CENTRAL TEXAS COLLEGE 2401-CLINICAL CHEMISTRY Enzymes I.

Enzymes - proteins that catalyze biochemical reactions without altering equilibrium, being consumed, or changing in composition A. Serum/plasma enzymes - often used in diagnosis of disease and physiologic abnormalities Made up of amino acids, the arrangement and structure determine the specific catalyst Each enzyme has an active site where substrate reacts Definitions 1. 2. 3. 4. 5. 6. Substrate - substance upon which enzyme reacts Isoenzyme - enzyme existing in different forms within one individual Cofactor - non-protein molecule necessary in some enzyme activity Coenzyme - organic cofactor (ex. NAD) Activator - inorganic cofactor (ex. Cl) Proenzyme - zymogen; structurally inactive form of enzyme; form most are originally secreted

B.

C. D.

E.

Naming 1. 2. arbitrarily - ex. trypsin suffix - ase added to: a. substrate it acted upon, ex. urease b. type of reaction catalyzed, ex. oxidase

F.

Relationship E+S ES E+P

E = enzyme, S = substrate, P = product, ES = complex G. Relationship influenced by: 1. Substrate concentration - first order kinetics (reaction rate is directly proportional to substrate concentration), then ... as product is formed, enzyme is freed and again reacts with free substrate zero order kinetics (reaction rate is dependent on enzyme concentration only)

2. 3.

Enzyme concentration - more enzyme = faster reaction pH - usually between 7-8; extreme pH will alter structure (denature the enzyme) Temperature a. increasing temperature speeds reaction until enzyme becomes denatured b. lowering temperature renders reversibly inactive Cofactor (binds to enzyme before catalysis occurs), activators (inorganic cofactor), and coenzymes (2nd substrate for reaction) Inhibitor - substance that interferes with reaction

4.

5.

6. H.

Measurement of enzymes - hard to isolate due to small quantities in body, so instead measure catalytic activity and relate this activity to concentration 1. Can measure photometrically by: a. increase in product concentration b. decrease in substrate concentration c. decrease in coenzyme concentration d. increase in concentration of altered coenzyme NADH is a coenzyme frequently measured in the lab a. it absorbs light at 340 nm (NAD is not) b. change in absorbance at 340 nm is readily measurable

2.

I.

Enzymes as reagents 1. Very specific 2. Used for glucose, cholesterol, and uric acid determinations

II.

Liver Enzymes A. Transaminases 1. Definition -- enzymes that catalyze the reversible transfer of an amine group (NH2) from an -amino acid to an -keto acid Transaminases participate in a.a. catabolism and biosynthesis. Other products formed are available for carbohydrate metabolic pathways and reactions that result in excretion of ammonia

2.

B.

Alanine Aminotransferase (ALT); (SGPT) 1. 2. Reaction: catalyzes formation of glutamate and pyruvate Tissue sources a. ALT is widely distributed in tissues 2

3.

b. Liver is the predominant source Clinical significance a. Increased ALT activity is primarily liver specific and rarely observed in other conditions Comparison of ALT and AST (ALT/AST ratio) 1) ALT activity usually exceeds AST activity in diseases that result in necrosis of liver tissue (ratio > 1) Cirrhosis and obstructive liver disease greater AST activity (ratio < 1)

b.

2)

4.

Specimen collection and processing a. b. Relatively unaffected by hemolysis Enzymes are stable for 3-4 days refrigerated; freeze for longer storage

5.

Laboratory determinations a. Coupled enzymatic reaction 1. 2. b. LD indicator enzyme reduces pyruvate to lactate with oxidation of NADH Read at 340 nm

Reference range: 6 - 37 U/L

C.

Aspartate aminotransferase (AST); (SGOT) 1. 2. Reaction: transfers an amino group between aspartate and alpha-keto acids Tissue sources -- widely distributed a. b. 3. 4. Cardiac, liver, and skeletal muscle are predominant tissue sources Smaller amounts in kidney, pancreas, and RBCs

Distribution in fluids - plasma/serum, bile, and CSF Clinical significance a. Elevated AST results are diagnostic for specific tissue damage only when compared with other measured enzyme activities (i.e. ALT, CK, LD) 3

b.

