Notes in CARBOHYDRATES, LIPIDS, PROTEINS, AND NPNs

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• Proteins are macromolecules composed of polymers covalently linked by amino acids

  • Amino acids are the building blocks of proteins and dictate their chemical properties and biological activity

• Proteins have various functions in cells and the extracellular matrix

  • They contribute to the structure of cells and the extracellular matrix

  • They transport materials and act as receptors for hormones and transcription factors

• Proteins are amphoteric and contain C, H, O, N, and S

• Proteins are synthesized in the liver and secreted into circulation

• Protein synthesis occurs in the ribosome

• Protein catabolism involves the disintegration of proteins into amino acids

• Nitrogen balance refers to the balance between protein uptake and excretion

  • Negative nitrogen balance occurs when protein catabolism exceeds protein anabolism

  • Positive nitrogen balance occurs when protein anabolism exceeds protein catabolism

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• Proteins have different levels of structure: primary, secondary, tertiary, and quaternary

• Primary structure refers to the specific sequence of amino acids in a protein

• Secondary structure is stabilized by hydrogen bonds and can be alpha helix or beta pleated sheet

• Tertiary structure is the overall conformation of the protein molecule due to the interaction of side chains

• Quaternary structure involves the interaction of multiple protein molecules or subunits

• Protein denaturation refers to the change in protein structure, resulting in loss of function and chemical characteristics

• Factors that promote denaturation include changes in temperature, hydrolysis, enzymatic action, exposure to certain substances, and UV light

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• Proteins can be classified based on their functions

  • Enzymes catalyze chemical reactions

  • Hormones control the actions of specific cells or organs

  • Transport proteins facilitate the movement of ions, molecules, or macromolecules across membranes

  • Immunoglobulins mediate immune responses

  • Structural proteins provide the structure of cells and tissues

  • Storage proteins serve as reserves of metal ions and amino acids

  • Plasma proteins serve as an energy source and participate in osmotic force, hemostasis, and acid-base balance

• Proteins can also be classified based on their structure

  • Simple proteins contain only amino acids

  • Conjugated proteins consist of a protein and a nonprotein prosthetic group

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• Simple proteins can be globular or fibrous

• Conjugated proteins include metalloproteins, lipoproteins, mucoproteins or proteoglycans, glycoproteins, and nucleoproteins

• Plasma proteins include albumin, globulin (α1, α2, β, γ), and are separated into fractions when subjected to electrophoresis

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Plasma Protein Function

• Prealbumin (Thyroxine binding protein)

  • Indicator of nutrition; first protein to decrease when the px is malnourish

  • Binds thyroid hormones (T3, T4)

  • Binds retinol-binding protein

  • Not seen in the typical electrophoresis; found before the albumin fraction

• Albumin

  • Binds bilirubin, steroids, fatty acids

  • Major contributor to oncotic pressure

  • Major transporter in the plasma; most abundant protein in the plasma

• α1-Globulins

  • α1 - Antitrypsin

    • Acute phase reactant – increases in acute stage of inflammation

    • Protease inhibitor – prevents the enzymatic activity of proteins

  • α1 - Fetoprotein

    • Principal fetal protein

    • Adults: low; if high, it is already abnormal and linked to liver cancer (hepatoma)

    • Newborns: high

      • Tumor marker for liver cancer

      • ↑ - spina bifida, neural tube defects, fetal distress

      • ↓ - Down syndrome, trisomy 18

  • α1 -Acid glycoprotein

    • Acute phase reactant

    • Increased in acute phase inflammation – happens when there is tissue damage and infection

  • α1 – Lipoprotein

    • Transport lipids (HDL)

  • α1 - Antichymotrypsin

    • Inhibits serine proteinases

  • Inter-α-trypsin inhibitor

    • Inhibits serine proteinases

  • Gc-globulin

    • Transports Vitamin D and binds actin

• α2-Globulins – present between a1 and beta

  • Haptoglobins

    • Acute Phase reactant

    • Binds hemoglobin (free hemoglobin)

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Plasma Protein Function (continued)

• α2-Globulins – present between a1 and beta (continued)

  • Ceruloplasmin

    • Acute Phase reactant, contains copper

    • ↓ - Wilson’s dse., Menkes syn. (kinkey hair dse.), Kayser Fleisher rings in cornea

    • Low ceruloplasmin level = free copper increases

  • α2 - Macroglobulin

    • Inhibits protease; enzyme inhibitor

• β-Globulin – found between a2 and gamma

  • Pre-β-lipoprotein

    • Transports lipids (VLDL triglyceride)

