Biomedical Science Notes

Metabolism and Enzymes

  • Metabolism: A sequence of enzyme-catalyzed chemical reactions in the cell, including both anabolic and catabolic reactions and regulated in various ways.
  • Chemical reactions occur as pathways, which are particular sequences of chemical reactions.

Anabolic Pathways

  • Involved in the assembly of complex organic molecules.
    • Biosynthesis (making molecules).
    • Reductive (requires electrons).
    • Requires energy.
    • Pathway is usually divergent.
  • Anabolic Reactions: Small molecules are converted into larger molecules through reduction reactions.
    • Examples: Carbohydrates, proteins, lipids, fatty acids, amino acids, acetyl CoA.
    • ATP is converted to ADP + Pi.
    • Reduced nucleotides (NADPH + H+) are converted to oxidized nucleotides (NADP+).

Catabolic Pathways

  • Related to the degradation of complex substances, resulting in the generation of energy.
    • Degradative (breaks molecules).
    • Oxidative (donates electrons).
    • Yields energy.
    • Pathway is usually convergent.
  • Catabolism: Large molecules are broken down into smaller molecules through oxidation reactions.
    • Examples: Carbohydrates, proteins, lipids are broken down into CO2, H2O, lactate, pyruvate, acetyl CoA.
    • Oxidized nucleotides (NAD+, FAD) are converted to reduced nucleotides (NADH, FADH2).
    • ADP + Pi are converted to ATP.

Anabolic and Catabolic Pathways

  • Catabolic and anabolic pathways proceed simultaneously in a dynamic steady state.

  • Energy-yielding catabolic pathways are balanced by anabolic pathways.

  • Dynamic Steady State: The rate of synthesis (r1) equals the rate of degradation (r2), hence the concentration of the intermediate [B] remains constant.

    r1 = r2

Enzymes

  • Enzymes are catalysts that regulate the chemistry of cells and organisms.
  • Catalysis is necessary to make essential biochemical reactions proceed at a useful rate under physiological conditions.

Characteristics of Enzymes

  • Mostly proteins, but some RNA molecules also act as enzymes.
  • Reaction-specific, with the ability to discriminate between molecules.
  • Highly regulated.
  • Essential for the proper function of cells.
  • Without enzymes, cellular reactions would proceed too slowly for life.

Why Study Enzymes?

  • Clinical pathologies: Monitor and diagnose diseases and pathologies.
  • Design and development of new drugs.
  • Understanding how cells work: Cells work because of enzyme-catalyzed reactions.

Chemical Reactions

  • A process where at least one new substance is produced by a chemical change.
  • The rate of a chemical reaction is the rate at which reactants are consumed or products are produced.

Reaction Coordinate Diagram

  • Shows the energy requirement of a chemical reaction going from reactant (R) to product (P).
  • Free energy of activation (DG^{\ddagger}) determines the rate (speed) of the chemical reaction.
  • Transition State: Arrangement of atoms during the reaction that has the highest energy. Reactants need to exceed this energy before being converted to products.

Chemical Reactions and Activation Energy

  • Each reaction has a specific activation energy or energy threshold that has to be reached before a reaction will occur, i.e., the Free Energy of Activation (DG^{\ddagger}).
  • The minimum energy required to get to the transition state.
  • The reactants need to form a transition state, which is an intermediate chemical stage between reactant and product.
  • Only reactant molecules that possess enough energy to exceed the Free Energy of Activation and pass through the transition state will proceed to products.

Free Energy of Activation (DG^{\ddagger})

  • Determines the rate (speed) of the reaction.
    • High activation energy → low rate (slower reaction).
    • Low activation energy → high rate (faster reaction).

Catalyst

  • A substance that increases the rate of a chemical reaction without being consumed in the reaction.
  • The catalyst is the same before and after the reaction.
  • Increases reaction rate by providing an alternative reaction pathway which has a lower activation energy than the uncatalyzed reaction.
  • Often exert their effects by forming a transient intermediate in the reaction called a transition state.

Enzymes as Nature’s Catalysts

  • Increase reaction rates up to 10^{20}. (Non-enzyme catalysts: 10^2 – 10^4).
  • Enzyme activity: the rate that an enzyme can catalyze a reaction.

Enzyme Terminology

  • Active site: The part of the enzyme that binds to the substrate; a 3D arrangement of residues that determines the catalytic rate.
  • Substrate: The reactant in an enzyme-catalyzed reaction; the substance acted upon by the enzyme.
  • Enzyme names usually end in -ase, -me, or -in (e.g., carboxypeptidase, lysozyme, chymotrypsin).
  • Enzymes are usually named according to function (e.g., hydrolase, lyase, isomerase, ligase, transferase, oxidoreductase).
  • Enzymes have different levels of selectivity for substrates; most enzymes are very specific for their substrate.

