Enzymes Notes

Acid Phosphatase (ACP)

• Hydrolase enzyme found in:

  • Prostate

  • Bone

  • Liver

  • Spleen

  • Kidney

  • Erythrocytes

  • Platelets

Diagnostic Significance

• Detection of metastatic prostatic carcinoma.

  • Prostate-Specific Antigen (PSA) is a newer marker for screening and diagnosis.

• Forensic cases involving rape:

  • ACP activity can persist for up to 4 days.

Increased in:

• Prostate cancer

• Benign prostatic hypertrophy

• Paget disease (bone disease)

• Breast cancer (with bone involvement)

• Gaucher disease

• Platelet damage

• Idiopathic thrombocytopenic purpura

Sources of Error

• Leakage of erythrocyte and platelet ACP.

  • Separate serum from red cells promptly after clotting.

• Standing serum at room temperature without preservative.

  • Serum activity decreases within 1-2 hours due to loss of CO_2 and increased pH.

  • Serum should be frozen or acidified to a pH less than 6.5.

    • ACP is stable for 2 days at room temperature under these conditions.

• Hemolyzed sample.

  • Avoid hemolysis due to contamination from erythrocyte ACP.

• RIA procedures for prostatic ACP require non-acidified serum samples.

  • Activity is stable for 2 days at 4°C.

Reference Range

• Prostatic ACP: 0 to 3.5 ng/mL

• Tartrate-resistant ACP:

  • Adults: 1.5 to 4.5 U/L (37°C)

  • Children: 3.5 to 9.0 U/L (37°C)

Test Methodology

• Chemical Inhibition Methods

  • Differentiate the prostatic portion via L-tartrate inhibition.

  • Thymolphthalein monophosphate specific substrates for prostatic ACP

  Serum + Substrate \rightarrow \text{incubated with/without L-tartrate}

  \text{Total ACP (without inhibition) - ACP (with tartrate inhibition) = Prostatic ACP}

  • Tartrate also inhibits lysosomal ACP, so the method is not entirely specific for prostatic ACP.

• Assay for Enzyme Activity

  • Total ACP procedures similar to ALP assays, but at an acid pH.

  • Phosphate ion = Colorless acid pH

  • Alkali stops the reaction

  • Products are converted to chromogens and measured spectrophotometrically.

• Immunochemical Techniques

  • RIA

  • Counterimmunoelectrophoresis

  • Immunoprecipitation

  • Immunoenzymatic assay (Tandem E)

    • Incubation with an antibody to prostatic ACP, followed by washing and incubation with p-nitrophenyl phosphate.

    • Formed p-nitrophenol is measured photometrically, proportional to the prostatic ACP in the sample.

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

• An oxidoreductase.

• First step in the pentose phosphate shunt of glucose metabolism, ultimately producing NADPH.

Tissue Source

• Adrenal cortex

• Spleen

• Thymus

• Lymph nodes

• Lactating mammary gland

• Erythrocytes

• Normal serum (little)

Diagnostic Significance in Erythrocytes

• G6PD maintains NADPH in a reduced form.

• NADPH is required to regenerate glutathione from its oxidized to reduced state.

• Reduced glutathione protects hemoglobin from oxidation.

• G-6-PD deficiency leads to inadequate NADPH supply and reduced glutathione levels.

• Exposure of erythrocytes to oxidizing agents causes hemolysis due to Hgb oxidation & cell membrane damage.

Increased in

• Megaloblastic anemia

• Myocardial infarction

Decreased in

• G6PD deficiency, inherited as a sex-linked trait (X-chromosome).

• G6DP deficiency leads to hemolysis.

  • Drug-induced hemolytic anemia (e.g., anti-malarial drugs like primaquine).

  • Infections

  • After ingestion of fava beans

Assay for Enzyme Activity

• G6PD deficiency requires analysis of a red blood cell hemolysate.

• Analysis of G6PD elevations requires a serum sample.

Reference Range

• 7.9 to 16.3 U/g Hgb (0.1 to 0.3 ukat/g Hgb)

Macroenzymes

• Serum enzymes with high-molecular-mass.

• Examples:

  • ACP

  • ALP

  • ALT

  • AMY

  • AST

  • CK

  • GGT

  • LD

  • LPS

• Bound to either an immunoglobulin (macroenzyme type 1) or a non-immunoglobulin substance (macroenzyme type 2).

• Found in patients with unexplained persistent increase of serum enzyme concentration.

• Increase with increasing age.