  • Transferrin

    • Transport iron, ↑-IDA, ↓-hemochromatosis

    • Inversely proportional in the iron

  • Hemopexin

    • Acute phase reactant, Binds heme

  • β-Lipoprotein

    • Transports lipids (LDL cholesterol)

  • β2-Microglobulin

    • Component of HLA molecules

  • C4, C3, C1q complement

    • Immune response

  • Fibrinogen (Factor I)

    • Precursor of fibrin clot

  • C-reactive protein

    • MAJOR acute phase reactants

    • Motivates phagocytosis in inflammation

• γ-Gamma-Globulins – least anodic

  • Immunoglobulin G

    • Antibodies; secondary response

    • Most abundant antibody in healthy human serum

  • Immunoglobulin A

    • Antibodies in secretions; primary response

    • Provides mucosal immunity

  • Immunoglobulin M

    • Antibodies in early response

    • Biggest antibody in the human serum

  • Immunoglobulin E

    • Antibodies (reagen, allergy)

    • A.k.a reaginic antibody; has the lowest concentration for healthy individuals

  • Immunoglobulin D

    • Surface antibody

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Other Plasma Proteins Function

• Myoglobin

  • Oxygen carrier in muscles

  • Cardiac marker (AMI)

  • ↑ 2-3 hours of onset, peak at 8-12 hours

  • ↑ crushing injury and muscle dystrophy

• Troponin (cTn)

  • Cardiac marker for acute coronary syndrome

• Brain Natriuretic peptide

  • Neurohormones that affect body fluid homeostasis and blood pressure

  • Marker of congestive heart failure

• Fibronectin

• Fetal fibronectin (fFN)

  • Cellular interaction (e.g., cell adhesion)

  • Adherence of the placenta to the uterus

  • ↑ - Preterm labor and delivery

• Cross-Linked C-Telopeptides

  • Proteolytic fragment of collagen I

  • Biochemical marker of bone resorption (promoted by osteoclast)

• β-Trace Protein

  • Syn: Prostaglandin D synthase

  • Marker for CSF leakage (e.g. head injuries)

• Cystatin C

  • Cysteine proteinase inhibitor

  • Serum marker for glomerular filtration rate (kidney function)

  • Being developed for acute/early renal damage marker

• Amyloid

  • Fibrous protein aggregates formed from alteration of β pleated sheaths

  • ↑ Amyloidoses

  • Differential diagnosis of Alzheimer

  • Low Aβ42 with high Tau proteins

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Total Protein Abnormalities

• Measures globulin and albumin

• If abnormal, further tests must be performed to identify which protein fraction is abnormal

• Reflect the following:

  1. Nutritional status of the px

  2. Renal or kidney disease

  3. Liver disease

  4. Many other conditions

• HYPOPROTEINEMIA

  • Negative balance is present

  • Excessive loss of protein

  • Total Protein

  • Albumin

  • Globulin

  • Disease

• Hepatic Damage

  • Cirrhosis β-γ bridging

  • Hepatitis ↑ γ-globulins

  • Obstructive jaundice ↑ α2

• Burns, trauma Infections

  • Acute α1 –, α2 – globulins

  • Chronic ↑ α1, α2 γ-globulins

• Malabsorption, Inadequate diet, nephrotic syndrome

  • ↑ α2-, β-globulins; ↓ γ-globulins

• Immunodeficiency syndrome

  • ↓ ↓ ↓

• Salt retention syndrome

• HYPERPROTEINEMIA

  • Not an actual disease state but a result of underlying cause (e.g. dehydration, excessive

  • Total Protein

  • Albumin

  • Globulin

  • Disease

• Dehydration

  • Multiple myeloma

  • Monoclonal and polyclonal gammopathies

Methods of Analysis Total Protein Measurement

• Reference Interval:

  • 6.5-8.3 g/dL (65-83 g/L)

  • 6.0-7.8 g/dL (60-78 g/L) in recumbent position

• METHOD

  • Kjeldahl

    • Digestion of protein; measurement of nitrogen content

    • Reference method. Assume average nitrogen content of 16%

  • Refractometry

    • Measurement of refractive index due to solutes in serum

  • Biuret

    • Formation of violet-colored chelate between Cu2+ ions and peptide bonds

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Notes in CARBOHYDRATES, LIPIDS, PROTEINS, AND NPNs by Mark Rodrigo D. Mendros, RMT, MT(ASCPi,), MSMT

• Dye Binding

  • Protein binds to dye and causes a spectral shift in the absorbance maximum of dye