Elevated activities 1) Cardiac disorders a. Myocardial Infarctions - peaks at 24 hours, normal within 5 days b. CHF c. Pulmonary embolism Hepatic disorders - cause highest elevations (100X ULN) a. Viral hepatitis b. cirrhosis Skeletal muscle disorders a. Progressive muscular dystrophy b. Muscle trauma (crush injury or surgery)

2)

3)

5.

Laboratory determinations a. Karmen method - coupled enzymatic assay using malate dehydrogenase as indicator and monitors change in absorbance at 340 nm as NADH is oxidized to NAD Reference range: 5 - 30 U/L

b. 6.

Specimen handling a. Avoid hemolysis; erythrocytes contain AST and can increase AST values tenfold Stable 3-4 days refrigerated

b. D.

glutamyltransferase (GGT) 1. Reaction: catalyzes the transfer of the -glutamyl group from one peptide to another a.a. Physiology a. Function: transport of a.a.s across cell membranes, protein synthesis, regulates glutathione levels Tissue sources 1) Primarily from: a) Kidney b) Pancreas c) Prostrate (88 levels) d) Liver e) Brain 4

2.

b.

c.

Metabolism 1) 2) Enzyme specific for -glutamyl groups Glutathione is common substrate of enzyme in body

d.

Clinical significance 1) In clinical applications, measured in serum mainly to evaluate liver and biliary disorders Liver -- most sensitive enzymatic indicator of hepatobiliary disease a) Enzyme activity is . 5 - 30 X above normal in patients with biliary obstruction Elevation occurs sooner and persist longer that other liver enzymes such as alk phos, ALT and AST Only moderate elevation (2-5 X normal) is seen in infectious hepatitis Small increases are seen with fatty livers or drug intoxication High conc.s of GT will be seen in neoplasms of liver and other tissues Cirrhosis of liver - enzyme of choice in detecting hepatic injury caused by alcohol-induced liver disease

2)

b)

c)

d)

e)

f)

3.

Methods for determination a. Sample 1) Hemolysis does not affect 2) Anticoagulants may depress GT activity by 10 15% 3) Stable 1 week refrigerated Methodology -- kinetic or fixed point 1) Principle - uses substrate -glutamyl-p-nitroanilide to transfer -glutamyl to glycylglycine yielding pnitroaniline (a chromogen) with strong absorbance at 405 nm Reference values: 1) Males: 6 - 45 U/L 5

b.

c.

2)

Females: 5 - 30 U/L (lower do to estrogen/progesterone suppression)

E.

Lactate dehydrogenase (LD); LDH 1. Reaction: catalyzes reversible conversion of lactate to pyruvate accompanied by reduction of NAD+ to NADH a. Lactate - from anaerobic metabolism; by product of emergency source of ATP in decrease oxygen state b. Pyruvate - from aerobic metabolism; normal end product of glycolysis Found in cytoplasm of most cells in the body (increased levels in diseases of the heart, liver, skeletal muscle, kidneys, and RBCs; therefore fairly non-specific and fractionation has more clinical significance) Importance of reaction -- In glycolysis, glucose is oxidized to pyruvate and NADH is produced. When oxygen is limited, reoxidation of NADH is impaired and this in turn impairs glycolysis. Under these conditions, NADH is reoxidized coupled with reduction of pyruvate to lactate and further glycolysis can occur Conversion of pyruvate to lactate when oxygen deficient leads to excess NADH; therefore laboratory testing for LD can be early indicator of oxygen deprivation (tissue hypoxia before pH change) LD isoenzymes a. b. c. d. e. Tetramers (contains four protein subunits) 5 different combinations possible H heart subunits M muscle subunits Interpretation of electrophoretic patterns 1) 2) f. LD1 - fastest migration to the anode in electrophoresis LD5 -slowest migration to the anode in electrophoresis

2.

3.

4.

5.

