- major natural proteins in blood serum globulins ...



- 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 total amounts of proteins (not activity)

- physicians concerned with release of normal cellular proteins in blood; direct correlation between activity and amount ( specific activity or Kcat

- Type I protein defects ( caused by altered amounts (reflected in altered activities); normal specific activity thalassemia

- Type II protein defect ( reduction 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 macrophages ( degradation 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 R1 used 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 positive decreases and number of false negatives increases

- sensitivity ( fraction of those with disease 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

- sensitivity decreased 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 distinct primary 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 membrane ( higher 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 liver ALP 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 creatine kinase; CPK for creatine phosphokinase

- 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

- polypeptides ( monomeric units ( dimers organize 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-3 may 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 troponins ( c-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

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