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!