Biochemistry and Clinical Biochemistry Notes

Introduction to Biochemistry and Clinical Biochemistry

• Biochemistry studies chemical processes in living organisms.
• Clinical biochemistry applies biochemistry in a clinical setting, focusing on the detection and study of diseases.
• It often involves the analysis of body fluids like serum.
• Clinical Biochemistry Labs perform many tests using diverse techniques and instruments.
• Key biomolecules studied include:
  • Amino acids and proteins
  • Lipids (fats)
  • Carbohydrates
• Example: Diabetes is an example of clinical biochemistry, specifically dealing with blood sugar levels.
• Instrumentation used, including spectrophotometry, is crucial in clinical biochemistry.
• Acknowledgement: Some lecture material adapted from MLS131, MLS132, Dr Paul Ellery, Dr Cyril Mamotte © Curtin University.

Biomolecules

• Fats:
  • Examples: cholesterol ("good" vs. "bad" cholesterol).
• Carbohydrates:
  • Examples: blood sugar levels.
• Proteins:
  • Examples: serum protein as an indicator of liver function.
• Enzymes:
  • (a type of protein)
  • Examples: cardiac enzymes.

Macromolecule Building Blocks

• Monomers: Amino acids, Sugars, Nucleotides, Fatty Acids
• Polymers: Proteins (from Amino Acids), Polysaccharides (from Sugars), Nucleic Acids (from Nucleotides), and Lipid Aggregates (from Fatty Acids)
• Today's focus: proteins, polysaccharides, and lipids.
• Next week: Nucleic acids.

Amino Acids and Proteins

Amino Acid General Structure

• Alpha (α) carbon: the central carbon atom.
• Carboxyl group: -COOH (may also be called the alpha (α) carboxyl group).
• Amino group: H_2N (may also be called the alpha (α) amino group).
• 'R' group (side chain): There are 20 different types.
• Side chains dictate the physical and chemical properties, leading to amino acid classification.

Amino Acid Classification

• Non-polar Amino Acids:
  • Include Glycine (Gly), Alanine (Ala), Leucine (Leu), Methionine (Met), Isoleucine (Ile), Proline (Pro), Valine (Val).
  • Characterized by even distribution of electrons and hydrophobic properties.
• Aromatic Amino Acids:
  • Phenylalanine (Phe) and Tryptophan (Trp) are non-polar and hydrophobic.
  • Tyrosine (Tyr) is polar and hydrophilic.
  • Aromatic R groups absorb UV light at 280nm, useful for determining concentration via spectrophotometry.
• Polar Amino Acids:
  • Asparagine (Asn), Glutamine (Gln), Serine (Ser), Threonine (Thr), Cysteine (Cys).
  • Hydrophilic due to hydroxyl, sulfhydryl, or amide groups in the side chain.
• Positively Charged Amino Acids:
  • Lysine (Lys), Arginine (Arg), Histidine (His).
  • Hydrophilic and basic.
• Negatively Charged Amino Acids:
  • Aspartate (Asp), Glutamate (Glu).
  • Hydrophilic and acidic.
• Classification of amino acids is useful for scientists because amino acids with similar R groups have similar properties.

Amino Acid Properties

• Ionization: Amino acids are ionized in solution, forming ions depending on pH.
  • Zwitterions predominate at pH 7 (neutral solution).
• Amphoteric Nature: Amino acids can act as acids (donate protons) or bases (accept protons).
  • Acidic structure occurs at low pH, basic at high pH.
• Chirality: Most amino acids are chiral compounds with a chiral center at the alpha carbon.
  • L and D stereoisomers exist, but the L form is primarily used by biological systems.
  • Chirality is important, with biological systems like enzymes distinguishing chemicals based on stereochemistry.

Importance of Amino Acids

• Basic building blocks of proteins.
• Occur in different proportions in different proteins.
• Joined together to form linear sequences, which constitute the primary structure of peptides and proteins.
• Form peptide (amide) bonds through condensation reactions between the carboxyl group of one amino acid and the amino group of another.
• Contribute 10-15% of metabolic energy (up to 90% in some carnivores).
• Inborn errors of metabolism can result from the absence of enzymes involved in amino acid metabolism.
• Precursors to other important biomolecules like histamine, serotonin, glutathione, and porphyrins.

Protein Synthesis

• The sequence of amino acids in proteins originates from DNA through transcription and translation.
• The central dogma of molecular biology:
  • DNA → mRNA → Protein.

