CC-2

Enzymes

Enzymes are biological proteins that catalyze biochemical reactions without altering the equilibrium point or being consumed. They are found in all body tissues and increase in serum after cell injury.

Function of Enzymes

• Hydration of Carbon dioxide (respiration)
• Nerve induction
• Muscle contraction
• Nutrient degradation
• Growth and reproduction
• Energy Storage and use

General Properties of Enzymes

Components of Enzymes

• Active site: A water-free cavity where the substrate binds and undergoes a chemical reaction.
• Allosteric site: A cavity other than the active site that binds a regulatory molecule (effector).
• Isoenzyme: Enzymes with similar enzymatic activity but differing in physical, biochemical, and immunologic characteristics.
• Isoform: Results from posttranslational modifications of an enzyme.
• Apoenzyme: The protein portion of the enzyme which is subject to denaturation and loses its activity.
• Holoenzyme: The active enzyme/substrate complex formed by the combination of a coenzyme and an apoenzyme.
• Zymogen/Proenzyme: Inactive enzyme precursor (e.g., coagulation cofactors, digestive enzymes).

Terms Associated with Enzymes

• Enzyme activity: The rate at which an enzyme converts substrate to product.
• Substrate: The substance acted upon by enzymes, specific for each enzyme.

General Form of Enzyme Reaction

$[E] + [S] = [ES] – [E] + [P]$

• Cofactor: A non-protein substance added to the enzyme-substrate complex to manifest enzyme activity.

  • Coenzyme/Prosthetic Group: An organic cofactor that binds tightly to the enzyme (e.g., NAD, NADP, Vitamins).
  • Activator: An inorganic cofactor, often a metal ion (e.g., Cl, Mg, Cu, Zn), that alters spatial configuration for substrate binding.

Enzyme Classification

• Oxidoreductase: Catalyzes REDOX reactions between two substances (e.g., Lactate dehydrogenase, Glucose-6-phosphate dehydrogenase).

• Transferase: Catalyzes the transfer of a group (other than hydrogen ion) from one substrate to another (e.g., ALT, AST, GGT, CK).

• Hydrolase: Catalyzes hydrolysis of bonds by adding a water molecule.

  • Esterase (e.g., ALP, ACP, Cholinesterase, Lipase) - splits esters into acid and an alcohol
  • Peptidase (e.g., trypsin, pepsin, leucine amino peptidase) - breaks down peptides into amino acids
  • Glycosidase (e.g., amylase, galactosidase) - catalyze the hydrolysis of glycosidic bonds in complex sugars

• Lyases: Catalyzes removal of groups of substances without hydrolysis (e.g., Aldolase, pyruvate decarboxylase, glutamate decarboxylase, tryptophan decarboxylase).

• Isomerase: Catalyzes the interconversion of geometric, optical, or positional isomers (e.g., Triphosphate isomerase, ribose phosphate isomerase, glucose phosphate isomerase).

• Ligase: Catalyzes the joining of two substrate molecules coupled with breaking of pyrophosphate bond in ATP or a similar compound (e.g., Glutathione synthase).

Enzyme Kinetics

Factors that Influence Enzymatic Reactions

1. Substrate concentration

   • First Order Kinetics when reaction rate is directly proportional to Substrate Concentration (Enzyme excess).

2. Enzyme concentration

   • Zero Order Kinetics when reaction rate depends on Enzyme Concentration (Substrate excess).

3. pH

   • Most physiologic reactions occur in the pH range of $7 – 8$, controlled by a buffer solution.

4. Temperature

   • Increased temperature typically increases the rate of a chemical reaction. For every $10$ degrees Celsius increase, the reaction rate will approximately double.
   • Enzymes are active at $25$, $30$, and $37$ degrees Celsius (analysis temperature for routine enzyme measurement).
   • $+/- 0.1$ degrees Celsius is important for incubation temperatures.
   • Temperatures between $40 – 50$ degrees Celsius can cause significant denaturation of enzymes.

5. Cofactor

   • Non-protein entities that must bind to enzymes before reaction.

   • Activator (inorganic): alters spatial configuration for substrate binding, linking, etc.

