[201x3] Unit 5: Enzymes

ACP Reference range

Acid Phosphatase : prostatic ACP: 0-3.5 ng/mL Large amounts found in seminal fluid; proved useful in forensic clinical chemistry, particularly vaginal washings are examined for the presence of seminal fluid in suspected rape cases.

ALT Reference range

Alanine Aminotransferase: 7-45 U/L (37°C).

ALP Reference range

Alkaline Phosphatase: 42-128 U/L (30°C) (M/F 20-50 y/o) Elevations found in pregnancy.

ALP Isoenzymes

Alkaline Phosphatase: Major isoenzymes found in: 1. Liver: fasted to migrate on ELP. 2. Bone: Most heat labile fraction. 3. Intestine. 4. Placenta.

AMS Reference range

Amylase: serum: 28-100 U/L; urine: 1-15 U/hour.

AST Reference range

Aspartate Aminotransferase: 5-35 U/L (37°C). AST levels 100 times normal in liver diseases

Describe the isoenzymes and tissue origins of the following: CK

CKMB: cardiac tissue contain significant amounts of this isoenzyme. Ordered within a CK total. Performed on an automated instrument using immunoassay methodology. CKMM: (healthy one) mostly muscle. major iosenzyme in healthy people. CKBB: brain. ELP can be ordered to detect the isoenzymes

CK Reference range

Creatine Kinase: Male: 46-171 U/L (37°C). Female: 34-145 U/L (37°C).

enzyme, substrate, product relationship relationship

E + S ➡ ES ➡ E + P

Describe how enzymes function.

Each contains an active and an allosteric site. Active site often a water-free cavity where the substance on which the enzyme acts, the substrate, interacts with particular charged amino acid residues. Allosteric site cavity other than the active site where regulator molecules may bind.

An elevated LD level would be a non-specific finding. WHY?

Elevation is not very specific; physician may order isos. to differentiate. Highest levels seen in Pernicious Anemia and Hemolytic disorders. Pernicious anemia: A decrease in red blood cells when the body can't absorb enough vitamin B-12. Hemolytic: relating to or involving the rupture or destruction of red blood cells.

Describe enzyme measurement

Enzymes are to isolate due to small quantities in body, so instead methods measure the catalytic activity and relate this activity to concentration. First Order and Zero Order Kinetics.

Enzymes catalyze reactions by ___________ activation energy level

Enzymes catalyze reactions by *lowering* activation energy level

Compare/contrast first order reaction with zero order reactions.

First-order Kinetics: Reaction rate is dependent on substrate concentration. Zero-order Kinetics: Reaction rate is dependent on enzyme concentration.

Found in all body tissues, enzymes frequently appear in the _________ following _________ _________, or sometimes, in smaller amounts, from ___________ _________.

Found in all body tissues, enzymes frequently appear in the *serum* following *cellular injury*, or sometimes, in smaller amounts, from *degraded cells*.

Describe enzyme reporting procedures

IUB International Union of Biochemistry System assigns name & code to each enzyme

Influencing Factors on Enzymatic Reactions: Cofactors

Influence the rate of reaction.

"LD flipped pattern"

LD1 will increase and be greater than the LD2. Note: Pernicious Anemia could also cause a flip and any preanalytical hemolysis in serum. LD is not used as a cardiac marker, but conditions involving cardiac damage OR intravascular hemolysis of rbcs the serum.

LD isoenzymes

LD1: fastest migrating on ELP; heart and RBCs (found in AMI and hemolytic anemia). LD2: normal healthy major isoenzyme. LD3: spleen, lungs, others. pulmonary and carcinomas (found in pulmonary edema). LD4, LD5: liver, skeletal and hepatic disorders (liver disorders).

Discuss what enzymes are useful in the diagnosis of various disorders, including cardiac, hepatic, bone, muscle, malignancies and acute pancreatitis.

LD: Lactate Dehydrogenase. AMS: Amylase. G-6-PD: Glucose-6-Phosphate Dehydrogenase AST(SGPT): Aspartate Aminotransferase. LIP, LPS: Lipase. ALT: Alanine Aminotransferase. CK: Creatine Kinase . GGT: γ-Glutamyltransferase. ALP: Alkaline Phosphatase. ACP: Acid Phosphatase.

LD Reference range

Lactate Dehydrogenase: 125-220 U/L (37°C).

LPS Reference range

Lipase: <38 U/L. Both amylase and lipase are hydolases and involved in the breakdown of starch and glycogen.

