cardiac muscle Screen for cTnI and cTnT Following AMI, levels begin to rise ~3 – 6 h, reach peak levels in 14-24 h, return to normal in 5 – 10 days Myoglobin For early detection (leaks 1-3 h of onset) of AMI. Peak is reached 5 – 12 hours. Myoglobin is a small molecule (kidney can freely filter) and thus returns to normal in 18 – 30 h after the AMI. Problem: NOT specific Present in all muscle cells so non-specific