     • Metallic (e.g., $Ca^{2+}$, $Fe^{2+}$, $Mg^{2+}$, $Mn^{2+}$. $Zn^{2+}$, $K^+$)
     • Non-metallic (e.g., $Cl^-$, Br^-$)

   • Coenzymes (prosthetic group, organic): serves as a secondary substrate (e.g., NAD)

6. Inhibitor

   • Competitive Inhibitor: Binds to the active site (reversible).
   • Non-Competitive Inhibitor: Does not bind to the active site but to the allosteric site (reversible or irreversible).
   • Uncompetitive Inhibitor: Binds to the Enzyme-Substrate Complex, increasing the substrate concentration and resulting in more ES complexes to which the inhibitor binds, increasing inhibition.
   • Mixed inhibitor: Binds to either E or ES complex at a different site from the substrate active site.

Measurement of Enzyme Activity

Enzyme Quantitation

• Measurement of catalytic enzymes/ activity then Activity is related to the concentration

• Common methods: Photometric

• Must be performed during linear phase of reaction

  • Increase in product concentration
  • Decrease in substrate concentration
  • Decrease in coenzyme concentration
  • Increase in concentration of altered coenzyme

General Methods of Measuring Enzymatic Reactions

• Fixed time (End point): Reagents are combined, and the amount of reaction is measured after a designated time; measures the concentration after the endpoint has been reached. The reaction is assumed to be linear; the larger the reaction, the more enzyme present.
• Continuous time/ Kinetic assay: Multiple measurements are made during the reaction at specific time intervals (usually 30-60 seconds); continuous measurement using a spectrophotometer to record.

Clinical Significance of Enzymes

A. MI (Myocardial Infarction) Profile

Includes:

• Creatine Kinase (CK)
• Aspartate aminotransferase (AST)
• Lactate Dehydrogenase (LDH)

1. Creatine Kinase (CK)

• Involved in storage of high energy creatine phosphate in muscle cells.

• Highest activity is in skeletal muscle, heart, and brain tissue.

• Used to assess Myocardial Infarction

• MW: 82,000

• Associated with ATP regeneration in contractile and transport systems

• Isoenzymes:

  • CK – 1 (CK-BB) Brain type

    • Migrates fastest toward the anode followed by CK-MB
    • Serum rarely contains CK-BB of brain because of its molecular size (80,000); its passage across the BBB is Hindered
    • Most techniques cannot detect CK-BB in normal serum
    • Increased in → CNS Shock, Seizures, childbirth → Lung and gastrointestinal diseases → Carcinoma of various organs; useful for tumor associated marker

  • CK – 2 (CK-MB) Hybrid type

    • Values for MB range should be <6=/ >6<6128,000

    • Increased levels are seen in:

      • Cardiac diseases
      • Hepatic diseases
      • Skeletal diseases
      • Renal diseases
      • Hematologic and Neoplastic disorders

    • Marker for AMI:

      • Rise: 12-24 hours
      • Peak: 48-72 hours
      • Return to Normal: 10 days (best indicator for delayed check-ups due to chest pain)

    ISOENZYME	TISSUE	DISORDER
    LDH-1	Heart RBC	Myocardial infarction Hemolytic anemia
    LDH-2	Heart RBC	Megaloblastic anemia Acute renal infarct
    		Hemolyzed specimen
    LDH-3	Lung	Pulmonary embolism Pulmonary pneumonia
    	Lymphocyte	Lymphocytosis Acute pancreatitis Carcinoma
    	Spleen pancreas
    LDH-4	Liver	Hepatic injury or inflammation
    LDH-5	Skeletal muscle	Skeletal muscle injury

    • Each isoenzyme contains 4 polypeptide chains with MW of 32,000 each

    • H = Heart, M = Muscle

    • LD-1 fastest; most anodal

    • LD-2

    • LD-3

    • LD-4

    • LD-5 slowest; most cathodal

    • Normal isoenzyme concentration of healthy individuals:

      • LD2 - LD1 - LD3 - LD4 - LD5

    • “LDH Flipped Pattern”

