6. Dysproteinaemias

  • 5.0-8.0% of total proteins are present in the blood plasma of mammalian animals

Protein Structure

  • primary
    • amino acid chain in which α carbons of amino acids are jointed together by peptide bonds
  • secondary
    • α helix, α- or β-sheet
  • tertiary
    • globular with a biological activity e.g. enzymes, albumin
    • linear e.g. fibrinogen
  • quarternary
    • several polypeptide chains are joined together producing one molecule with a biological activity, e.g. creatin kinase - dimer, LDH - tetramer

Chemical Classification

  • Simple proteins
    • only amino acids are present
  • Conjugated proteins
    • non-proteinogenic structures are present e.g. metal ions, prosthetic groups
    • metalloproteins (ferritin – Fe is present)
    • phosphoproteins (phosphoric acid residue, e.g casein)
    • lipoproteins (lipids are bound)
    • glycoproteins (glycohemoglobin, fructosamine)

Physical Properties

  • water and salt solubility
  • molecular weight
  • mobility in electric field
  • net electric charge (acidic, basic)

Functions of Proteins

  • the basic structures of cells, organs and tissues;
  • they maintained coloid osmotic pressure
  • biocatalysts (enzymes)
  • having been buffer systems, they maintain acidobasic homeostasis;
  • regulators of metabolism (hormones);
  • function in the clotting of blood, and in body   defence (antibodies);
  • source of aminoacids (alimentary function);
  • transport a variety of substances (albumin)

Synthesis of Proteins

  • 20 different amino acids are present in the vertebrate proteins. Some of them are
    • synthetized by organisms themselves – so     called nonessential AMAs (Ala, Arg, Asp,     Asn, Cys, Gly, Glu, Gln, Pro, Ser, Tyr)
    • Other AMAs must be present in the foodstaff to     prevent disorders in the synthesis of proteins     because organism does not have enzymes     for their synthesis – so called essential     amino acids (His, Ile, Leu, Lys, Met, Phe,     Thr, Trp, Val)
  • In the molecule of DNA (cell nucleus) an exact sequence of nucleotides forms template (chemical scheme) where replication of DNA and transcription of mRNA take place. This process is catalyzed by DNA-directed-RNApolymerases. Newly formed mRNAs move into cytoplasm, where it is bound to ribosomes.
  • Translation processes:
    • in cytoplasm transfer RNAs (tRNAs) bind their specific AMAs. These tRNAs (anticodon) with their AMA move to their complementary segment (codon) on mRNA. Now new peptide bonds are produced between AMAs, and synthesis of protein chain proceeds untill the protein molecule reaches so called terminatory sequence on mRNA. Finally, newly synthetized protein chain is released from a given mRNA segment and passes for its postranslational modifications.This mechanism enables a synthesis of a large amount of molecules with the same structure
  • In certain disease states a single cell or a family of genetically homogeneous cells (clone) might exibit uncontrolled proliferation. E.g. plasma cells in multiple myeloma excessively produce a single species of protein – IgM

Sites of Plasma Protein Synthesis

  • liver (main site)
  • immune system
    • monocyte-macrophage system
    • lymphoid
    • plasma cells
  • peripheral cells
    • these proteins are present in plasma in minor quantities as a result of physiological cell turnover

Degradation of Proteins

  • deamination
    • obtain carbon skeleton (high energy value)
  • Tissue and plasma proteins are constantly degradated to their constituent amino acids (AMAs). Carbon skeletons of these AMAs in turn are sources of energy via their conversion to fat (lipogenesis) or carbohydrate intermediates or glucose (glukoneogenesis and glycolysis) having been finally oxidized into CO2 a H2O in Krebs cycle and respiratory chain. Carnivores derive as much as 40 – 50% of their energy from dietary proteins; omnivores and herbivores less 10% -20%.
  • Ammonia is detoxicated in the urea cycle (exclusivelly only in the liver) through carbamoyl phosphate with the urea as the final product. In this form it is excreted from organism

Rate of Degradation

  1. turnover number
  2. fractional clearance
  3. half-time
  • Clearance half-times may range from a few hours (some enzymes) to as long as 160 days for hemoglobin in cow red blood cells.
  • The clearance half-times of most plasma proteins range between 1 to 3 weeks