Influencing Factors on Enzymatic Reactions: pH

Most reaction occur in range of 7.0 to 8.0. Changes in pH can denature an enzyme

Influencing Factors on Enzymatic Reactions: Temperature

Most reactions performed at 37 oC. Increasing temp increases rate of reaction. High or low temps denaturation enzyme.

Influencing Factors on Enzymatic Reactions: Inhibitors

Presence can interfere with a reaction. Can be reversible or irreversible.

Describe the enzyme structure

Primary structure composed from a specific amino acid sequence. Resultant polypeptide chains twist to form secondary structure, then folds into tertiary structure. Enzyme may have more than 1 polypeptide unit; spatial relationship between those is quaternary structure.

Zero-order kinetics

Reaction rate dependent on enzyme concentration only: When product forms, excess enzyme combines with excess free substrate. A plateau is reached where the reaction rate is independent of substrate concentration; The enzyme cannot work any faster, all the enzyme is bound to substrate, and the reaction is based on enzyme activity only.

Enzymes

Specific biologic proteins that catalyze biochemical reactions without altering the equilibrium point of the reaction or being consumed or changed in composition.

Name and describe the factors that influence enzyme reactions.

Substrate Concentration. Enzyme Concentration. pH. Temperature. Cofactors. Inhibitors.

Influencing Factors on Enzymatic Reactions: Enzyme Concentration

The higher the enzyme level, the faster the reaction

First-order Kinetics

The reaction rate is directly proportional to the substrate concentration. With enzyme excess, the reaction rate steadily increases as more substrate is added until the substrate saturates all available enzymes

Amylase (AMS)

Tissue source: acinar cells of pancreas & salivary glands. Diagnostic significance: acute pancreatitis, disorders causing salivary gland lesions (mumps, parotitis), intraabdominal diseases. Method: four main approaches: amyloclast, saccharogenic, chromogenic, continuous monitoring. Reference range: serum: 28-100 U/L; urine: 1-15 U/hour.

Glucose-6-Phosphate Dehydrogenase (G-6-PD)

Tissue source: adrenal cortex, spleen, thymus, lymph nodes, lactating mammary gland, erythrocytes. Diagnostic significance: G-6-PD deficiency (an inherited sex-linked trait), which can be clinically manifested in drug-induced hemolytic anemia; myocardial infarction, megaloblastic anemias.

Aspartate Aminotransferase AST; old name SGPT

Tissue source: cardiac tissue, liver, skeletal muscle. Diagnostic significance: hepatocellular disorders (viral hepatitis, cirrhosis), skeletal muscle disorders (muscular dystrophies, inflammatory conditions), pulmonary embolism. Assay for enzyme activity: based on Karmen method. Source of error: hemolysis; stable in serum for 3-4 days at refrigerated temperatures. Reference range: 5-35 U/L (37°C). AST levels 100 times normal in liver diseases

Lactate Dehydrogenase (LD)

Tissue source: heart, liver, skeletal muscle, kidney, erythrocytes (found in many tissues) Elevated in variety of disorders, cardiac, hepatic, skeletal and renal diseases (non-specific enzyme) also erythrocytes. Diagnostic significance: pernicious anemia, hemolytic disorders, viral hepatitis, cirrhosis, acute myocardial infarction, pulmonary infarct, skeletal muscle disorders, leukemia. Assay for enzyme activity: catalyzes interconversion of lactic & pyruvic acids using coenzyme NAD, in either forward or reverse direction. Source of error: any degree of hemolysis; instability in any temperature. Reference range: 125-220 U/L (37°C).

Alkaline Phosphatase (ALP); non-specific enzyme.

Tissue source: intestine, liver, bone, spleen, placenta, kidney. Diagnostic significance: hepatobiliary (biliary tract obstruction) & bone (Paget's disease, osteomalacia, rickets, hyperparathyroidism, osteogenic sarcoma) disorders. Assay for enzyme activity: various methodologies are used, including a continuous-monitoring technique (Bowers & MaComb). Source of error: hemolysis; assays should be run as soon as possible after collection; high-fat meal. Reference range: 42-128 U/L (30°C) (M/F 20-50 y/o) Elevations found in pregnancy.

γ-Glutamyltransferase (GGT)

Tissue source: kidney, brain, prostate, pancreas, liver. Diagnostic significance: hepatobiliary disorders (biliary tract obstruction), hepatic parenchyma, alcoholism, acute pancreatitis, diabetes mellitus, myocardial infarction. Assay for enzyme activity: y-glutamyl-p-γ nitroanilide is most widely accepted substrate used in GGT analysis; -glutamyl residue is transferred γ to glycylglycine, releasing p-nitroaniline. Source of error: stable for 1 week at 4°C; hemolysis not a concern. Reference range: male: 6-55 U/L (37°C); female: 5-38 U/L (37°C). GGT is very good indicator of liver damage from alcoholism also in chronic alcoholism.