      • LDH1 > LDH2 - LD3 - LD4 - LD5
      • Occurs in:
        • AMI
        • Intravascular Hemolysis
        • Hemolyzed Specimen

    MI PROFILE	RISE	PEAK
    CK	4 – 8 HOURS	12 – 24 HOURS
    AST	8 – 12 HOURS	24 HOURS
    LDH	12 – 24 HOURS	48 – 72 HOURS

    Liver Enzymes

    Includes:

    • Alanine aminotransferase (ALT)
    • Alkaline phosphatase (ALP)
    • Gamma-glutamyltransferase (GGT)
    • Lactate dehydrogenase (LHD)
    • Aspartate aminotransferase (AST)

    1. Alanine Aminotransferase (ALT)

    • Formerly called – Serum glutamic-pyruvic transaminase (SGPT or GPT)
    • Similar activity to AST, catalyzes transfer of an amino group from alanine to a-ketoglutarate with the formation of glutamate and pyruvate
    • Distributed in many tissues, highest concentration in the liver. It is the more liver specific enzyme among the transferases.
    • Used to evaluate hepatic disorders (ALT elevations are higher than AST)
    • Cardiac tissue contains small amount of ALT activity.

    2. Alkaline Phosphatase (ALP)

    • Catalyzes hydrolysis of phosphomonoesters at an alkaline pH

    • Cleaves inorganic phosphate group of the phosphomonoesters from organic phosphate ester with concomitant of alcohol and producing phosphate ion

    • Not specific, can react with many different substrate

    • More active in an alkaline pH (optimal pH: 9.0 – 10.0)

    • Requires Mg^{2+} activator

    • High concentrations are found in intestine, liver, bone, spleen, placenta, and kidney

    • Used in the evaluation of hepatobiliary and bone disorder

    • Electrophoresis – most useful single technique for ALP isoenzyme

    • Isoenzymes

      • Liver ALP isoenzymes

        • Increased in liver diseases
        • Migrates fastest

      • Bone ALP isoenzymes

        • Increased in bone disease, healing of bone fractures, and physiologic bone growth

      • Placental ALP isoenzyme

        • Increased in pregnancy

      • Intestinal ALP isoenzyme

        • Increased in GIT disorders

    • ABNORMAL FRACTIONS = “Cardinoplacental ALPs”

    ISOENZYME	COMMENTS	CONDITION
    Regan	Most heat stable	Malignancy Carcinomas (lung, breast, ovarian,
    	65C for 30 minutes
    Nagao	Regan variant	Cancers of pleural surfaces Adenocarcinoma
    	and phenylalanine, and L-
    	leucine inhibition

    3. Gamma-Glutamyltransferase (GGT)

    • Catalyzes transfer of the gamma-glutamyl residue from peptides to amino acids or water molecules

    • Present in cells in bile and hepatic duct. Also, in brain, prostate, and pancreas

    • Increased in:

      • Hepatobiliary disorders (biliary tract obstruction)
      • Chronic alcoholism
      • Patients in enzyme inducing medications: warfarin, phenobarbital, phenytoin
      • Smoking; 10% increase for moderate smokers, 20% increase for heavy smokers

    • Useful in differentiating the source of an elevated ALP because:

      • GGT levels are increased in liver disorders
      • GGT levels are normal in skeletal disorders and pregnancy

    Pancreatic Enzymes

    Includes:

    • Amylase
    • Lipase

    1. Amylase (AMY)

    • Belong in hydrolases; breakdown of starch ang glycogen

    • Found in acinar cells of pancreas and salivary glands

    • Increased in acute pancreatitis but also a non-specific

    • Requires calcium and chloride for activation

    • Smallest enzyme (MW: 50,000 – 55,000) that is why it is easily filtered and the ONLY protein that can be cleared by the kidneys

    • Amylase levels during acute pancreatitis

      • Rise: 2-12 hours (5 – 8 book)
      • Peak 24 hours
      • Normalize 3-5 days

    • Other elevations are seen in:

      • Mumps and parotitis (salivary gland lesions)