• Bind to immunoglobulins in a nonspecific manner.

• Enzyme-Ig complex is formed by specific interactions between circulating autoantibodies and serum enzymes.

Test Methodologies

• Protein electrophoresis

• Gel filtration

• Immunoprecipitation

• Immunoelectrophoresis

• Counter-immunoelectrophoresis

• Immunofixation

• Immunoinhibition (macro-CK)

Clinical Significance

• Accumulate in plasma due to high molecular masses (not filtered by kidneys).

• Presence can cause difficulty in interpreting diagnostic enzyme results.

• Can cause false elevations in plasma enzymes or falsely decrease the activity by blocking the bound enzyme.

Drug-Metabolizing Enzymes

1. Transform xenobiotics into inactive, water-soluble compounds for excretion through the kidneys.

2. Transform inactive prodrugs into active drugs, convert xenobiotics into toxic compounds, or prolong the elimination half-life.

3. Catalyze addition or removal of functional groups by:

   • hydroxylation

   • oxidation

   • dealkylation

   • dehydrogenation

   • reduction

   • deamination

   • desulfurization reactions

Theories of Anti Enzyme antibodies formation:

1. Antigen-driven Theory

   • The self-antigen becomes immunogenic by being altered or released from a sequestered site and reacts with an antibody that is initially formed against a foreign antigen.

2. Dysregulation of “immune tolerance theory”

   • Formation of enzymes with autoantibodies in patients with autoimmune disorders

Phase I (transformation reactions)

• Mediated by cytochrome P450 (CYP 450) enzymes

Phase II (enzyme-mediated conjugation reactions)

• Xenobiotics become transformed into more polar compounds.

• Xenobiotics are conjugated with:

  • Glucuronide (UDP-glucuronosyltransferase 1A1 [UGT1A1])

  • Acetate (N-acetyltransferase [NAT])

  • Glutathione (glutathione-S-transferase [GST])

  • Sulfate (sulfotransferase)

  • Methionine groups

CYP 450

• Enzymes are a superfamily of isoenzymes.

• Contain heme molecules named CYP 450 (absorb max. amount of light at 450 nm).

• Location of CYP 450 (CYP1, 2,3, and 4) families:

  1. Liver

  2. Extrahepatic tissues: lung, kidney, GIT, skin, placenta

• Activity of drug-metabolizing enzymes altered by food, nutritional supplements, or other drugs.

Alteration of activity of drug-metabolizing enzymes

• Inducers

  • Stimulate increased synthesis of CYP 450 enzymes.

  • Increase the metabolism of drugs.

  • Reduce the bioavailability of the parent compound.

• Inhibitors

  • Reduce the expression or activity of drug-metabolizing enzymes.

  • Compete with substrates for the active site of CYP 450.

  • Decrease the metabolism of drugs.

  • Increase the bioavailability of the parent compound.

  • Block activity or expression through anticompetitive means.

Functions of CYP450

1. Involved in xenobiotic metabolism of more than 50% of all drugs

   • E.g., Patients who are poor metabolizers for the CYP2D6 enzyme are at risk for therapeutic failure when inactive prodrugs such as tamoxifen require CYP2D6 for drug activation.

2. Involved in the biosynthesis of endogenous compounds

   • CYP5 family: thromboxane synthase catalyze reaction → platelet aggregation

   • CYP7 and CYP27 families: catalyzed hydroxylation of cholesterol for bile acids biosynthesis

   • CYP24 family: catalyzes the hydroxylation and inactivation of vitamin D3.

3. Synthesize steroid hormones from cholesterol (CYP11, 17, 19, and 21).

Other Drug-Metabolizing Enzymes

1. NAT

   • N-acetyltransferase 2 (NAT2) - primary enzyme in the acetylation of isoniazid (tuberculosis drug)

2. UGT1A1

   • Has polymorphisms leading to nonfunctional enzyme

   • Responsible for the metabolism of bilirubin; patients with nonfunctioning UGT1A1 at risk for hyperbilirubinemia.

3. GST (glutathione-S-transferase)

4. Thiopurine methyltransferase (TPMT)

   • Found in bone marrow & erythrocytes

   • Functions to inactivate chemotherapeutic thiopurine drugs like azathioprine and 6-mercaptopurine.

   • Patients with low TPMT activity are at risk of developing severe bone marrow toxicity

CYP Enzymes, Substrates, Inducers, and Inhibitors

Reference table of various CYP enzymes with their substrates, inducers and inhibitors: