- major natural proteins in blood serum globulins (antibodies), albumins, and proteins of the clotting cascade such as fibrinogen
- blood often contains proteins that are not endogenous, but released upon damage to cells
- most diagnostically useful proteins are enzymes; conversion of substrates and/or generation of products can be monitored; enzyme signals can be amplified
2. Type I and Type II protein defects
- radioimmunoassay (RIA), enzyme linked immuno-sorbent assays (ELISA) and electrophoresis/Western blotting other proteins have diagnostic utility; measure of totalamounts of proteins (not activity)
- physicians concerned with release of normal cellular proteins in blood; direct correlation between activity and amountspecific activity or Kcat
- Type I protein defects caused by altered amounts (reflected in altered activities);normal specific activity thalassemia
- Type II protein defectreduction in enzyme activity occurring without reduction in amount; specific activity is reduced due to defect in enzyme
1.Importance of protein glycosylation (reference to sialic acid, in regulating levels of exogenous proteins in blood serum)
- proteins exposed to extracellular environment (interstitium/blood) have sugar to prevent proteolysis and aid in recognition
- glycosylation is post-translational modification; sugars are small chain of 5-8 monosaccharide units connected to proteins via asparagine (N-linked) or threonine (O-linked) covalent bonds
- common sugars are glucose and galactose; N-acetyl glucosamine, N-acetyl galactosamine, and sialic acid (N-acetyl neuraminic acid, NANA) is a common terminal sugar for most oligosaccharide chains
- release of intercellular proteins into blood is continuous because of natural tissue turnover caused by damage; proteins cleared through hepatobiliary system
- small proteins filtered by glomeruli appear in urine
- liver cells/macrophages contain asialoglycoprotein receptors (asgR) recognized proteins that lack sialic acids; intracellular/foreign proteins do not contain sialic acids recognized by asgR, which binds them and induced receptor-mediated endocytosis proteins internalized by hepatocytes and macrophagesdegradation by lysosome or packaged/excreted in bile
3.AST/ALT
- diagnostic potential for serum component determined by:
1.) tissue specificity
2.) concentration in tissue
3.) size of source organ
- Aspartate transaminase (AST) and alanine transaminase (ALT); catalyze interconversion of amino acids and keto acids
- AST level higher 8000X in heart, liver, SKM, and kidney relative to serum; small damage to these organs large increase in serum levels of AST; AST less diagnostically useful for damage to pancreas, spleen or lung
- ALT is highest in kidney and liver (low in all other organs); if both AST and ALT elevated aid in differential diagnosis of liver or kidney damage; if only AST is elevated cardiac/SKM damage; ALT/AST ratio diagnostic for liver disease
- some enzymes used in pathodiagnosis are tissue-specific, prostate specific antigen (PSA); PSA is a serine protease present in seminal fluid and secreted by prostate parenchyma; levels elevated in prostatic hyperplasia and prostate cancer
6. Sensitivity, Specificity, PPV, NPV and Receiver operator characteristic
- PPV is positive predictive value, NPV is negative predictive value
- define normal range series of lab testes obtained from diseased (D) and healthy (H) patients
- assumption #1 H patients are healthy and D patients have disease
- diseased population has subscript i because different diseases can present with different ranges and this can aid in differential diagnosis
- for most tests overlap between H and D is substantial (with x-axis being distribution of serum analyte level)
- choice can be made of any threshold to distinguish H from D
- normal ranges: R1, R2, and R3; if R1used then there will be a lot of healthy subjects with values out of the normal range (false positives), but a correspondingly small number of patients with disease who have normal values (false negatives)
- range 1 used if one wants to discriminate those patients who clearly are not disease for those who may by disease
- as range expands from R2 and R3 number of false positivedecreases and number of false negatives increases
- sensitivity fraction of those withdisease correctly identified as positive by test; sensitivity = TP/(TP + FN)
- specificity fraction of those without disease correctly identified as negative by test; specificity = TN/ (TN + FP)