Proteins - General Properties

• Building blocks of the cell.
• Linear polymers of amino acid monomers linked by peptide bonds.
• A protein chain has directionality, from the amino end (N-terminus) to the carboxyl end (C-terminus).
• Peptide: Less than 50 amino acids.
• Polypeptide or protein: More than 50 amino acids.
• The sequence of alpha carbons, carboxyl carbons, and amino nitrogens forms the 'backbone' of the polypeptide.
• The linear sequence specifies the complete three-dimensional (3D) structure of a protein, which folds spontaneously into its lowest energy conformation.
• Proteins act through 3-D stereospecific interactions.
• There are various levels of protein structure: primary, secondary, supersecondary, tertiary, and quaternary.

Protein Structure

• Primary Structure: Linear sequence of amino acids.
• Secondary Structure: Regular geometric structures formed by the polypeptide backbone. Examples: α-helix, β-sheet, turns.
• Tertiary Structure: The final folded form of the protein, also known as the 'native' form.
• Quaternary Structure: Association of two or more polypeptide tertiary structures. Applies only if there is more than one polypeptide chain (e.g., Hemoglobin).
• Bonds Responsible for Protein Structure
  • Covalent bonds (peptide bonds) provide the backbone structure (primary structure).
  • Non-covalent bonds (hydrogen bonds, hydrophobic forces, van der Waals interactions) dictate secondary, tertiary, and quaternary structures and provide the dynamic 3-D structure.
• General Facts
  • Proteins can be classified into families based on amino acid sequence and 3D structure.
  • Proteins interact with each other, essential for cellular function.
  • Proteins are dynamic and can change conformation in response to environmental stimuli.
  • Proteins can be comprised of domains, which are independently folded structures within the tertiary structure.
  • Proteins with more than one domain are called 'mosaic' proteins.
  • Proteins can be globular or fibrous. Fibrous proteins include collagen, α-keratin, and elastin.
  • The 3D folded state can be unfolded or denatured by high temperature, pH, or detergents – a process that can be reversible or irreversible.
  • Proteins can act as buffers due to ionizable functional groups in their side chains.
  • Proteins have varied flexibility – some are rigid, others flexible.
  • Prosthetic groups (metal or organic molecules) are sometimes critical for protein function.
  • Proteins contain a wide range of functional groups (e.g., acids, amines, thiols).

Importance of Proteins

• Enzymatic Catalysis: e.g., enzymes.
• Transport: e.g., hemoglobin.
• Storage: e.g., ferritin for iron.
• Motion: e.g., myosin in muscle.
• Structural Support: e.g., collagen in tendons and ligaments, tubulin in cells.
• Immunity: e.g., antibodies.
• Growth: e.g., growth hormone.
• Communication: e.g., hormones and growth factors.
• Sensing: e.g., receptor proteins.
• Regulation: e.g., transcription factors.
• Other: e.g., green fluorescent protein.

Lipids

• Relatively simple molecules forming large structures through non-covalent associations.
• Heterogenous class of molecules, e.g. fats, waxes, and oils, found in both animals and plants.
• Insoluble in water but soluble in organic (nonpolar) liquids.
• Essential for life with many functions:
  • constructed from fatty acids, glycerol, phosphoric acid, monosaccharides, oligosaccharides, amines, amino acids, and isoprenes.

Functions of Lipids

• Fuels
• Structural: Biological membranes.
• Specific Biological Actions: Messengers, cofactors, carriers, etc.
• Insulation
• Protection
• Heat production
• Storage

Types of Lipids

• Triacylglycerols (fats & oils).
• Waxes.
• Phospholipids.
• Sterols.
• Eicosanoids.
• Glycerophospholipids.
• Glycolipids.
• Sphingolipids.
• Specific Action Lipids: Phosphatidylinositol, Isoprenoids, Vitamins A, D, E, K

Lipid Components - Fatty Acids

• Carboxyl group and hydrocarbon chain (saturated or unsaturated).
• Naming Fatty Acids
  • Usual numbering from carboxy end.
  • Omega system from methyl end.
    • Omega-3 fatty acid: Linolenic Acid
    • Omega-6 fatty acid: Linoleic Acid
• Structures of lipids
  • glycerol, fatty acids, phosphoric acid, sphingosine, and different functional groups.
  • Triacylglycerols, phosphoacylglycerols, and sphingolipids.

Lipid Aggregates

• Not true macromolecules, as individual monomers (fatty acids) are not covalently bound.
• Form large aggregates through non-covalent interactions.
• Examples: micelles, liposomes, membranes, lipoproteins.
• In micelles all hydrophobic groups are sequestered from water to minimize the ordered H₂O shell.
• Lipoproteins:
  • Chylomicrons: Transport dietary lipids from the intestine to peripheral tissues.
  • VLDL (very low density lipoproteins): Transport lipids from the liver to peripheral tissues.
  • LDL (low density lipoproteins): Transport cholesterol to peripheral tissues and are taken up by the liver.
  • HDL (high density lipoproteins): Collect cholesterol and transport it to the liver.

