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Blood - Biochemical Aspects Functions. Respiratory Transport O 2 from lungs to tissues Transport CO 2 from tissues to lungs Nutrition Transport “food” from gut to tissues (cells) Excretory Transport waste from tissues to kidney (urea, uric acid, water). Regulatory

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blood biochemical aspects functions
Blood - Biochemical AspectsFunctions
  • Respiratory
    • Transport O2 from lungs to tissues
    • Transport CO2 from tissues to lungs
  • Nutrition
    • Transport “food” from gut to tissues (cells)
  • Excretory
    • Transport waste from tissues to kidney (urea, uric acid, water)
slide2
Regulatory
    • Water Content of Tissues
      • Water exchanged through vessel walls to tissue (interstitial fluid)
  • Body Temperature
    • Water- high heat capacity, thermal conductivity, heat of vaporization
    • Typical heat generation is 3000 kcal/day
  • Protective
    • Antibodies, antitoxins, white blood cells (WBC)
slide3
Blood composition
    • 5-6 L in an adult
    • 70 mL/kg of body weight
    • Suspension of cells in a carrier fluid (plasma)
      • Cells - 45% by volume
      • Plasma - 55% by volume
  • Cells
    • Red cells (erythrocytes)
      • 5x106/mL
    • White cells (leukocytes)
      • 7x103/mL
    • Platelets (thrombocytes)
      • 3x105/mL
slide4
Plasma composition
    • Water - 90% of plasma volume
    • Proteins - 7% of plasma volume
    • Inorganic - 1% of plasma volume
      • Na+, K+, Mg2+, Ca2+, PO43-…
    • Organic - 2% of plasma volume
      • urea, fats, cholesterol, glucose ...
slide5
Male versus female
    • Hematocrit (% volume that is red cells)
      • 40-50% in males
      • 35-45% in females
proteins see lehninger chapter 3 6
ProteinsSee Lehninger Chapter 3-6
  • Proteins are polyamino acids
  • Macromolecules - MW 5000 - several million
    • Insulin - MW = 6000
    • Hemoglobin - MW = 68 000
slide7

O

O

~NHCHC-NHCHC~

R1

R2

Peptide bond

R

NH2CH

COOH

Amino Acid Structure

Protein Structure

slide8
20 common amino acids (AA)
  • Classified based on the properties of the R groups

Acidic Glutamic Acid

Basic Lysine

slide9

Polar Tyrosine

Apolar Glycine

amino acids and proteins
Amino Acids and Proteins
  • Acidic and basic groups are charged at blood / physiologic pH
  • Proteins are polyelectrolytes
  • pH of zero net charge (pI or isoelectric point) depends on amino acid composition of protein
  • Blood proteins negative at pH 7.4
    • more COO- than NH3+, pI < 7.4
slide11
pI
  • Protein has many negative charges
  • Requires H+ to neutralize
  • Therefore low pI
  • Consider a protein with pI = 4
    • If pH increases above pI protein becomes?
    • If pH decreases below pI protein becomes?
  • Higher the pI the more +,- is protein?
slide12
Need to go to a higher pH to neutralize or compensate for + charges
  • Minimum solubility occurs at pI since there is no intermolecular repulsion
  • At pH 7.4 (blood pH), all blood proteins are negative and therefore have pI’s less than 7.4
protein structure
Protein Structure
  • Four levels
    • Primary structure: sequence of amino acids
      • 20 amino acids in long chain molecules
      • many possible combinations
    • Secondary structure: arrangement of the chains in space (conformation of chains)
      • a-helix: coil shape (due to H bonding)
      • b-sheet: stretched zig-zag peptide chain (H bonding
      • random coil: similar to synthetic polymers
slide18
Tertiary structure: folding of chains into 3 dimensional shape due to H bonding, S-S bonds and hydrophobic interactions
      • Several different types of secondary structure within the full three dimensional structure of a large protein
    • Quaternary structure: present in proteins with several polypeptide chains, arrangement and interelationship of the chains due to S-S bridging
  • Four levels result in well defined shape and chemical structure essential for function of protein
plasma proteins
Plasma Proteins
  • More than 200
  • Most abundant
    • Albumin - 4-5 g/100 mL
    • g-glubulins - ~1 g/100 mL
    • fibrinogen - 0.2-0.4g/100 mL
  • Original classification by zone electrophoresis at pH 8.6
  • Separation by pI with several molecular weight species within each group
zone electrophoresis of plasma proteins
Zone Electrophoresis of Plasma Proteins