Sources and normal serum activity 1) LD1 (HHHH) a) heart b) 14-26% of serum LD activity 2) LD2 (HHHM)

a) b) 3)

kidney (acute renal infarction), RBC (hemolytic anemia, hemolyzed specimen), and heart 29-39% of serum LD activity

LD3 (HHMM) a) b) lung (pulmonary embolism, pneumonia), pancreas(pancreatitis), lymphs, and spleen 20-26% of serum LD activity

4)

LD4 (HMMM) a) b) brain, spleen, liver, skeletal muscle 8-16% of serum LD activity

5)

LD5 (MMMM) a) b) liver (liver inflammation), skeletal muscle (skeletal muscle injury) 6-16% of serum LD activity

g.

Isoenzyme separation techniques 1) -hydroxybutyrate dehydrogenase (-HBD) 2) Immunoinhibition 3) Electrophoresis -- best method that allows quantitation of all 5 isoenzymes

6.

Laboratory determinations: Reversible reaction; clinical assays can use either direction. U.S. uses Lactate to Pyruvate; Europe uses Pyruvate to Lactate a. LD-L test 1) Principle: a) b) c) LD catalyzes oxidation of L-lactate to pyruvate with coenzyme NAD+ being reduced to NADH and H+ Rate of change in absorbance is measured at 340 nm LD activity is % to rate of NADH production

7.

8.

Specimen: a) Non-hemolyzed serum; RBCs contain high LD levels, any degree of hemolysis is unacceptable b) Store at room temperature; stable only 48 hours [LD5 most labile if fractionation required test within 24 hours] Reference range: Forward (L to P): 100 - 225 U/L 7

Reverse (P to L): 80 - 280 U/L 9. Clinical significance a. LD1 and/or LD2 1) 2) 3) 4) 5) Hemolysis -- either in vivo or in vitro Anemias -- megaloblastic, hemolytic, pernicious, or acute sickle cell anemias Renal disease or tumors Germ cell tumors ([ LD1) Myocardial infarction (MI) 1 LD (peaks 48-72 hrs) after CK peaks (24-48 hrs) and returns to normal in 10-14 days LD1/LD2 flip a b 3 LD3 1) Massive platelet destruction (transfusions and pulmonary embolism) 2) Lymphatic system involvement a) Inf. mononucleosis (LD5 also ) b) Lymphomas c) Lymphocytic leukemias LD4 and LD5 1) Liver diseases a) Cirrhosis b) All hepatitis c) Passive congestion (congestive heart failure) d) Liver carcinomas LD1 level exceeds LD2 level Occurs in 80% of patients after infarction

Patients with pernicious anemia and renal infarction may have same serum isoenzymes as those with MI

c.

d.

2)

e.

Skeletal muscle a) Injuries - extreme exercise or crushing injuries b) Inflammatory or degenerative diseases - progressive muscular dystrophy in all LD isoenzymes - "isomorphic pattern" 1) Pattern of LD isoenzymes appears normal but total serum LD increases 8

2)

Seen in multisystem diseases or injuries a) Hypoxia b) Extreme hyperthermia c) Congestive heart failure

f.

LD in cerebrospinal fluid 1) 2) 1/10 of serum activities Used for differential diagnosis in bacterial or viral meningitis a. b. High LD activities in 90% of bacterial meningitis High LD activities in 10% of viral meningitis

F.

Alkaline phosphatase (ALP) 1. Reaction: catalyze hydrolysis at an alkaline pH (between 9-10) to liberate phosphate and produce an alcohol ALP presents in most tissue; forms of ALP with specific characteristics are found in: 1) Liver and bone (major sources) 2) Spleen and kidney 3) Intestine 4) Placenta (2nd and 3rd trimester) Precise metabolic function of ALP is not known; however it may be associated with lipid transport in intestine and calcification process in bone Clinical significance of ALP Hepatobiliary disease (obstructive disorders) (activities 2.5 X upper limit) b. Bone disease (highest activities of ALP) c. Hyperparathyroidism (sl. to mod. elevations) d. Physiological elevations (transient elev.s) a. fracture healing b. growth spurts e. Hypophosphatasis - yields 9 in serum activity Method of determination: p-Nitrophenylphosphate (colorless) is hydrolyzed to p-nitrophenol (yellow), the increase in absorbance at 405 nm is proportional to ALP a.

