Use of enzyme (and other) markers in diagnostics of selected pathophysiological states Evaluation of LDH isoenzymes Ústav patologické fyziologie LF MU Aim and importance of laboratory examination • biochemical investigations • used in medicine for a variety of purposes • measurement of a substance in a body fluid • reflect pathological processes • 70 – 80 % of medical decisions depend on tests • low costs (3 – 5 % of total) • last 5 years increase of • glucose measurements (10 – 15 % increase) • molecular biology (25 – 35 % increase) • point-of-care testing • aims • confirming a clinical suspicion (glucose – diabetes) • assessment of severity (creatinine) • monitoring disease/treatment (HbA1c) • providing of prognosis • screening • research of the disease Classification of laboratory examinations • by availability • basic • quickly accessible • specialized • highly specialized • centralized • by demands of processing • routine • statim • by 60 minutes since delivery • vital indication • by 30 minutes since delivery • point-of-care testing • on-site examination • acid-base, oxygen metabolism, diagnostic strips – blood and urine Reference intervals • rather than normal interval (what is normal?) reference interval of values is used in clearly defined reference group of subjects • what is reference group? • healthy or better people without state of health which directly affects/interferes with measured variable • determination • historical: x  2 SD • i.e. 95% of values with normal distribution • distribution might be influenced by • age, gender, race, diet, … • values outside reference interval • statistical/methodological variability • biological variability • 5% of healthy population outside Sample collection and analysis • preanalytic phase • sample collection, storage and transport • as many as 60% of errors • analytic phase • follow the good laboratory practice conditions • internal and external quality control – elimination of errors • postanalytic phase • results interpretation Factors affecting preanalytic phase • biological • influenceable • weight – correlation of cholesterol, TAG, cortisol, uric acid with obesity • eating habits • high-protein diet – increase in urea, cholesterol, phosphates • smoking – cholesterol, TAG, cortisol, vitamins B12 and C • alcohol • chronic abuse – increase in ALT, AST, cortisol • mild doses – temporary increase of HDL • pharmaceuticals and drugs • impact on biological processes (induction of enzymes, cytotoxicity), interference • physical load • depends on the duration and intensity • environment – altitude, temperature, travel across time zones • mechanic effects • muscle trauma – increase in ALT, AST and CK, myoglobin Factors affecting preanalytic phase • biological • uninfluenceable • race – different enzymatic activities • gender • minimal differences in childhood • in adulthood values ofter higher in men • age • ALP – high activity in childhood, then decrease, ferritin • pregnancy • biological rhythms – circadian – hormones, iron, urea • sample management • labelling – all stages of handling, request forms • sampling material • technique of sampling – nurses vs. doctors • sample transport • storage of samples Examples of biased results Enzymes • proteins with catalytic properties • virtually all reactions in the cell depend on enzymes • decrease activation energy • not being consumed • change only the rate at which equilibrium is established • enzyme molecules are larger than their substrates • exception - proteases Enzymes • structure • primary • secondary • conformation of limited sequences of polypetide chain • tertiary • quaternary • holoenzyme • apoenzyme + cofactor • cofactor • prosthetic group • coenzyme • active site • relatively small • 3D structure formed as a result of the tertiary structure Cellular localization of enzymes/markers • extracellular • intracellular • membrane-bound • cytosolic • in organelles Plasma enzymes • specific • blood clotting enzymes, ceruloplasmin, lipoprotein lipase • nonspecific • secreted • amylase, lipase • celullular enzymes • enzymes of main metabolic pathways Factors affecting plasma enzyme concentration • intracellular enzyme activity • intracellular localization • permeability of plasma membrane • the extent of cell damage • the mass of the damaged cell • the rate of enzyme elimination Example Different forms of enzymes • proenzyme (zymogen) • inactive enzyme precursor • requires a biochemical change • angiotensinogen, pepsinogen • isoenzymes • multiple forms of an enzyme that catalyze the enzyme‘s characteristic reaction but that differ in structure • primary • more than one gene locus coding for the structure • secondary (=isoforms) • modification of polypeptide chains • examples • glucokinase, LD, CK, PKC, cytochrome P450 Diagram of the origin of isoenzymes a bStructural genes mRNA