+

-

globulins

albumin

g

b

a1

a2

pI

6.0

5.6

5.1

4.7

protein separation
Protein Separation
  • Size Exclusion Chromatography (SEC)
    • Porous matrix (sephadex)
slide22
Affinity chromatography
    • molecule attached to a column that specifically binds the protein of interest
      • Coenzyme / enzyme
      • Antigen / Antibody
slide23
SDS-PAGE (polyacrylamide gel electrophoresis)
    • Separates by size
    • Proteins are complexed with SDS to give the same charge density
two dimensional electrophoresis
Two Dimensional Electrophoresis

Decreasing Mr

Decreasing pI

functions of plasma proteins
Functions of Plasma Proteins
  • Maintenance of:
    • Colloid osmotic pressure (p)
    • pH
    • electrolyte balance
  • COP relates to blood volume

DP = p

Protein

sol’n

Water

slide27
If membrane present p important
  • “Isotonic” - same osmotic pressure
  • Human blood - 300 milliOsmoles /L
  • Normal saline - 0.9% NaCl by weight
    • 0.15 mol/L
    • 0.30 mol/L of particles
  • Calculate osmotic pressure from concentration?
slide28
By analogy with the ideal gas law
  • In blood, which protein contributes most to p?
  • Low molecular weight, high concentration
slide29
Colloid - large particle that cannot easily cross a membrane
    • Stays in the compartment
    • In blood pprotein = 20-30 mmHg
    • Total ~ 5000 mmHg
  • Protein stays in the blood as p is maintained in the blood
  • Water content is therefore maintained
slide30

H2O

Hb

  • Hypotonic - lower p than normal
    • Hemolysis of RBC

H2O

Ghost Cells

  • Hypertonic - higher p than normal
    • Hemolysis of RBC

Crenated Cells

Hypertonic

1.5% NaCl

functions of plasma proteins cont d
Functions of Plasma Proteins (cont’d)
  • Transport of ions, fatty acids, steroids, hormones etc.
    • Albumin (fatty acids), ceruloplasmin (Cu2+), transferrin (Fe), lipoproteins (LDL, HDL)
  • Nutritional source of amino acids for tissues
  • Hemostasis (coagulation proteins)
  • Prevention of thrombosis (anticoagulant proteins)
  • Defense against infection (antibodies, complement proteins)
function and properties of selected plasma proteins
Function and Properties of Selected Plasma Proteins
  • Consider three abundant plasma proteins
  • Structure, function
  • Coagulation, fibrinolysis, complement
albumin
Albumin
  • MW 66 000
  • Single chain, 580 amino acids, sequence is known
  • Dimensions - Heart shaped molecule
  • 50% a helix[He and Carter, Nature, 358 209 (1992)]
  • Modeled as:

80 Å

30 Å

slide34
Synthesis
    • Mainly liver cells then exported
    • Assembly time on ribosome ~ 1-2 min
    • t0.5 in circulation - 19 days
    • 14 g lost per day
    • 0.4 mg synthesized per hour per g of liver
    • Need liver of approximately 1.5 kg in weight to maintain
slide35
Functions
    • “Colloid” osmotic pressure of blood is 80% due to albumin
      • relatively low molecular weight
      • regulates water distribution
    • Transport of fatty acids
      • Liver to tissues, binding
    • Source of amino acids for tissue cells (pinocytosis)
      • 60% albumin in tissue (interstitial) fluid
g globulins
g-Globulins
  • 20% of plasma proteins
  • “g” refers to electrophoretic mobility
  • Represents a group of proteins of variable structure
    • immunoglobulins
  • Main functional task is immunochemical
    • Antibodies - combine with specific antigens
slide37
Basic 4 chain structural unit
    • MW = 2x55000 +2x27000 = 160000
slide38
Variable region varies with respect to primary, secondary and tertiary structures
  • Basis of specificity of antigen binding (106 average number)
  • 5 classes of immunoglobulins
    • IgG, IgA, IgM, IgD, IgE
    • Different structures of constant regions of heavy chains
    • Some are polymers (multiples of 4 chain unit - IgA - dimer - MW 350 000, IgM - pentamer - MW 900 000
    • See any immunology book for more details
slide39
Functions
    • Primary function is antigen binding (immune response)
    • Secondary function is complement binding (after antigen)
    • Each class has different functions
      • IgE - allergic reactions (defence)
      • IgA - secretory protein, high concentration in external fluids (saliva, tears)
      • IgD - ? Involved in differentiation of B lymphocytes (found on the surface of B-lymphocytes)
slide40
Synthesis
    • In lymphocytes (T and B)
    • Made in response to presence of antigen (“foreign” macromolecule, virus particle etc.)
fibrinogen
Fibrinogen
  • Coagulation
  • Structure
    • MW 340 000
    • Sequence of amino acids is known (3000)
    • 4y, 3y structure
      • 6 polypeptide chains, 2a (67,000), 2b (56,000), 2g (47,000)
slide43

a

b

g

disulfide

Triple dumbell model (EM)

450 Å

90 Å

D

E

D

a’s, b’s and g’s are intertwined

slide44

Thrombin

Fibrinogen

Fibrin

Plasmin

Fibrin

Degradation (FDP)

  • Function
    • Blood coagulation (clotting)
  • Plasmin is end product of fibrinolytic system
  • Clot needs to be removed
    • Not needed forever
    • Could embolize to lungs, brain
sickle cell anemia
Sickle Cell Anemia
  • Occurs because of a minor variation in one amino acid in the b chain of Hb
  • Results in Hb that, when exposed to low O2 concentrations precipitates into long crystals
  • Elongate cell
  • Damage cell membrane
  • Decrease in amount of RBC
cellular elements of blood
Cellular Elements of Blood
  • Red cells
    • 40 - 50% of blood volume
    • 5 x 106 cells /mL
    • “bag” of hemoglobin
      • non-nucleated
      • no proliferation
      • cell membrane in excess so that deformation does not rupture
    • Shape
      • Biconcave disc
      • 8 mm in diameter, 2.7 mm thick, volume ~ 90 mm3, area ~ 160 mm2
slide48
Why this shape?
    • Area to volume ratio is high (maximal?)
    • Facilitates diffusion of O2 and CO2
      • minimal distance of contents from surface
      • Originates in bone marrow (hematopoiesis)
    • Molecular explanation based on the properties of the proteins in the cell membrane is found in Elgsaeter et al. Science, 234, 1217 (1986)
oxygen binding of hb
Oxygen Binding of Hb
  • Blood must carry 600 L of O2 from lungs to tissues each day
    • Very little carried in plasma since O2 only sparingly soluble
    • Nearly all bound and transported by Hb of RBC
    • Possible for Hb to carry four O2 molecules, one on each a chain, one on each b chain
slide50
O2 depleted Hb solution placed in contact with O2(g)
  • Equilibrium reaction
  • Fraction (s) of Hb converted to oxyhemoglobin
slide51
Described by empirical equation

K depends on ionic strength and pH of Hb solution

n generally given as 2.5 -2.6

slide52
Binding of O2 to 4 heme sites given by:
  • Equilibrium constants for different reactions different
    • Binding of first O2 relatively low affinity
    • 2nd, 3rd and 4th - much higher affinity
  • Cooperative effect
slide54

At equilibrium

  • Myoglobin - oxygen reaction
acid effect o 2 dissociation
Acid Effect - O2 Dissociation
  • O2 binding causes release of H+
  • pH decreases, [H+] increases then the equilibrium moves to left
  • % saturation decreases, more dissociation for a given pO2
  • Tissues are at a lower pH than the lungs due to CO2 which facilitates release of O2 to tissues
hb versus mb
Hb versus Mb
  • Hb carry O2 to tissues where it is released
    • Releases quickly in tissues where pO2 is lower
  • Mb store O2 in the muscle, make available to cells
    • Releases very little in tissues