Factors Affecting Plasma Proteins

  • age
    • at birth, plasma proteins of most animals are quite low due to the minimal quantities of immunoglobulins and albumin.
    • As the new born animal ingests colostrum, a rapid rise in the immunoglobulins occurs as a result of the absorbed maternal immunoglobulins (as far as 48 hours after birth)
    • neonatal rapidly gains immunocompetence and begins to synthesize its own immunoglobulins.
    • Upon reaching young adulthood, adult levels of the albumin and globulins are reached (20 % Ig of the TPP)
  • pregnancy and lactation
    • During gestation total plasma protein decreases due to an albumin decrease.
    • Near term, there is a sharp rise in the γglobulins. In lactation the total plasma protein decreases again due to an albumin decrease
    • In cows, the total serum protein, γ- and     β2-globulins begin to increase at 2 months before term, reach a maximum at 1 month, and then rapidly decline toward term. This suggests that the immunoglobulins rapidly leave the plasma when colostrum is being formed in mammary gland

Quantitative Determination of Total Serum Proteins in Animals

  1. Chemical Methods (colorimetry)
  2. Physical Methods (refractometry)
  3. Fractionations (separation using salts; electrophoresis)

Chemical Methods

  • biuret method;
  • Folin-Ciocalteau;
  • Bicinchinonic acid;
  • Bradford method

Fractionations - Electrophoresis

  • Electrophoretic methods for serum proteins separations become a rutin laboratory method for an assesment of their pattern in different diseases of animals. Several types of serum protein electrophoresis (SPE) are at disposal for this purpose.
  • They differ ususally each other by the type of separation - support medium (gel):
  • agarose; acetatecellulose; starch;
  • polyacryl amide (PAA) (homogeneous, gradient of   PAA)
Principle of Electrophoresis
  • based on a different rate of proteins migration in the electric field in dependence to their different net molecular charge,shape and size
  • important factors influence this mobility also, e.g. intensity of the electric field, type of buffer, type of the separation medium (acetate cellulose vs gradient PAA) and temperature
  • Most serum proteins posses negative net charge on their molecule at the pH value 8,6. This is the reason why prevalence of serum proteins are acidic and migrate to the anode producing species characteristic serum proteins pattern
  • most rapidly migrate

  

  1. prealbumins
  2. albumin
  3. globulins
  • Negative charge of globulins determine their slower migration in three basic fractions as α-, β- and γ-globulins.
  • γ-Globulins having been basic proteins show an unwillingness to move in the electric field at the pH 8.6, and produce broad diffuse zone on the electrophoretoram.
  • Depending on biological species subfractions of serum proteins appears on the electrophoretogram: α1-, α2-; β1-, β2-
  • Serum proteins are not visible after their electrophoretic separation. That is why they must be visulized using a colorproducing method (Coommassie Brilliant Blue R).
  • Using colorimetric method a pattern of serum proteins is obtained, and it is possible to quatify the individual fraction densitometrically, i.e. to express them as a percentage of the total protein concentration

Prealbumin

  • most rapidly migrated fraction which cannot be detected using acetate cellulose as support medium. In the gradient of polyacrylamide (8-25%) it was found in dog, horse, rabbit, pig and cattle.
  • The main function of prealbumin is a transport of tyroxine

Albumin

  • fast moving fraction due to its strong electronegativity to the anode
  • most concentrated fraction with the 35%- 50% of the total serum protein
  • Its main function is maintain osmotic pressure and   general transport (of different molecules - solubilization of hydrophobic substances)
  • site of albumin synthesis is liver
  • degratdation takes place in all metabolically active tissues
  • rate of metabolism is reflected in the half-times for clearance which is quite species characteristic and ranges from 8.2 to19.4 days.

α1- and α2-globulins

  • α1-antitrypsin
  • α1-antichymotrypsin,
  • α1-acid glycoprotein (orosomukoid, seromucoid)
  • α1-lipoprotein,
  • α2-lipoprotein,
  • α2-macroglobulin,
  • ceruloplasmin (copper transport)
  • haptoglobin (hemoglobin binding)

β-globulins

  • β-lipoprotein,
  • transferrin,
  • ferritin,
  • hemopexin (heme transport),
  • plasminogen (plasmin proenzyme - fibrinolysis),
  • fibrinogen (precursor of fibrin – blood coaggulation),
  • C-reactive protein (activates complement)

γ-globulins

  • IgG (major antobody formed in response to infectious and toxin agents);
  • IgA (antibodies of the respiratory, gastrointestinal, and genitourinary tracts);
  • IgE (antobodies in allergy);
  • IgM (the first antibody against antigen)

Interpretation of Serum Protein Profiles

  • Before the electrophoretic separation of serum proteins it is necessary to determine concentration of total serum protein and the concentration of albumin
  • Thus it is posssible to calculate albumin-to-globulin ratio(A:G).
  • A change in the value of A:G is often the first signal of a protein dyscrasia (pathologically changed serum protein composition in blood of animals)