Polypeptides A A B B Subunits Possible dimers Possible tetramers Distribution of isoenzymes • distribution of isoenzymes is not uniform • variation in the activity at the organ, cellular and subcellular levels • the basis for organ-specific diagnosis • through isoenzyme measurement • certain loci may be expressed exclusively in a single tissue • lactate dehydrogenase – 2 loci • third locus active only in mature testes • third type of subunit X or C – isoenzyme LD-X or LD-C • adaptation of metabolic patterns to the changing needs of different organs and tissues • pathological conditions may be associated with alterations in the activity of specific isoenzymes Changes in isoenzyme distribution during development and disease • several sets of isoenzymes change during normal development • changes result from changes in the relative activities of gene loci • skeletal muscle – LD, CK • liver • 3 aldolase isoenzymes A, B and C are present during embryogenesis • isoenzyme B is predominant in the adults • changes in the number of cells containing respective isoenzyme • increased number and activity of osteoblast • elevation of total serum ALP in young people • malignant tumors • LD – shift in the balance of isoenzymes Detection of isoenzymes • isoenzymes can be distinguished • difference in various physical properties • electrophoretic mobility, resistance to chemical or thermal inactivation • physical-chemistry • electrophoresis • chromatography • immunohistochemistry • chemical • determination of reaction rate in different settings (pH, t, substrate concentration) Li Q et al. J. Biol. Chem. 1999;274:3764-3771 Diagnostic enzymology • changes in the activity in the serum of enzymes that are predominantly intracellular and that are normally present in the serum at low activities • changes in activities of these enzymes in disease – location and nature of pathological changes in the tissues • the measured levels of an enzyme in blood – result of the balance between • the rate at which it is entering the circulation from the cells of origin • the rate at which it is inactivated or removed • existence of multiple forms of enzymes • increase in diagnostic specificity and sensitivity Leakage of enzymes from cells • plasma membrane retains enzymes within the cell • its integrity depends on the availability of ATP • any process impairing ATP production promotes deterioration of the cell membrane • very high concentration of enzymes within cells • ICF/ECF ratio • small amount of enzyme can be detected • an increase of enzyme activity in plasma is sensitive indicator of cellular damage Causes of cell damage or death Clearance of enzymes • urinary excretion • few enzyme molecules are small enough to pass through the healthy glomerulus (α-amylase) • receptor-mediated endocytosis • many enzymes are removed by the reticuloendothelial system (spleen, liver, bone marrow) • in lesser extent by all cells in the body • Kupffer‘s cell • LD5, CK-MM, AST • half-life of enzymes • few hours to several days • average 6 – 48 hours Cardiac markers • the ideal cardiac marker  high sensitivity – high concentration in myocardium – rapid release for early diagnosis – long half-life in blood for late diagnosis  high specificity – absent in non-myocardial tissue  analytical characteristics – measurable by cost-effective and simple method  clinical characteristics – ability to influence therapy and to improve patients outcome • the ideal cardiac marker does not yet exist Cardiac markers • Creatine kinase (CK) • cytoplasmic and mitochondrial enzyme • catalyzes reversible transfer of phosphate from ATP onto creatine • ATP + creatine → ADP + creatine phosphate • dimeric – M (muscle) and B (brain) • 3 isoform • CK-BB – smooth muscle, brain, prostate • CK-MB – myocardium (also in skeletal muscle) • CK-MM – skeletal muscle, myocardium • CK-MB – diagnosis of acute myocardial infarction and monitoring of reperfusion in the course of trombolytic treatment of AMI • Myoglobin • intracellular protein found in cardiac and skeletal muscle cells concerned in aerobic metabolism • released quickly from damaged cells into circulation (small size, 0,5 – 2 hours) • the smallest cardiac marker – quick propagation and degradation • non-specific marker (present also in skeletal muscle) Cardiac markers • troponins • troponin complex – part of the structural proteins, which participates on muscle contraction • heterotrimer consisting of troponins I, T and C • tightly connected with contractile apparatus – low levels of cardiac troponins in the circulation • TnI level is undetectable if the heart is not injured (even in the presence of skeletal muscle damage) • cardiac isoform troponin I (TnI) differs from skeletal muscle isoform specific determination Troponin I • benefits • absolute cardiospecifity • long period of liberation – monitoring of