Alanine Aminotransferase (ALT)

Tissue source: liver. Diagnostic significance: hepatic disorders. Assay for enzyme activity: coupled enzymatic reaction using lactate dehydrogenase as indicator enzyme, which catalyzes reduction of pyruvate to lactate with simultaneous oxidation of NADH. Source of error: stable for 3-4 days at 4°C; relatively unaffected by hemolysis. Reference range: 7-45 U/L (37°C).

Lipase (LPS)

Tissue source: primarily in pancreas; also in stomach & small intestine. Diagnostic significance: acute pancreatitis, other intra-abdominal diseases (penetrating duodenal ulcers, perforated peptic ulcers, intestinal obstruction, acute cholecystitis). Assay for enzyme activity: estimation of liberated fatty acids (triolein is substrate), turbidimetric methods, colorimetric methods. (old method Cherry-Crandall used olive oil substrate) Reference range: <38 U/L. Both amylase and lipase are hydolases and involved in the breakdown of starch and glycogen.

Acid Phosphatase (ACP)

Tissue source: prostate, bone, liver, spleen, kidney, erythrocytes, platelets. Diagnostic significance: prostatic carcinoma, hyperplasia of prostrate, prostatic surgery, osteoclasts, Paget's disease, breast cancer with bone metastases, Gaucher's disease. Assay for enzyme activity: same techniques as in alkaline phosphatase, except performed in an acid pH. Source of error: Serum should be separated from red cells as soon as blood has clotted; serum should be used immediately, frozen, or acidified. Reference range: prostatic ACP: 0-3.5 ng/mL Large amounts found in seminal fluid; proved useful in forensic clinical chemistry, particularly vaginal washings are examined for the presence of seminal fluid in suspected rape cases.

Creatine Kinase (CK)

Tissue source: skeletal muscle, heart muscle, brain tissue. Diagnostic significance: acute myocardial infarction, muscular dystrophy; cerebral vascular accident, seizures, nerve degeneration, shock; hypothyroidism, malignant hyperpyrexia, Reye's syndrome. Assay enzyme activity: Catalyzes both forward & reverse reactions involving phosphorylation of creatine or ADP. Source of error: Hemolysis of serum samples may elevate CK activity Serum should be stored in a dark place, because CK is inactivated by light. Reference range: Male: 46-171 U/L (37°C); Female: 34-145 U/L (37°C).

What reactions in the clinical lab are measured in.

Zero-order Kinetics; The rates are independent of substrate concentration, and are equal to some constant k.

cofactor

a nonprotein molecule that may be necessary for enzyme activity

active site

a region on an enzyme that binds to a protein or other substance during a reaction.

substrate

a substance or layer that underlies something, or on which some process occurs, in particular; the substance on which an enzyme acts.

product

a substance produced during a natural, chemical, or manufacturing process.

catalyst

a substance that increases the rate of a chemical reaction without itself undergoing any permanent chemical change.

coenzyme

an enzyme activator

1. Oxidoreductases

catalyze an oxidation-reduction reaction between two substrates

3. Hydrolases

catalyze hydrolysis of various bonds

5. Isomerases

catalyze interconversion of geometric, optical, or positional isomers

6. Ligases

catalyze joining of 2 substrate molecules, coupled with breaking of pyrophosphate bond in ATP

4. Lyases

catalyze removal of groups from substrates without hydrolysis; product contains double bonds

2 Transferases

catalyze transfer of a group other than hydrogen from one substrate to another

isoenzyme

different forms of an enzyme that may originate from genetic or nongenetic causes and may be differentiated from each other based on certain physical properties, such as electrophoretic mobility, solubility, or resistance to inactivation.

Activation energy

energy required to raise all molecules in 1 mole of a compound at certain temperature to transition state at peak of energy barrier

Catalyzed reactions are frequently specific and essential to physiologic functions such as ____

hydration of carbon dioxide, nerve conduction, muscle contraction, nutrient degradation, energy use, *coagulation*.

international unit (IU)

the amount of enzyme that will catalyze the reaction of 1 um of substrate per minute, under specified conditions of temperature, pH, substrates, and activators.

GGT Reference range

γ-Glutamyltransferase: male: 6-55 U/L (37°C). female: 5-38 U/L (37°C). GGT is very good indicator of liver damage from alcoholism also in chronic alcoholism.