2.

3.

4.

5.

6.

Multiple forms of alkaline phosphatase -- at least 9 isoenzymes of ALP I.D.'d in human serum Reference range: 30 - 90 U/L Specimen: a. Avoid hemolysis (false increase) b. Run ASAP (increases upon standing) c. High fat diet may increase by 25%

7. 8.

G.

Pseudocholinesterase (PChE) 1. Most important applications a. Organophosphate (insecticide) poisoning b. Patient's with reactions to anesthetic (may be genetic); susceptible to prolonged apnea Reaction: PChE acetylcholine <========> choline + acetic acid 3. Sources a. Serum b. Pancreas c. Liver d. Heart e. White matter of the brain (CNS) Specimen: a. Avoid hemolysis b. Stable days refrigerated Reference values a. b. c. d. 7 - 19 U/mL Decreased concentrations are clinically significant PChE concentrations dependent on rate of protein synthesis Indicator of prognosis of CA patients, starvation, burn victims

2.

4.

5.

III.

Cardiac Enzymes A. Aspartate aminotransferase (AST)

10

B. C.

Lactate dehydrogenase (LD) Creatine phosphokinase (CPK) (CK) 1. Reaction catalyzed: reversible phosphorylation CK Creatine + ATP <===========> Creatine phosphate (CrP) + ADP 2. Functions a. CK catalyzes above reaction, which is a source of ATP in muscle metabolism Creatine phosphate provides enough energy to reform high-energy bond of ATP mainly in muscle contraction Reactions involving ADP and ATP require Mg as an activator for maximum activity

b.

c.

d.

3.

Inhibitors include following metal ions: 1) Magnesium (in excess) 2) Manganese 3) Calcium 4) Zinc 5) Copper Sources of creatine kinase a. b. c. d. Skeletal muscle - highest activity Heart Brain Other tissues 1) Kidney 2) Diaphragm Liver and RBCs - NO CK activity Isoenzymes a. b. c. Muscle MM or CK-3 Heart MB or CK-2 Brain BB or CK-1 Normal % 94 - 100 (slowest) <6 0 (fastest)

e. 4.

5.

Clinical significance: when total CK is elevated it is non-specific; separation of isoenzyme. much more significant to diagnosis 11

a.

Marked elevation 1) Myocardial infarction (MI) a) CK is released from heart muscle earlier than any of other enzymes CK-MB > 6% of total CK = AMI CK begins in 4-8 hrs after infarction and peaks at 24-36 hrs

b)

2) 3) 4) 5) NOTE:

Muscular dystrophy (Duchenne type) Polymyositis -- inflammation of many muscle cells at once Convulsive seizures Surgical procedures

Strenuous exercise will increase CK total for up to 48 hours 6) Brain disease a) Cerebrovascular accidents (strokes) will have an CK activity due to release of brain CK into blood b) In contrast to MI, rise will be slower, reaching a peak in 3 days and remaining for about 2 wks

6.

Laboratory determinations a. Colorimetric 1) Phosphorylation of creatine or ADP, read change of NADH to NAD at 340 nm 2) Creatine phosphate from CK reaction is hydrolyzed to yield PO4 a) PO4 formed is quantitated by Fiske-Subbarow method, and read at 620-700nm b) Amt of PO4 formed % to CK activity

b.

Ultraviolet method 1) ATP from CK reaction reacted with glucose using hexokinase and G6PD 2) Change of NADP to NADPH read at 340 nm 3) Reverse reaction using a coupled enzyme Electrophoresis methods - visualizes and measures all of typical and atypical CK isoenzymes Immunochemical methods 1) CK-MB by immunoinhibition 12

c.

d.

a)

Performed on many automated clinical analyzers(Kodak Ektachem) Immunoinhibition = inhibition of enzyme activity of a specific polypeptide subunit by its reaction with Ab M units of CK-MM and CK-MB are inactivated by rxn with anti-M Ab, remaining B subunit activity is measured POS results obtained from this technique should be confirmed by more definitive method electrophoresis

b)

c)

d)