course • sensitivity – detection of smaller injury • not affected by chronic renal insufficiency • limitation • slower onset than myoglobin (nonspecific) Cardiac markers - comparison Biochemical markers of liver function • indicators of hepatocyte damage • ALT, AST, LDH • indicator of bile ducts obstruction • ALP, GMT • indicators of synthetic liver function • albumin, CHE, LCAT, PT • tests of conjugation and liver transport of organic anionts • bilirubin, urobilinogen Markers of hepatocyte damage • Alanin aminotransferase (ALT) • L-alanin+2-oxoglutarate pyruvate+L- glutamate • reaction is reversible, it proceeds in the syntesis, degradation and transformation of aminoacids • cytoplasmatic enzyme • the most abundant in hepatocytes, plasmatic level elevated as early as in the disorder of membrane permeability • Aspartate aminotransferase (AST) • L-aspartate+2-oxoglutarate oxalacetate+L- glutamate • reaction is reversible, it proceeds in the syntesis, degradation and transformation of aminoacids • cytoplasmic and mitochondrial isoenzymes • occurs in liver, myocard, skeletal muscle, kidney and pancreas • plasmatic level of cytoplasmic isoenzyme elevated as early as in the disorder of membrane permeability, releasing of mitochondrial isoenzyme accompanies hepatocellular necrosis Interpretation of ALT/AST elevation • increased activity of both ALT and AST in many liver diseases • extremely high values (10-100x) in toxic and acute viral hepatitis and shock conditions • plasmatic aminotransferase activity does not tell us anything about excretoric or metabolic function of hepatocytes • correlation between level of amino transferases and the extent of liver lesions is not the rule • De Rittis index = AST/ALT • less than 0,7…good prognosis • 1 and more…bad prognosis (necrosis) • physiologically and in majority of liver diseases ALT > AST • exception - AST/ALT >2 • alcoholic damage • postnecrotic cirrhosis Markers of bile ducts obstruction • Alcaline phosphatese (ALP) • membrane bound enzyme catalyzes hydrolysis of phosphate esters at alkalic pH • tetramer, into the circulation released as dimer • widespread - occurs primarily in liver, gut and bones (different isoenzymes) • plasmatic ALP level – diagnosis of bone and hepatobiliar disorders • considerable part of liver ALP is localized membranes of cells covering bile ducts • membranes are disturbed in cholestasis and ALP is released • elevated also in other conditions (liver tumors, cirrhosis) • -glutamyl transferase (GMT) • membrane bound enzyme found in liver, kidney, pancreas, gut and prostate • catalyzes transfer of  -glutamyl from glutathione on aminoacid and enables the aminoacid transport through membrane • serum GMT activity determination is used for evaluation of hepatobiliar diseases Markers of synthetic liver function • albumin • synthesized in liver, plasmatic level determination • long half-life – does not fall in acute disorders • exclusion of another causes of decline (malabsorption, reduced intake of proteins, kidney disease) → liver disease • significant decline in alcoholic cirrhosis • cholinesterase • enzyme generated in hepatocytes and released into blood (secretory enzyme) • catalyzes hydrolysis of cholin esters in plasma • enzyme production (thereby plasmatic activity) is decreased when liver parenchyme is damaged or in malnutrition • irreversibly inhibited by organophosphates Synthetic liver function • coagulation factors • produced in liver, short half-life – quick changes • Quick test – extrinsic coagulation system • values are changed in disorders of liver parenchyma accompanied by proteosynthesis failure or in obstructive icterus with disorder of lipid and lipid soluble vitamins uptake Lactate dehydrogenase (LDH) • tetramer  M (gene LDHA, ch.11)  H (gene LDHB, ch.12) • LDH1 (HHHH) 31-49% • heart, liver, erythrocytes • LDH2 (HHHM) 38-58% • reticuloendothelial system • LDH3 (HHMM) 5.5-16.5% • lungs • LDH4 (HMMM) 0-0.7% • kidney • LDH5 (MMMM) 0-1.5% • skeletal muscle, liver Lactate dehydrogenase  LDH 1 and LDH 2  converts lactate into pyruvate in tissues with aerobic metabolism  LDH 4 and LDH 5  converts pyruvate into lactate in tissues with anaerobic glycolysis Changes in plasma LDH levels • myocardial injury • elevated LDH1 and LDH2 • ratio LDH1/LDH1>1 (in healthy <1) • myocardial infarction (peak 3-4 after MI) • liver injury • elevated LDH4 and LDH5 • hepatitis, cirrhosis, organic solvent intoxication • hemolysis • elevated LDH2 • hemolytic anemia, incompatible blood transfusion Electrophoretic separation of LDH isoenzymes • agarose gel, TBE buffer • staining solution • lithium lactate • NAD+ • stain nitroblue tetrazolium • phenazine methosulphate – carrier of electrons between NADH and the dye • 5 % acetic acid Isoenzymes detection • lactate + NAD+ → pyruvate + NADH + H+ • NADH + H+ + NBT → NAD+ + formazan