Reference: Science 255 54 (1992)

rbc reversible shape changes
RBC - Reversible Shape Changes
  • Surfactants result in cells becoming more spherical
  • Mechanical stress - deformation in capillaries to allow for passage of cells
  • Disease eg. Sickle Cell Anemia
  • Hemolysis - release of Hb from the cell
    • Osmotic swelling
    • Surface collisions with artificial organs
white blood cells leukocytes
White Blood Cells (Leukocytes)
  • Total count - approximately 7000/mL
  • Various types
    • Neutrophils 62%
    • Eosinophils 2.3%
    • Basophils 0.4%
    • Monocytes 5.3%
    • Lymphocytes 30%
    • Plasma cells (mainly in the lymph)
  • Monocytes in tissue become macrophages
slide59
Function
    • Defense against foreign invaders
      • bacteria
      • viruses
      • foreign materials (including biomaterials)
  • Phagocytosis
    • Neutrophils, macrophages
    • Move to foreign particle by chemtaxis
      • Chemicals induce migration
      • Toxins, products of inflamed tissues, complement reaction products, blot clotting products
    • Response is extremely rapid (approx 1 h)
slide60
Lymphocytes
    • B cells - responsible for humoral immunity
    • T cells - responsible for cell mediated immunity
  • B cells responsible for production of antibodies
    • Receptor matches antigen
    • Cells multiply
    • Antibodies
  • Abs are just immunoglobulins discussed earlier
slide61
T cells
    • Cytotoxic T cells (Killer T cells)
      • Bind to cytotoxic cells (eg infected by virus)
      • Swell
      • Release toxins into cytoplasm
    • Helper T cells
      • Most numerous
      • Activate B cells, killer T cells
      • Stimulate activity by secretion of IL2
      • Stimulate macrophages
    • Suppressor T cells
      • Regulate activities of other cell types
slide62
AIDS
  • HIV - attacks many cell types
    • epithelial cells
    • macrophages
    • neurons
    • lymphocytes (helper T)
  • Infected helper T cells when stimulated, produces viral proteins which kill the cell
  • Helper T cell population disappears
platelets
Platelets
  • Non-nucleated disk shaped cells
  • 3-4 mm diameter
  • Volume 10 x 10-9 mm3
  • 250 000 cells/mL
  • 10 day circulation time
  • Surface contains membrane bound receptors (GP Ib and IIb/IIIa)
    • mediate surface adhesion reactions, aggregation reactions
    • interact with coagulation proteins
slide64
Contain muscle proteins actin and myosin which contract when platelet is activated
  • Also a granules, dense granules, lysosomal granules
  • Platelets activated by minimal stimulation
    • Become sticky
    • Shape change
    • Release of cell contents
  • Stimulate other platelets
slide65
Function
    • Initially arrest bleeding through formation of platelet plugs
    • Stabilize platelet plugs by catalyzing coagulation reactions leading to formation of fibrin
slide66
Platelet Adhesion
    • Site of injury - exposure of connective tissue elements (eg collagen)
    • Artificial surfaces through forming thrombi (clots)
  • Platelet Aggregation
    • Caused by ADP, collagen, thrombin, epinephrine, PAF, TXA2
  • Release of cell contents
    • Induced by ADP, collagen, thrombin, TXA2 and epinephrine
coagulation
Coagulation
  • Maintenance of hemostasis (prevention of blood loss)
  • At least 12 plasma proteins interact in series of reactions
  • Cascade of reactions
  • Inactive factors become enzymatically active following surface contact, proteolytic cleavage by other enzymes
  • Amplification is rapid
  • Reactions are localized
slide72
Extrinsic system
    • Blood comes in contact with traumatized vascular wall or extravascular tissues
  • Intrinsic system
    • Initiated by surface contact (often negatively charged surface)
  • Most reactions are Ca++ dependent
  • Chelaters of Ca++ effective anticoagulants
fibrinolysis
Fibrinolysis
  • Results in dissolution of fibrin clot