- positive predictive value (PPV) fraction of people with positive tests who actually have the condition; PPV = TP / (TP + FP)
- negative predictive value (NPV) fraction of people with negative tests who actually do not have the condition; NPV = TN / (FN + TN)
- specificity increased from R1 R3; ratio of healthy to diseased populations to right of threshold
- sensitivitydecreased from R1 R3; determined by diseased population to right of threshold, divided by total disease population
- optimal choice for Normal range determined by a receiver-operator characteristic (ROC) plot of the likelihood ratio asks, If you have a positive test, how many times more likely are you to have the disease?; likelihood ratio of 6 someone with positive tests is six times more likely to have the disease than someone with a negative test
- likelihood ratio = sensitivity/ (1.0-specificity)
- ROC analysis sensitivity plotted against (1.0 – specificity) ; best diagnostic tests are those with greatest area under curve; optimum choice is takes as point at which ROC curve crosses diagonal
- sometimes many false positive OK, if there is high sensitivity small Normal range (R1 used); false positives then separated from true positives by CT/MRI
4. ISOZYMES
- isozymes are two or more forms of enzymes that catalyze the same reaction yet have distinctprimary sequences; products of different genes; pancreatic (P) and salivary (S) amylases both catalyze hydrolysis of starch or glycogen in the chain (endo-saccharidases)
- amylases are small enzymes and is one of few serum proteins that can be cleared by kidney (urine activity is measurable); kidney disease increase in urine amylase without change in serum amylase
- both P and S amylase undergo post-translational modification (glycosylation); S-form glycosylated, P-form is not
Electrophoresis
- electric current makes protein samples move according to size, shape, and charge; small, negatively charged proteins move faster; separation can be determined just by molecular mass by adding denaturants to impart a constant shape and charge/mass ratio
- transfer of proteins from electrophoresis gel to a membranehigher specificity; then stained with antibodies (Western blot)
Alkaline phosphatase (ALP)
- ALP is a plasma membrane protein both liver/bone (same MW) forms are sialidated (glycosylated with sialic acid residues at termini)
- sialic acid negative charge; since both are same MW both liver and bone forms migrate as a single band; treatment of serum with neuraminidase (sialidase) removes sialic acid termini and native differences in charge are revealed; bone isozymes travel more slowly than liver isozymes towards anode
- ALP is diagnostically important in hepatobiliary disease and bone diseases associated with increased osteoblastic activity
- hepatobiliary disease serum liverALP rises 10X upon extrahepatic obstruction, such as bile stone are cancer; serum levels of bone ALP increase during bone remodeling; very ALP high levels in bone cancer
5.
Creatine kinase
- CK for creatinekinase; CPK for creatinephosphokinase
- used to diagnose damage to heart muscle (MI)
- CK present in all muscle tissues and in brain
- specific isoforms for brain, skeletal muscle and cardiac muscle
- CK is a dimer comprised of two polypeptide chains derived from either brain (B) or skeletal muscle (M) isoforms
- three possible combinations: BB (CK-1) in brain, MB (CK-2) in cardiac muscle, MM (CK-3) in skeletal muscle
- B subunit from chromosome 14; M subunit from chromosome 19
- polypeptidesmonomeric units dimersorganize according to expression pattern in given cell
- brain only B gene active; in skeletal muscle only M gene is predominant
- in heart both M and B genes active; isozyme pattern in heart is 1:2:1 MM:MB:BB
- CK-2 isozyme increases in serum 3 hour following a myocardial infarction and persists for 3 days; CK-3may increase, as well; CK-1 not detectable in serum
- cardiac involvement is indicated y area under curve
Troponins
- CK-2 increases upon onset of MI, but takes 3-4 hours before serum levels exceed normal range
- cardiac-specific troponins; troponins are proteins involved in functioning of myofibril (not enzymes)
- cardiac specific troponinsc-TnT and c-TnI elevate in serum at same rate as CK-2; however, levels of cTnT and cTnI are undetectable in normal patients takes little time for cardiac troponins to exceed normal values
- release of cardiac specific troponins takes occurs over days and have a longer half life good markers for late detection
- CK-2, cTnT, and cTnI reach maximum diagnostic potential at 6 hours