2)

CK-MB by immunoprecipitation a) Immunoprecipitation techniques use monoclonal anti-MM and anti-MB Abs linked to magnetizable particles After centrifugation, anti-CK-B sera precipitate all CK-1 (BB) and CK-2 (MB) from a serum sample, leaving only CK-3 (MM) activity Also, anti-CK-M sera precipitate all CK-3 and CK2, leaving only CK-1 (if present) to be detected Serum content of CK-2 can be measured by calculating difference between total serum CK activity and sum of CK-1 and CK-3 activities

b)

c)

d)

7.

Specimen a. CK is unstable above 370C b. CK is unstable in vitro and has half-life of 6 hrs therefore; serum should be stored at 40C, preferably frozen c. Long term storage at -200C will result in minimal loss of activity over 2 months d. CK activity not affected by slight hemolysis Reference values (serum) a. b. Males: up to 160 IU/L Females: up to 130 IU/L

8.

D.

Troponin I 13

1. 2.

Very specific for cardiac damage First appears 2-6 hours after cardiac damage and continues to rise 24 - 48 hours Measurable levels present about 7 days Reference range: a. 0.3 - 2.0 ng/mL

3. 4.

Values above 2.0 due to: 1) unstable angina 2) congestive heart failure 3) myocarditis 4) cardiac surgery 5) myocardial infarction

5. III.

Specimen - serum or heparinized plasma

Pancreatic Enzymes A. Amylase 1. Hydrolyzes complex carbo.s molecules (starch, glycogen, and dextrins) into maltose molecules (then maltose glucose by enzyme maltase) Starch = amylose and amylopectin Characteristics a. Stability -- stable at rm temperature. at pH 5-11 b. Activator -- chloride Sources of serum amylase Pancreas (primary) -- produced by acinar cells and secreted into intestinal tract by way of pancreatic duct system b. Saliva (secondary) -- produced by salivary gland and initiates hydrolysis of starches in mouth (inactivated in stomach due to acidity) c. Amylase is smallest enzyme, it is filtered by glomerulus, therefore is found in urine Methods of analysis a. Iodometric (Caraway) 1) Principle: Degradation of starch by serum amylase reduces reaction of iodine with starch: reduction in iodine-starch product (Amax = 600 nm) is inversely related to amylase activity 14 a.

2. 3.

4.

5.

2)

Reaction: Starch + I2 -----------> Blue Color Complex amylase Blue color complex -----------> Decreased Color

3)

Common usage but are limited by protein-iodine interaction, require blanks and may lack consistency because of substrate variability

b.

Saccharogenic (Somogyi)- classic reference method 1) Principle: Measure of serum amylase utilizing reducing substance formed by action of amylase on starch substrate. Enzyme's action is directly % to amount of reducing substrate produced Reaction: amylase Starch Substrate -----------> Maltose 3) Most reliable but time consuming and have high sample blanks and variable substrates, hindered by glucose interference and utilizes expensive reagents

2)

c.

Chromolytic - principle: starch + insoluble dye + amylase = soluble dye complex; increase in color (soluble dye) is proportional to amylase conc Specimen 1) Either serum or heparinized plasma 2) Nonacidified urines (random or timed) 3) Stable 1 week at RT; 2 months refrigerated

d.

6.

Clinical significance a. serum levels 1) Acute pancreatitis 2) Carcinoma of pancreas 3) Mumps 4) Administration of opiates/morphine serum levels - not diagnostically helpful 15

b.

B.

Lipase 1. Definition: Enzyme that hydrolyzes ester linkages of triglycerides to yield glycerol and fatty acids a. b. Emulsification -- process by which fat globules are broken into small sizes so that lipase enzyme can act Sources 1) Pancreas (primary source) 2) Tongue -- lingual Lipase Lipase more specific than amylase, but less sensitive

c. 2.