Enzyme Activity

  • Enzyme-substrate complex: Enzyme initially interacts with substrate to form an energetically favorable intermediate in the active site.
  • Enzyme active sites are usually complementary to the transition state of the reaction.

Factors Affecting Enzyme Activity

  • Temperature
  • pH
  • Concentration of substrate
  • Concentration of enzyme
Temperature
  • Active only over a limited range of temperatures.
  • Have an optimal temperature where their catalytic activity is optimum.
  • Differ widely in their temperature sensitivity.
pH
  • Active only over a limited range of pH.
  • Have an optimal pH where their catalytic activity is optimum.
  • Vary widely in their pH sensitivity.
Substrate Concentration
  • Michaelis-Menten Plot: Km (Michaelis constant) refers to the strength of association between the enzyme and substrate. At Km, the reaction rate will be half of the maximum reaction rate (Vmax).
Enzyme Concentration
  • Reaction Rate is directly proportional to Enzyme Concentration.

Biological Specimens

  • Different biomedical science disciplines may be used in the diagnosis, treatment, and control of disease.
  • Different biological specimens can be collected for analysis by these different disciplines.

Types of Biological Specimens

  • Blood
  • Urine
  • Feces
  • Solid tissue
  • Cerebrospinal fluid
  • Swabs
  • Less common specimens

Blood

  • Solid particles (cells) suspended in a liquid (plasma).
  • Also referred to as “whole blood”.
  • Cellular components: Red blood cells (RBCs), white blood cells (WBCs), platelets.
  • Plasma components: Water, albumin, immunoglobulins, coagulation proteins, electrolytes, and more.
  • Blood will clot when removed from the body.
  • Complex clotting process with many components: Damage to blood vessel, Ca^{2+}, platelets, clotting proteins (e.g., fibrinogen).

Serum vs Plasma

  • Blood will clot when taken from the body due to a series of reactions forming fibrin (clot).
  • The remaining liquid component is serum.
  • Serum is used for many tests, e.g., clinical biochemistry.
  • Plasma is the liquid portion of whole blood.
  • Serum = essentially same as plasma minus clotting proteins.
  • Serum may not be suitable for some analyses; in such cases, plasma is used instead.

Anticoagulants

  • Anticoagulants allow plasma to be obtained.
    • Heparin: Inhibits clotting proteins (e.g., thrombin) - used for some Clin Biochem.
    • EDTA (ethylene-diamine-tetra-acetic acid): Complexes divalent cations - used in Haem.
    • Sodium citrate: Chelates calcium (reversible) - used for coagulation testing samples.
    • Oxalate: Chelates calcium (irreversible) - used for some Clin Biochem.
    • Sodium fluoride: Weak anticoagulant (additive) - preserves glucose.

Considerations for Biological Specimens

  • If plasma, ensure correct anticoagulant and amount, with proper mixing.
  • Correct collection by phlebotomist.
  • Identification.
  • Separation (e.g., centrifugation).
  • Preservation/storage.
  • Transport.
  • Patient factors: Posture, immobilization, exercise, circadian variation, underlying medical condition, food/drugs/stimulants.

Chromatography

  • Important tool used to both analyze and purify proteins, DNA, RNA, and other organic molecules.
  • Separates a mixture into its components or isolates one component from a mixture of others.
  • Two types: Preparative vs analytical.

Principle of Chromatography

  • Two phases: Stationary and mobile.
  • Components of a mixture are carried through the stationary phase by the flow of the mobile phase.

Types of Chromatography

  • Adsorption: Solid stationary phase and liquid mobile phase.
  • Partition: Liquid stationary phase and liquid or gas mobile phase.
  • Ion Exchange: Solid stationary phase and liquid mobile phase.
  • Size Exclusion: Liquid stationary phase and liquid mobile phase.
  • Affinity: Stationary ligand and liquid mobile phase.

Ion Exchange Chromatography

  • Matrix-C^- …A^+ + X^+ \rightleftharpoons Matrix-C^- …X^+ + A^+
    • Where:
      • Matrix-C^- is the ion exchange resin.
      • A^+ is the counterion.
      • X^+ is the sample ion.
  • Cation exchanger: Exchanges positive ions; can be strong or weak.
  • Anion exchanger: Exchanges negative ions; can also be strong or weak.

Size Exclusion Chromatography

  • Also known as Gel Permeation or Gel Filtration.
  • Separates molecules depending on molecular size and shape.
  • Resolves molecular components of a mixture and estimates molecular weights.

