Enzymology and Clinical Chemistry Notes

INTRODUCTION TO ENZYMOLOGY

  • Biological protein catalysts that accelerate biochemical reactions.

  • Lower activation energy, found in all tissues.

  • Elevated serum enzyme levels indicate tissue damage.

SUMMARIZED CLASSES OF ENZYMES

  • Based on EC code:

    • Oxidoreductases: Oxidation-reduction (e.g., LDH, G-6-PD).

    • Transferases: Transfer of functional groups (e.g., CK, AST/ALT).

    • Hydrolases: Hydrolysis (e.g., Amylase, Lipase).

    • Lyases: Double bond formation by group removal (e.g., Aldolase).

    • Isomerases: Isomer interconversion (e.g., Triose phosphate isomerase).

    • Ligases: Bond formation with ATP hydrolysis (e.g., Glutathione synthetase).

ENZYME CLASSES

1. OXIDOREDUCTASES

  • Catalyze oxidation-reduction reactions.

  • Examples: LDH, G6PD, MDH, GLDH.

2. TRANSFERASES

  • Catalyze the transfer of functional groups.

  • Clinically notable subclasses:

    • Kinases: Transfer phosphate groups (e.g., CK, PK).

    • Aminotransferases: SGOT/AST, SGPT/ALT.

    • Transpeptidases: GGT.

3. HYDROLASES

  • Catalyze hydrolysis reactions.

  • Examples: Amylase, Lipase, Trypsin, ALP, ACP, ACE.

4. LYASES

  • Catalyze group removal to form double bonds.

  • Example: Aldolase.

  • Functions include intramolecular rearrangement and cleavage of bonds without hydrolysis.

5. ISOMERASES

  • Catalyze interconversion of isomers.

  • Not clinically useful.

6. LIGASES

  • Catalyze joining of molecules with ATP breakdown.

  • Not clinically useful.

  • Example: Glutathione Synthetase

ENZYME KINETICS

Michaelis-Menten Hypothesis:

  • Enzyme-substrate complex formation leads to product conversion.

  • Enzymes are not consumed.

  • V=V<em>max×[S]K</em>m+[S]V = \frac{V<em>{max} \times [S]}{K</em>m + [S]}

  • Km is the substrate concentration at Vmax/2V_{max}/2.

  • First-order kinetics: Rate depends on substrate concentration.

  • Zero-order kinetics: Rate is at Vmax, independent of substrate.

Lineweaver-Burke Plot

  • Double reciprocal plot of Michaelis-Menten equation.

  • 1V=K<em>mV</em>max×1[S]+1Vmax\frac{1}{V} = \frac{K<em>m}{V</em>{max}} \times \frac{1}{[S]} + \frac{1}{V_{max}}

FACTORS AFFECTING ENZYME LEVELS

a. Substrate and Enzyme Concentration

  • First-order: [E] > [S], rate proportional to [S].

  • Zero-order: [S] < [E], rate depends on enzyme concentration.

b. pH

  • Optimum: pH 7-8 (except ACP and ALP).

  • ACP: pH 4-5.

  • ALP: pH 9-10.

c. Optimum Temperature

  • Rate doubles for each 10°C increase, up to denaturation (40-50°C).

d. Cofactors

  • Activators: Inorganic (e.g., Mg, Zn, Cl-).

  • Coenzymes: Organic (e.g., NAD, Pyridoxal phosphate).

e. Inhibitors

  • Competitive: Bind to active site, ↑ [S] reverses inhibition.

  • Non-competitive: Bind elsewhere, no effect from ↑ [S].

  • Uncompetitive: Bind to enzyme-substrate complex, ↑ [S] increases inhibition.

MEASUREMENT OF ENZYME ACTIVITY

  • Measured as activity (rate) rather than concentration.

Activity Units:

  • International Unit (IU): 1 µmol/min.

  • Katal: 1 mol/sec.