Methods of analysis a. Cherry-Crandall (Classical technique) 1) Olive oil substrate; measure liberated fatty acids by titration after 24 hour incubation (phenolphthalein indicator) Other methods 1) Colorimetric - dye reacts with liberated fatty acids 2) Emulsion clearing -- monitored at 340 or 400 nm, turbidity of oil-water emulsion is reduced as lipase hydrolyze triglycerides; rate of clearing estimates lipase activity Specimen 1) Serum store at 4o C; avoid repeated freezing and thawing 2) No urine lipase 3) Stable 1 week at RT, 3 weeks refrigerated 4) Avoid hemolysis (decreases values)

b.

c.

3.

Clinical significance a. serum levels 1) Acute pancreatitis 2) Pancreatic carcinoma serum - no diagnostic importance

IV.

b. Other Enzymes A.

Acid phosphatase (ACP) 1. Reaction similar to alkaline phosphatase; major difference is pH of activity, optimally at 5.0 ACPs are intracellular enzymes, located inside cell organelles, principally lysosomes 16

2.

3.

Name refers to a group of similar or related enzymes rather than one particular enzyme Sources of ACP a. Prostate gland - Richest source of ACP and 1/3 to 1/2 of enzyme present in sera from healthy males b. Liver c. Spleen d. Bone marrow e. Erythrocytes f. Platelets g. Lysosomes h. Kidney Clinical significance of ACP a. b. c. d. e. f. Carcinoma of prostate - major use is aid in detection of prostate cancer Paget's Disease Hyperparathyroidism with skeletal involvement Malignant invasions of bone Benign (nodular) hypertrophy of prostate concentrations of non-prostatic ACP found in patients with: 1) Gaucher's and Niemann-Pick disease 2) Myelocytic leukemia 3) Hematological disorders with increased destruction of platelets ACP is present in very high concentration in semen. ACP is used in forensic medicine in investigation of rape cases and similar offenses

4.

5.

g.

6.

Measurement of ACP same as alk phos but at acid pH a. End point: Thymolphthalein monophosphate (TMP) is hydrolyzed by prostatic ACP at a pH of 5.4 at 37 oC. Reaction is stopped after 30 minutes by addition of NaOH-NaCO3 solution Immunological methods for prostatic ACP (PAP) 1) Radioimmunoassay (RIA) 2) Enzyme immunoassay = immunoprecipitation

b.

7.

ACP activity in serum Males < 60 years: 30-90 U/L Females < 60 years: 20-80 U/L Adults > 60 years: 30-90 U/L

B.

Type I Cholinesterase; (ACHe) 17

1.

2.

3.

Nomenclature a. Acetylcholine acetylhydrolase b. "True" cholinesterase c. RBC cholinesterase d. Neurotransmitter Sources a. Nerve endings (major source) b. Red blood cells c. Lungs d. Spleen e. Gray matter of brain Laboratory determinations -- manual method (pH change) a. Specimen - no hemolysis allowed b. Reaction: measure decrease in pH CHE Acetylcholine -------> choline + acetic acid

4.

Clinical significance a. b. c. Cholinesterase concentrations in serum may be used as test for liver function May indicate possible insecticide poisoning May detect patients with atypical forms of enzyme

C.

Glucose-6-Phosphate Dehydrogenase (G-6-PD) 1. Catalyzes oxidation of G-6-phosphate 2. First step in pentose phosphate shunt of glucose metabolism in production of NADPH 3. Tissue sources: a. adrenal cortex b. spleen c. thymus d. lymph nodes e. RBC (very little in normal serum) 4. Role in RBC: maintain NADPH in its reduced form, in order to regenerate glutathione (in reduced state) to protect hemoglobin from oxidation Decreased G-6-PD = decreased NADPH = decreased glutathione = increased oxidation = increased hemolysis in presence of oxidizing drug such as primaquine (anti-malarial) Deficiency = Sex linked, more prevalent in African American population Increased levels associated with 18

5.

6. 7.

a. b. 8.

Myocardial infarctions megaloblastic anemias

Assay serum for elevations a. Reference range 0.0 0.18 U/L Assay hemolysate for deficiencies a. Reference range 10-15 U/g hgb

9.

19