Cholesterol

• A sterol (modified steroid).
• Synthesized by cells and ingested from food.
• Essential component in animal cell membranes.
• Precursor of other important molecules.

Carbohydrates

• 'Hydrate of carbon' with the formula (CnH{2n}O_n), where n ≥ 3.
• Sugars: Monosaccharides (simple monomers) and polymers.
  • Oligosaccharides: 2 to 20 monosaccharides.
  • Polysaccharides: ≥20 monosaccharides.
• Sugar derivatives: Glycoproteins, proteoglycans, glycolipids.
• Wide distribution in nature as abundant organic molecules.

Functions of Carbohydrates

• Energy source.
• Cell recognition (e.g., blood group).
• Cell-to-cell communication.
• Cell adhesion.
• Structure.
• Antibiotics.
• Coenzymes (NAD+).
• Activated carriers.
• Nucleic acids (DNA and RNA).

Carbohydrate - Structure

• General name according to number of carbons: trioses, tetroses, pentoses, hexoses, heptoses.
• Aldoses: aldehyde group.
• Ketoses: ketone group.
• Sugars with a free aldehyde or ketone group can reduce cupric ions (Cu^{2+}) to cuprous ions (Cu^{+}) and are called reducing sugars.
• D sugars are most prominent.
• Stereoisomers possess a chiral center at one (or more) asymmetric carbon atom(s).
• Named according to Glyceraldehyde - D and L configuration.

Carbohydrate - Mutarotation:

• Aldotetroses and monosaccharides with five or more carbon atoms rarely exist in linear form.
• They form a cyclic structure where the aldehyde or ketone covalently bonds to a hydroxyl group within the chain.
• Anomers – isomers that differ in their structure around the anomeric carbon.
  • Alpha - OH group of the anomeric carbon is below the ring.
  • Beta - OH group of the anomeric carbon is above the ring.

Important Monosaccharides

• D-Glucose: Most abundant monosaccharide, principal energy molecule.
• D-Fructose: Common in honey and fruit.
• D-Ribose: Common pentose sugar, component of DNA, RNA, NADH, etc.

Disaccharides

• Monosaccharides joined by a glycosidic bond.

Important Disaccharides

• Sucrose: Table sugar (α-D-glucose + D-fructose).
• Lactose: Milk sugar (β-D-glucose + galactose).
• Maltose: Malt sugar (α-D-glucose + D-glucose).

Oligosaccharides

• For example blood groups (also example of glycolipid).
  • Type O, A, and B blood groups.

Polysaccharides

• Composed of monosaccharides linked by glycosidic bonds with branch points.
• Homopolysaccharides consist of the same monomer.
• Heteropolysaccharides consist of two or more different monomers.

Diabetes

• A disease state with major effects on glucose metabolism.
• Glucose is an important carbohydrate, major fuel, abundant dietary carbohydrate and a precursor for other sugars.

Terminology

• Diabetes Mellitus vs. Diabetes Insipidus
• Diabetes Mellitus
  • State of chronic hyperglycaemia from genetic or environmental factors.
  • Type 2 DM: Insulin resistance.
  • Type 1 DM: Absolute insulin deficiency (loss of β cells).

Diabetes (DM) in Australia

• ~8% of men, ~7% of women affected.
• 50% undiagnosed.
• Prevalence increasing worldwide, linked to lifestyle.
• A significant issue for certain groups, including indigenous Australians.

Clinical Biochemistry

• Variety of diagnostic and management tests used.
• Glycosuria is suggestive but not diagnostic.
• Blood Glucose: Random, fasting, glucose tolerance test.
• Diagnosis based on plasma glucose:
  • e.g., >7.0 mmol/L fasting.

Instrumentation: Spectrophotometer

• Photometry uses radiant energy (UV, visible, or IR) to measure its interaction with matter.
• Can determine:
  • Structure of a molecule (UV, IR).
  • Concentration of a solution (UV, visible).
• Electromagnetic radiation consists of particles and waves with:
  • Wavelength (λ)
  • Frequency (ν)
  • Intensity (I)
• Electromagnetic Spectrum:
  • Different types of radiation (γ rays, X rays, UV, Visible, Infrared, Microwaves, Radio waves) with varying wavelengths and energies.
• ER interacts with matter through reflection, transmission, or absorption, altering the internal energy states of molecules.
• Absorption Spectra: Different wavelengths of light are absorbed differently.
• The color of objects is determined by the wavelengths of light that are reflected, transmitted, or absorbed.