  • 1IU=0.0167μkat1 IU = 0.0167 \mu kat

Methods

  • Fixed Time (Endpoint).

  • Continuous Monitoring (Kinetic).

  • Multipoint Analysis.

CLINICAL ENZYMOLOGY

A. CREATINE KINASE

  • Isoenzymes: BB (Brain), MB (Heart), MM (Muscle).

  • CK-MB: ↑ in myocardial infarction.

  • CK-MM: ↑ in Duchenne’s muscular dystrophy.

  • Specimen considerations: Avoid hemolysis and chelators.

Laboratory Methods

  • UV methods measure absorbance change at 340 nm.

  • Tanzar Gilvarg Assay: Forward reaction, pyruvate kinase, pH 9.

  • Oliver-Rosalki Assay: Reverse reaction, hexokinase-g-6-p-d-NAD pH 6.8.

B. LACTATE DEHYDROGENASE

  • Not tissue-specific, least clinically useful among cardiac biomarkers.

  • Isoenzymes: LD1 (Heart, RBCs), LD2, LD3 (Lungs), LD4 (Liver), LD5 (Skeletal muscle).

  • Flipped ratio LD1 > LD2 indicates MI.

  • Specimen considerations: Hemolysis increases values.

C. AMINOTRANSFERASES

1. ALT/SGPT

  • Tissue source: Liver.

  • Diagnostic Significance: Acute hepatocellular disorder.

2. AST/SGOT

  • Tissue source: Liver, heart, skeletal muscles.

  • Diagnostic significance: Acute hepatocellular disorders, MI.

  • De Ritis Ratio: <1 in acute hepatocellular disorder.

D. ALKALINE PHOSPHATASE

  • Isoenzymes: Liver, Bone, Placenta, Intestinal.

  • Isoenzyme Analysis: Electrophoresis, Heat Stability, Chemical Inhibition (Phenylalanine,Levamisole).

  • ALP Determination: Bowers-McComb (p-nitrophenylphosphate).

E. PANCREATIC ENZYMES

1. AMYLASE

  • Tissue sources: Salivary glands, Pancreas.

  • Diagnostic significance: Acute pancreatitis.

2. LIPASE

  • Tissue source: Pancreas.

  • Diagnostic Signficance: Acute Pancreatitis.

  • More pancreas-specific than amylase.

F. MISCELLANEOUS ENZYMES

1. GAMMA GLUTAMYL TRANSFERASE

  • Hepatobiliary disorders, alcoholic liver disease.

  • Used to identify the source of increased ALP

3 ACID PHOSPHATASE

  • Major sources: Prostate (richest source), RBCs

  • Significance:Prostatic carcinoma

ELECTROLYTES AND BLOOD GASES
Regulatory Factors

1. Antidiuretic Hormone (ADH)

  • Stimulates water reabsorption in the collecting duct of the nephrons

2. Atrial Natriuretic Peptide (ANP)

  • Promotes excretion of sodium

3. Renin-Angiotensin-Aldosterone System (RAAS)

  • Increases plasma sodium

Important Electrolytes

1. Sodium

  • Major extracellular cation

  • Reference range: 136 - 145 mmol/L

2. Potassium

  • Major intracellular cation

  • Reference values: 3.5 - 5.0 mmol/L

3. Chloride

  • Major extracellular anion

  • Reference range: 98 - 106 mmol/L

4. Bicarbonate

  • Major buffer in blood

  • Reference range: 22 - 28 mmol/L

5. Calcium

  • Essential for blood coagulation and muscle contraction

  • Reference range: 8.5 - 10.5 mg/dL

6. Magnesium

  • Involved in numerous enzymatic reactions

  • Reference range: 1.5 - 2.5 mg/dL

BLOOD GASES

  • Include measurements of oxygen and carbon dioxide levels in blood.

  • Important for assessing respiratory and metabolic function.

  • pH, pCO2, pO2, and HCO3 are the key parameters analyzed.