  • KJELDAHL

    • Digestion of proteins with sulfuric acid at 340 deg C

    • Potassium sulfate is introduced to increase the boiling point and improve digestion

    • Acid precipitation (TCA or tungstic acid) of protein with measurement of total nitrogen

  • Kjeldahlization

    • Conversion of nitrogen to ammonia

    • Nitrogen Ammonia measurement H2SO4à NH3

    • Nessler’s reaction (double iodide of Hg and K)

      • Ammonia + Nessler’s rgt Gum ghatti à yellow solution (Ammonium dimercuric iodide)

    • Berthelot reaction

      • Ammonia + alkaline hypochlorite

      • Na nitroprusside à Indophenol blue

  • REFRACTOMETER

    • Measurement of refractive index (velocity of light in air and water) due to solutes in serum

    • ADVANTAGE: small volume of sample is needed

  • BIURET

    • Formation of violet-colored chelate between Cu2+ ions and peptide bonds (measured at 540 nm)

    • There must be 2 or more peptide bonds

    • Composition:

      • Cupric ions – breaks the peptide bonds

      • Tartrate salt – keeps copper in solution

      • Potassium iodide – stabilizes cupric ions

  • DYE BINDING

    • Protein binds to dye and causes a spectral shift in the absorbance maximum of dye.

    • Bromphenol blue

    • Ponceau S

    • Amido black 10B

    • Lissamine green

    • Coomassie brilliant blue

  • ALBUMIN MEASUREMENT METHOD

    • PRINCIPLE

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Notes in CARBOHYDRATES, LIPIDS, PROTEINS, AND NPNs by Mark Rodrigo D. Mendros, RMT, MT(ASCPi,), MSMT

• Salt Precipitation

  • Uses sodium sulfate as precipitating agent

  • Globulins are precipitated in high salt concentrations

  • Albumin in supernatant is quantitated by biuret reaction

• Dye binding

  • Methyl orange

    • Nonspecific for Albumin

  • HABA (2,4-hydroxyphenyl benzoic acid)

    • Many Interferences (salicylates, bilirubin)

  • BCG (Bromcresol green)

    • Sensitive

    • Overestimates low albumin levels

    • Most commonly used dye

  • BCP (Bromcresol purple)

    • Specific, Sensitive and Precise

• Electrophoresis

  • Proteins separated based on electric charge densities

  • Give overview in relative changes in different protein fraction

• ELECTROPHORESIS

  • Support media: Cellulose acetate/agarose gel

  • After separation, protein fractions are immersed in acid solution then stained by dyes (e.g. Coomassie blue)

  • Best method to fractionate proteins

  • The medium is placed in scanning densitometer which compute the area under the absorbance.

  • Reference values:

    • Albumin: 53-65% (3.5-5.0 g/dL)

    • α1 – Globulin: 2.5-5% (0.1-0.3 g/dL)

    • α2 – Globulin: 7-13% (0.6-1.0 g/dL)

    • β – Globulin: 8-14% (0.7-1.1 g/dL)

    • γ – Globulin: 12-22% (0.8-1.6 g/dL)

    • Monoclonal increase (M-spike; seen in multiple myeloma)

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Notes in CARBOHYDRATES, LIPIDS, PROTEINS, AND NPNs by Mark Rodrigo D. Mendros, RMT, MT(ASCPi,), MSMT

• ACUTE PHASE REACTANT

  • Fibrinogen

  • Haptoglobin

  • Ceruloplasmin

  • Serum amyloid

  • CRP – major acute phase reactant; first to increase

• HIGH RESOLUTION PROTEIN ELECTROPHORESIS

  • Uses higher voltage couple with a cooling system and more concentrated buffer

  • More than 5 bands are demonstrated, specific protein bands are also seen

• PROTEIN IN OTHER BODY FLUIDS TOTAL PROTEINS

  • Oligoclonal bonding – in CSF protein; multiple sclerosis

• METHOD

  • Turbidimetric methods (SSA, TCA, benzethonium chloride)

    • Proteins are precipitated as particles, turbidimetry is measured spectrophotometrically rapid and easy to use; accurate in sensitive methods

  • Biuret

    • Proteins is reacted with Cu2+ forms colored complex with peptide bonds

  • Folin-Lowry

    • Initial biuret reaction; oxidation of AA (tyrosine, etc.) residues by Folin Phenol reagent; measurement of resultant blue color.

  • Dye binding (Coomassie blue, Ponceau S)

    • Protein binds to dye, causes shift in absorption maximum.