Influence of Age and Development of an Animal

  • generally, in the fetus, the concentration of total protein progressively increases in the absence or low concentration of γ-globulins;
  • in newly born baby pigs and calves a large amounts of γ-globulin appears in the blood up to 24 hours after ingestion of colostrum (passive transport of γ-globulin across the permeabile gut).
  • The absorption continues for up to 48 hours after birth when gut permeability ceases.
  • in colostrum-deprived calves, imunoglobulin increases only minimaly
  • in foals from bird to 12 months of age albumin,
  • globulins and total proteins progressively increase;
  • Summary   In animals, there is a general
  • increase in total protein,
  • increase in globulins, and
  • decrease in albumin   with advancing age

Effects of Inflammation on Serum Proteins

  • The changes in a select group of plasma proteins occur in the early stages of inflammatory process in all animals.
  • This group is classified as acute phase proteins (APPs). The main APPs differ each other from one animal species to another:
    • cattle: haptoglobin (Hp), serum amyloid A (SAA);
    • dog: C-reactive protein (CRP), SAA;
    • horse: SAA
    • pig: CRP
  • Specific methods have been developed for   detection of individual APPs, e.g. radial   immunodifusion, ELISA, immunoturbidimetry

Acute Phase Proteins (APP) as Indicators of Inflammatory Process

  • positive APPs
    • α-globulins (α1-antitrypsín, α1-kyslý glykoproteín; α2-makroglobulín, ceruloplazmín, SAA -serum-amyloid A, haptoglobulín)
    • β-globulins: (fibrinogen, complement, C3, C4, Creactive protein, feritin, amyloid A)
  • negative APP: prealbumin, albumin, transferin

Dysproteinaemias

  • disorders in protein status of blood plasma/serum which rise from disorders/diseases of some organs (liver, kidney, skin, gastro-intestinal tract, also due to presence of internal parasites, tumors.etc.)
  • The first approach is determination of albumin:globulin ratio (A:G). This can be obtained
    • a) by qualitative colorimetric determination of TP and albumin, which is subtracted from TP and globulin concentration is obtained
    • b) A:G can be determined using serum proteins electrophoresis. Due to a disorder, the pattern is changed and together with the changed A:G it can serve as a useful indicator in diagnosis of a patient´s disease

  

  1. Normal A:G ratio and normal SPE profile

      1. a) hyperproteinemia: occurs with simple dehydratation with water loss. All fractions increase proportionately including albumin.         That is why A:G ratio is not changed 2. b) hypoproteinemia: overhydratation, e.g. vigorous fluid therapy, excess water intake (acute blood loss). As the dilution of plasma proteins is proportional the A:G ratio is not changed

  1. Decreased A:G ratio and abnormal SPE profile

      1. decresed albumin: this is quite a common form of dysproteinemia.         Reasons: either loss of albumin or failure in its synthesis: the only site of albumin synthesis is liver. Thus the hypoalbuminemia is an important feature of chronic liver disease

         1. Hypoalbuminaemia also observed in diseases of:

            1. kidney (glomerulonefritis, nephrosis) – renal loss of albumin due to the small size of its molecule; 2. gastrointestinal tract (diarhea) 3. intestinal parasitism

  1. increased globulins and SPE pattern

   

  1. α-globulins: in the acute inflammatory process α2 -globulins increase predominantly (α2-lipoprotein, α2-macroglobulin, ceruloplasmin, haptoglobulin, protein C)
  2. \       
    1. β-globulins: increase in β-globulins (β2 lipoproteín, transferín, feritín, hemopexín) is infrequent, and may occur at:

          1. acute hepatitis (transferin, hemopexin); 2. nefrotic syndrome (β2-lipoprotein, transferin); 3. suppurative (acute) dermatitis (IgM, C3) 3. γ-globulins – polyclonal gammopathy. SPE pattern:          diffuse, intensive and/or broad zone in the area of γ-globulins as a result of synthesis of a heterogeneous mix of immunoglobulins, especially IgM, IgG and/or IgA. This pattern is accompanied with hypoalbuminemia as a result of decreased synthesis. This pattern of plasma proteins appears in chronic inflammatory diseases, malignancies and immunologically mediated processes:          chronic hepatitis, hepatic abscesses, diseases of immunological system, acute dermatitis, tumors of the reticuloendothelial system (lymphoosarcoma).          The diffuse and broad zone of γ-globulins is manifested as a broad peak on the electrophoretogram (densitogram). It          is quantitatively far intensive then that of albumin. The A:G ratio is also changed in favour for globulins