Intensity

• Amount of energy transmitted through an area over time.
• Light Transmitted (T) = \frac{It}{Io}
• Decrease in intensity results from more molecules interacting with the incident light beam, depending on concentration and pathlength.

Laws of Photometry

• Beer's Law
• Lambert's (Bougher's) Law
• Combined (Lambert-Beer) Law: A = \varepsilon cl
  • Where:
    • A = Absorbance
    • \varepsilon = Molar absorptivity constant (L·mol⁻¹·cm⁻¹)
    • c = Concentration (M)
    • l = Pathlength (cm)

Transmission

• Solution containing no absorbing species in a container of fixed dimensions
  • Such that when concentration = 0.0 M, T = 1.0, or 100%
• Solution containing a compound at a concentration of 0.1 M which interacts with the light so that only half is transmitted
  • Such that T = 1.0 x 0.5 = 0.5 or 50%
• Solution containing a compound at a concentration of 0.2 M. There is now twice as many molecules capable of interacting with the beam
  • Such that T = 1.0 x 0.5 x 0.5 = 0.25 or 25%
• Solution containing a compound at a concentration of 0.3 M. There is now three times as many molecules capable of interacting with the beam
  • Such that T = 1.0 x 0.5 x 0.5 x 0.5 = 0.125 or 12.5%

Concentration increases ➩transmission decreases

%Transmission and Absorbance

• More convenient to define a directly proportional relationship

• Use absorbance

  • The difference between the intensity of the incident light beam Io and the intensity of the light transmitted by the solution It.

• Involves a log to linear transformation of the transmission equation.

• Absorbance and Transmission

A = - log \frac{It}{Io} = -log T = log \frac{1}{T}

A = log \frac{100}{\%T} = log 100 – log %T = 2 – log %T

Therefore there is a directly proportional relationship between absorbance and concentration

• The Biochem lab session will show:

  • Directly proportional relationship between analyte concentration and absorbance.
  • Can use Standards and Standard Curve to determine analyte concentration.
  • Require the selection of an appropriate blank.
  • Require the selection of optimal analytical wavelength.
  • May need to plot “Line of Best Fit” for standard curve.

• Should be aware:

  • Are exceptions when Beer’s Law will not be obeyed.
  • Are other ways to determine concentration using absorbance.

Feedback Study Questions – Biochem / Biomolecules

1. What are the “building blocks” for protein macromolecules?
2. What are the “building blocks” for polysaccharides?
3. What are the “building blocks” for nucleic acids?
4. What are the “building blocks” for lipid aggregates?
5. What macromolecule/s can be made up of branched chains?
6. How many standard amino acids are there?
7. What is the general structure of an amino acid (diagram is ok).
8. How do the standard amino acids differ from one another?
9. Amino acids can be classified according to their R group. Why might it be useful to use this classification? (Hint, might some amino acids with similar R groups have similar properties?)
10. What is a peptide bond?
11. Explain why amino acids are important.
12. BRIEFLY explain what is meant by each of the terms: Protein Primary Structure, Protein Secondary Structure, Protein Tertiary Structure, Protein Quaternary Structure.
13. List two examples of terms that describe different secondary structures that can be found in proteins.

Feedback Study Questions Biochem / Biomolecules

14. List four of the major roles of Protein.
15. Lipids should not be considered true macromolecules because of their structure. Briefly explain.
16. Name three major functions of lipids.
17. List some examples of lipid aggregates.
18. Give one example of an important Monosaccharides.
19. Give one example of an important Disaccharides.
20. List four of the major functions of carbohydrates in a cell.
21. Can two sugar molecules have different configurations despite having the same molecular formula? BRIEFLY explain.
22. Explain the term Diabetes mellitus
23. Discuss the statement “The diagnosis and management of Diabetes mellitus is only of concern to a very small portion of the health care industry”. (Hint, you should first decide if this statement is true or false, then BRIEFLY explain why it is true or false.)
24. How is Diabetes mellitus diagnosed?

Feedback Study Questions – Instrumentation / Spectro.

1. Spectrophotometers can be used to measure the absorbance of light of a particular wavelength by a certain material. BRIEFLY explain what is meant by the term absorbance.
2. What is Beer's Law?
3. What is Lambert's Law?
4. What is the combined Lambert-Beer Law?
5. Is it more convenient to use Absorbance or Transmittance to determine the concentration of a substance?
6. Why is the selection of optimal analytical wavelength important for spectrophotometric measurement? (Hint, material to be discussed in the Biochemistry Practical may assist you to answer this).
7. Why is the selection and use of an appropriate Blank important for spectrophotometric measurement? (Hint, material to be discussed in the Biochemistry Practical may assist you to answer this).