Affinity Column Chromatography

  • Column contains a polymer-bound ligand specific for the protein of interest.
  • Ligand forms complex with the analyte.
  • Unwanted proteins are washed through the column.
  • Add solution of more ligand to elute protein of interest.

Laboratory Considerations

  • Laboratories perform tests to obtain accurate results.
  • Consideration of test variables includes cost, sensitivity, specificity, and reliability.

Sensitivity and Specificity

  • Sensitivity: Measure of the amount of an analyte that can be quantitated. The lower the amount that can be measured, the more sensitive the test.
  • Specificity: Represents what is being measured. A test with high specificity measures only the substance to be measured.

Test Reliability

  • Combination of accuracy and precision.
  • Quality of results needed to make informed decisions about a patient’s condition.

Measurements

  • Not perfect; degree of uncertainty due to limitations of instruments and human error.
  • Recorded as Certain ± Uncertainty (e.g., 68 ± 0.5 ml).

Accuracy

  • Closeness of measured value to true value. Reflects ‘true value’.
  • Result reported should not vary and affect the clinical interpretation (± 5% is achieved for many routine tests).
  • Comparison of test result with result obtained from commercial reference quality control material. Result for a quality control sample should be within specified limits (± 2 SD of the known value).

Precision

  • How close together a set of measurements (of the same parameter) are to each other. Nothing to do with the ‘true value’ of a measurement.
  • High level of precision improves ability to detect small changes in a patient's results over a period of time.

Accuracy and Precision

  • Independent of one another.
  • Measure different properties for a particular set of results.
  • Accuracy: Mean value of data should reflect ‘true value’; can be estimated with only one measurement; measure of reliability of a result.
  • Precision: Related to dispersion of data around a point (standard deviation); can only be calculated with multiple measurements; measure of reproducibility (uniformity) of a result; uncertainty analyzed statistically.

MEDI1000 End Of Semester Test

Exam Format

  • Exam date and time as per examination timetable.
  • Two components: Theory Component (Part 1) and Laboratory Component (Part 2).
  • Take both Part 1 and Part 2, one after the other.
  • Each is an invigilated Blackboard test through Respondus LockDown Browser.

Theory Component

  • 60 minutes.
  • Lectures from Weeks 8, 10, 11, 12, 13 (Anato Path, Haem, Biochem, Mol Biol, Specimens/A&P/Chrom/Metabolism).
  • 3 Short Answer Questions (1 mark each).
  • 37 Multiple Choice Questions (1 mark each).

Laboratory Component

  • 45 minutes.
  • Laboratory sessions from Weeks 7, 10, 11, 12, 13 (Immuno, Anato Path, Haem, Biochem, Mol Biol).
  • 30 Multiple Choice Questions (1 mark each).

Exam Question Examples

  • Demonstrate your broad knowledge of Biomedical Science by briefly summarizing part of a relevant article that you have read about (from a text, scientific journal, or similar).
    • Must be more than what has been discussed in the lectures (shows some broad knowledge beyond what was provided in the lectures).
    • Must be Biomedical Science-related (relevant to this biomed unit).
    • Brief! One sentence can be sufficient. It does not need to summarize the whole article – you just need to demonstrate you have read it.
    • Must be from a scientific source (good idea to include the author, source, and date).
    • Preferably something interesting to you!
  • Relevance of biomedical sciences to your degree or potential career…
  • Consider how the different areas of Biomedical Science relate to each other…

Preparation Tips

  • Practical: Review practicals, notes made during class discussions, and the lab note questions. Understand the concepts from the practicals. Understand how to interpret results. Discussions and review of practicals with self-formed study groups can be very valuable.
  • Theory: Make your own summaries. Repetition is useful. “Active” study techniques are more likely to help (e.g., self-written cards, write/cover/test, etc.). Discussions with self-formed study groups can be very valuable. Use the lecture feedback questions to test your knowledge once you have studied that topic.
  • Read the students’ Questions and Answers document when it is released.
  • Take the Practice Test (Part 1 and Part 2) once they become available. You must take them well before the actual assessment to check that Respondus LockDown Browser is working correctly for you.
  • If any other info needs to be communicated, it will be announced on Blackboard – check regularly.
  • Good time management of study-load helps A LOT. Plan your study schedule for each of your remaining assessments for each unit. The sooner the better – so do it now!
  • There is sufficient time, so don’t rush the Medi1000 EOS Test Components (note that most students finished the Mid Sem Theory test with plenty of time to spare).
  • READ THE QUESTIONS!