      • Diabetic ketoacidosis

      • Renal insufficiency

      • Intraabdominal diseases

        • Peptic ulcer, intestinal obstruction
        • Cholecystitis, acute appendicitis
        • Ectopic pregnancy, mesenteric infarction

    • Macroamylasemia

      • Results when AMS molecules combine with immunoglobulins to form a complex that is too large to be filtered across the glomerulus
      • Serum AMS levels increase because of the reduction in renal clearance of the enzyme

    • Isoenzyme

      • P – type (amylopsin)
        • Derived from pancreatic tissue
        • Pancreatic isoenzyme, migrates more slowly
        • P3 is the most dominant in acute pancreatitis
      • S – type (thyalin)
        • Derived from salivary gland tissue, fallopian tube, and lung
        • Salivary isoenzyme migrates most quickly
        • S – type represents two-thirds of AMS activity in serum

    2. Lipase (LPS)

    • Hydrolyzes ester linkages of fats to produce alcohol and fatty acids

    • Found in pancreas, some in stomach and small intestine

    • Most specific marker for acute pancreatitis

    • Large molecule remains in the circulation for up to 7 days

    • During acute pancreatitis

      • Rise: 4 – 8 hours
      • Peak: 24 hours
      • Normalize: 8-14 days

    Other Enzymes

    Acid Phosphatase (ACP)

    • Prostate enzyme
    • Catalyzes hydrolysis of phosphomonoesters
    • Evaluation of metastatic carcinoma of prostate
    • Forensic investigation of rape cases: 12 hours up to 4 days
    • Non specific
    • Found in: Liver, spleen, kidney, bone, RBCs, platelets, and Prostate – Richest source (prostatic carcinoma)

    Methods of Determination

    Creatine Kinase

    • Tanzer-Gilvarg

      • Forward reaction
      • pH = 9.0
      • Measures decrease in absorbance at 340 nm
      • CK Creatine + ATP → Creatine phosphate + ADP

    • Oliver and Rosalki (modified by this guy)

      • Reverse reaction
      • pH = 6.8
      • 2 – 6X faster than the forward reaction
      • Measures increase in absorbance at 340 nm
      • CK Creatine phosphate + ADP → Creatine + ATP

    Aspartate Aminotransferase

    • Karmen method

      • pH = 7.3 – 7.8
      • Uses malate dehydrogenase (MD) as the indicator enzyme
      • Measures decrease in absorbance at 340 nm
      • Used to diagnose hepatocellular disorders
      • AST Aspartate + α-ketoglutarate → glutamate + oxaloacetate

    Lactate Dehydrogenase

    • Wacker method

      • Forward reaction
      • pH = 8.3-8.9
      • Measures increase in absorbance at 340 nm
      • Source of error
        • Hemolysis – RBCs contain 100X LD concentration than that found in serum
        • Cold – loss of LD-5 activity

    • Wrobleuski-Ladue

      • Reverse reaction
      • pH = 7.2-7.4
      • Measures decrease in absorbance at 340 nm
      • Utilized by dry-slide methods

    Alanine Aminotransferase

    • Coupled enzymatic reaction

      • Uses lactate dehydrogenase (LDH) as indicator enzyme
      • pH = 7.3–7.8
      • Measures change in absorbance at 340 nm
      • ALT Alanine + α-ketogluterate → pyruvate + glutamate

    Alkaline Phosphatase

    • Bower’s and McComb

      • Based on molar absorptivity of p-nitrophenol (yellow color)
      • Uses p-nitrophenylphosphate (colorless) as substrate and hydrolyzes to p-nitrophenol (yellow)
      • Optimal pH = 10
      • Measures increase in absorbance at 405 nm
      • ALP P-nitrophenyl-phosphate → p-nitro-phenol + phosphate ion

    • Methods to determine ALP isoenzymes

      • A. Heat stability

        • Heating serum at 56C for 10mins
        • Placenta – most heat stable (resist heat denaturation at 65C for 30mins)
        • Intestine
        • Liver
        • Bone – heat labile
        • ALP activity is measured before and after heating serum at 56 degrees Celsius for 10 mins.
        • If the residual activity after heating is less than 20% of the total activity before heating, then ALP elevation is assumed to be a result of bone phosphatase.
        • If greater than 20% of the activity remains, the elevation is probably a result of liver phosphatase.
        • Imprecise method due to
          • Correct temperature control
          • Timing
          • Analytic methods sensitive enough to detect small amounts of residual ALP activity

      • B. Electrophoresis – mobility

        • Liver: most anodal
        • Bone
        • Placenta
        • Intestine: least anodal

      • C. Chemical inhibition

        • Phenylalanine – inhibits placenta and intestine
        • L-leucine – inhibits the abnormal nagao isoenzyme
        • Levamisol – inhibits liver and bone
        • 3M urea (synthetic urea) – inhibits bone

    Gamma-Glutamyltransferase

    • SZASZ assay

      • Uses γ-glutamyl-p-nitroanilide as substrate
      • Measures absorbance at 405 – 420 nm
      • GGT activity is stable with no loss of activity for 1 week at 4C
      • Not affected by hemolysis

    Amylase

    • Amyloclastic (Iodometric)

      • Measures disappearance of starch
      • Substrate: starch molecule with iodine
        *Decrease in color is proportional to AMY concentration

    • Saccharogenic (Reference method)

      • Substrate: starch
      • Measures appearance of product

    • Chromogenic

      • Substrate: starch attached with chromogenic dye

    • Continuous coupled

      • Coupling of several enzymes to measure AMS activity
      • Absorbance of NAD+ at 340 nm

    Acid Phosphatase

    • Same as ALP but done in an acid pH

    • Inhibitors

      • L-tartrate ions
      • Formaldehyde and cupric ions

    • Quantitative

      • Thymolpthalein monophosphate

    • Continuous monitoring

      • Alpha-napthyl-phosphate
      • pH 5.0

    Electrolytes

    • Ions capable of carrying an electric charge

    • Can be classified as:

      1. Cations
         • Have positive charge
         • Migrate toward the cathode
         • Examples: Mg^{2+}$,$Ca^{2+}$,$Zn^{2+}$,$Na^+
      2. Anions
         • Have negative charge
         • Migrate toward the anode
         • Examples: Cl^−$,$HCO3^-$,$PO4^-

    Function of Electrolytes

    1. Volume and osmotic regulation

       • Na, K, Cl

    2. Myocardial rhythm and contractility

       • Mg, Ca, K

    3. Important cofactors in enzyme activation

       • Mg, Ca, Zn

    4. Regulation of ATP ion pumps

       • Mg

    5. Maintenance of acid-base balance

       • HCO_3^-, Cl, K

    6. Blood coagulation

       • Mg, Ca

    7. Neuromuscular excitability

       • Mg, Ca, K

    8. Production and use of ATP from glucose

       • Mg, PO_4$$

    Water

    • 40-75% is the average water content of the human body

    • Excess fluid supress ADH

    • Lack of fluid stimulate ADH.

    • Extracellular Fluid (ECF)

      • Represents 1/3 of total body

        • Intravascular ECF (plasma): 93% water
        • Interstitial fluid: Water around the cells, 7%.

    • Intracellular Fluid (ICF)

      • Represents 2/3 of total body water.

    • ADH (Antidiuretic Hormone)

      • AVP (Arginine Vasopressin Hormone)

    Sodium (Natrium)

    • Major extracellular cation (90% of all extracellular cations).

    • Principal osmotic particle outside the cell.

    • Plasma concentration depends greatly on the intake and excretion of water.

    • Reference values: 135-145 mmol/L

    • Threshold critical value:

      • 160 mmol/L
      • 120 mmol/L

    • Hypernatremia: >145 mmol/L

    • Hyponatremia: <135 mmol/L

    Hypernatremia

    1. Excess water loss

       • Diabetic insipidus
       • Renal tubular disorder
       • Prolonged diarrhea
       • Profuse sweating
       • Severe burns

    2. Decreased water intake

       • Older person
       • Infant
       • Mental impairment

    3. Increased intake or retention

       • Hyperaldosteronism
       • Sodium bicarbonate excess
       • Dialysis fluid excess

    Hyponatremia

    1. Excess water loss

       • Hyperadrenalism
       • Potassium deficiency
       • Diuretic use
       • Ketonuria
       • Salt-losing nephropathy
       • Prolonged vomiting or diarrhea

    2. Increased water retention

       • Renal failure
       • Nephrotic syndrome
       • Hepatic cirrhosis
       • Congestive heart failure

    3. Water imbalance

       • Congestive heart failure
       • Excess water intake
       • SIADH Symptoms of inappropriate antidiuretic hormone
       • Pseudohyponatremia With pseudohyperkalemia

    Methods of Determination

    • Flame Emission Photometer
    • Ion Selective Electrode – most common
    • Atomic Absorption Spectrophotometry
    • Colorimetry

    Potassium (Kalium)

    • Major intracellular cation (only 2% of total body potassium circulates in plasma)

    • Single most important analyte in terms of an abnormality being immediately life threatening

    • Function:

      • Heart contraction
      • Neuromuscular excitability
      • Intracellular fluid
      • Volume regulation and hydrogen ion concentration.

    • Reference value: 3.5-5.2 mmol/L

    • Specimen consideration:

      • Hemolysis

      • Plasma levels are lower (0.1-0.7mmol/L) compared to serum levels

      • Muscular activity: Exercise and prolonged standing, 10-20% increase.

        • Mild to moderate exercise: 0.3-1.2mmol/L
        • Vigorous exercise; fist clenching: 2-3mmol/L

      • Prolonged contact of serum and red cells

      • Prolonged tourniquet application

    • Hemolysis of 0.5% RBC can increase levels by 0.5mmol/L (30% increase in gross hemolysis)

    • Because of the released of platelets into serum during clot formation

    • Fist clenching increases potassium

    Hyperkalemia

    1. Decreased renal excretion

       • Acute or chronic renal failure
       • Hyperaldosteronism
       • Addison’s disease

    2. Extracellular shift

       • Acidosis
       • Muscle/cellular injury
       • Chemotherapy
       • Leukemia
       • Hemolysis

    3. Increased intake

       • Oral or IV infusion

    4. Artifactual

       • Prolonged tourniquet application
       • Hemolysis

    Hypokalemia

    1. Gastrointestinal loss

       • Gastric suction
       • Vomiting and diarrhea
       • Intestinal tumor
       • Malabsorption
       • Cancer therapy

    2. Intracellular shift

       • Alkalosis and insulin overdose
       • Insulin promotes acute entry of K ions into skeletal muscle and liver

    3. Renal loss

       • Duiretic use (thiazide)
       • Nephritis
       • Renal tubular acidosis
       • Hyperaldosteronism
       • Cushing syndrome
       • Hypomagnesium
       • Acute leukemia

    Chloride

    • Major extracellular anion (counterpart of Na in extracellular fluid)

    • Promotes maintenance of osmolality, blood volume and electric neutrality

    • Only anion to serve as an enzyme activator

      • Angiotensin-converting enzyme

    • Excreted in urine and sweat

    • Reference values: 98-107mmol/L

    • Specimen consideration:

      • Hemolysis
      • Slightly lower values in post prandial specimen
      • Low levels are observed in considerations with high bicarbonate levels

    Hyperchloremia

    • Renal tubular acidosis
    • Diabetis insipidus
    • Salicylate intoxication
    • Primary hyperparathyroidism
    • Metabolic acidosis
    • Prolonged diarrhea

    Hypochloremia

    • Prolonged vomiting
    • Aldosterone deficiency
    • Metabolic alkalosis
    • Salt-losing nephritis

    Methods of Determination

    • Mercurimetric Titration
    • ISE (Ion Selective Electrode)
    • Amperometric - Coulometric Titration
    • Colorimetry

    Magnesium

    • Prolonged tourniquet application

    • Second most abundant intracellular cation

    • 4th most abundant cation

    • Vasodilator and cause decrease uterine hyperactivity in eclampsic state and increase blood flow

    • Eclampsia

      • Onset of seizures (convulsions) in a woman with pre-eclampsia

    • Pre-eclampisa

      • Disorder of pregnancy in which there is high blood presure and either large amount of protein in the urine

    • Function: