Exam B Blakes flashcards
What are the functions of proteins
Nutrition
Maintenance of oncotic pressure
Transport- lipids, bilirubin, calcium, iron, some hormones, some drugs, chylomicrons
Catalytic function- enzymes
Immunity: Specific-Immunoglobulin
Non-Specific-Complement, Cytokines, Interferon, CRP, alpha trypsin
Some hormones
Coagulation Factors
Buffering
Salt fractionation
It is the process of purification of protein using salt. The protein competes with
the salt to bind with the water molecules. As the concentration of the salt increases, the solubility of the protein decreases and the protein is precipitated out. Different proteins have different group on their surface and thus different solubility. So they precipitate out at different concentrations of salt. Highly water soluble salt is used. Ammonium sulfate is the most commonly used salt.
Zwitterions
dipole ion containing both positive and negative charged groups. Thus they have both acidic and basic group in their surface. This makes them highly soluble in water and less soluble in organic solvent. Amino acids are the example of zwitterions due to the presence of positively
charged amino group and negatively charged carboxyl group on same molecule.
Zeta potential
It is the potential produced by the effective charge on a surface of macromolecule (colloids), usually taken at the boundary between what is moving with the macromolecule and the
rest of the solution.
polysaccharide
long chain of repeating units of carbohydrates joined together by glycosidic bond. The repeating units may be monosaccharide or disaccharide and can be branched.
polypeptide
It is a chain or a complex structure of amino acids joined together by peptide bond. Proteins are polypeptide molecules. They constitute of multiple polypeptide subunits.
oligoclonal banding
When various proteins share the same electrophoretic mobility, they are
called oligoclonal bands. For example, immunoglobulins from cerebrospinal fluid appear in a
same γ-range in an agarose gel.
Briefly describe the Kjeldahl techniques for determination of protein and nonprotein nitrogen
Kjeldahl: this is the oldest approach for quantifying total serum proteins. This method has good accuracy and precision but is very time consuming and tedious. In this method nitrogen containing compounds are oxidized to NH4+, then in alkaline conditions NH4+ Is converted to NH3. Then NH3
is titrated with HCL.
Hypoalbuminemia causes
Low production:
Malnutrition (low AA)
Liver disease
Loss or use after synthesis:
GI leakage from inflammation and disease of GI mucosa
Renal disease (glomerulonephritis, nephrotic syndrome)
Burns
Inflammation
Neoplasm
Pregnancy
Edema → loss of intravascular oncotic pressure
Ascites
Hyperalbuminemia
Dehydration - Hemoconcentration
Due to other pathologies (Three Cs):
Cancer
Chronic infection (high protein synthesis)
CHF (low GFR so more protein retention)
The globulin level may be elevated in
Chronic infections (parasites, some cases of viral and bacterial infection)
Liver disease (biliary cirrhosis, obstructive jaundice)
Carcinoid syndrome
Rheumatoid arthritis
Ulcerative colitis
Multiple myelomas, leukemias, Waldenstrom’s macroglobulinemia
Autoimmunity (systemic lupus, collagen diseases)
Kidney dysfunction (nephrosis)
The serum globulin level may be decreased in
Nephrosis (a condition in which the kidney does not filter the protein from the blood and it leaks into the urine)
Alpha-1 Antitrypsin Deficiency (Emphysema)
Acute hemolytic anemia
Liver dysfunction
Hypogammaglobulinemia/Agammaglobulinemia
What is the theory of refractometry? What are major interfering substances in the determination of serum protein by refractometry?
This method is based on refraction of incident light by dissolved solids. The refractive index of water at 20 C is 1.3330. The refractive index increases proportionally with increasing amounts of a particular solute. It is a relatively simple, easy and fast method. The disadvantage is that the assumption that concentrations of inorganic electrolytes and nonprotein organic compounds are not changing from sample to sample. Lipemia and hemolysis may cause falsely increased plasma protein values.
Name three ways to separate albumins from globulins.
Gel electrophoresis
Column Chromatography
Ultracentrifugation
Others: precipitation, plasma fractionation
Discuss the reasons for determining spinal fluid protein and glucose. What are normal values? What results are expected in meningitis? Encephalitis? Neurosyphilis?
Spinal fluid protein and glucose is determined as a part of CSF total protein test. CSF is a clear fluid that occupies the subarachnoid space and the ventricular system around and inside the brain and circulates in the space surrounding the spinal cord. Any abnormal protein level in CSF tells us that there is an abnormal process occurring in the central nervous system or if there is subarachnoid hemorrhage.
Plasma proteins and Glucose levels in
Meningitis
Increased CSF proteins for Bacteria
normal CSF proteins for Viral
Decreased CSF glucose for Bacterial and normal for viral
Plasma proteins and Glucose levels in
Encephalitis
Increased CSF protein and normal glucose
Plasma proteins and Glucose levels in
Neurosyphilis
Increased CSF protein and normal CSF glucose
What is more sensitive BCP or BCG in the BCG method for Albumin
BCP is more sensitive
What is the Biuret reagent? Explain its function in determination of total protein. What are the major interfering substances?
Biuret reagent contains 2 reactants: 0.15% cupric sulfate, 3% sodium hydroxide and stabilizers. This is a widely used colorimetric reaction in which cupric ion complexes with peptide bonds to produce a violet colored complex in an alkaline solution which can be measured at 540 nm. Since copper reacts with the peptide bonds, the more peptide bonds, the darker the sample will be. The biuret reagent has specificity for backbone of proteins.
What are the major interfering substances in the Biuret reaction
BSP dye, phenazopyridine, hemolysis, lipemia, radiopaque dyes, plasma
expanders that contain: proteoses, peptones, gelatins and dextrans These all falsely elevate the results
What is an A/G ratio? How is it used diagnostically?
The A/G ratio is the Albumin/Globulin ratio. A low A/G ratio suggests overproduction of globulins such as is seen in multiple myeloma and autoimmune disease, or underproduction of albumin such as occurs with cirrhosis, or selective loss of albumin from the circulation as occurs with nephritic syndrome. A high A/G ratio suggests underproduction of immunoglobulins as may be seen in some genetic deficiencies and leukemias.
Normal Albumin/Globulin ratio = 1 - 2.5
How to calculate the A/G ratio
Albumin-[total proteins-Albumin]
Which proteins increase in acute phase? Where (which zones) do these proteins migrate upon electrophoresis?
Positive acute phase reactants increase during inflammatory responses and are usually involved in immune and protective functions
Alpha 1: alpha 1-antitrypsin, alpha 1-antichymotrypsin
Alpha 2: alpha 2 macroglobulin, prothrombin, ceruloplasmin, haptoglobin
Beta: fibrinogen, complements
Mid Beta to post Gamma: CRP
Others: serum amyloid P component, MBL, factor VIII, vWF, PAI-1, Ferritin
Negative acute phase reactants decrease during inflammatory responses as the amino acids required for synthesis of these proteins are being prioritized elsewhere
Examples: albumin, antithrombin, transferrin, retinol-binding protein
What are their iGs relative concentrations in serum
Order from increased serum concentration to decreased serum concentration
IgG= 800-1600mg/dl
IgA=150-400mg/dl
IgM=50-200mg/dl
IgD=1.5-40mg/dl
IgE=.002-.05mg/dl
Alpha-1-antitrypsin- (ATT or A1TT)
is a protein that irreversibly inactivates or neutralizes trypsin enzyme in the duodenum and as such is also referred to as a protease inhibitor. This prevents uninhibited breakdown of tissue that could lead to liver cirrhosis. Lack of this protein leads to pulmonary emphysema and liver cirrhosis. Increases are seen in acute phase reaction, pregnancy, and oral contraceptive use.
Question asked in simple diffusion assay, to look for Ig concentration the patient’s serum is added to gel that contains:
Antibody
Ceruloplasmin has what enzymatic activities and is in what disease states
This has enzymatic activities such as copper oxidase, histaminase, and ferroxidase. It is
the major copper binding protein in the plasma. Lowered levels could indicate Menke’s syndrome or Wilson’s copper storage disease. Higher levels may indicate pregnancy, lymphoma, infections, or rheumatoid arthriti
Alpha 2 macroglobulin is produced in the and neutralizes what
Large protein produced in liver that functions to neutralize enzymes (particularly thrombin, trypsin, pepsin and plasmin), carry zinc and it is also involved in the innate immune response.
An increase in this protein is associated with nephritic syndrome and is also linked to increased risk of Alzheimer’s disease.
Beta-2 Microglobulin
This protein is part of the light chain of HLA antigen found on the surface of most nucleated cells, especially lymphocytes. It is increased in tubular damage due to decreased clearance by
the kidney, overproduction of certain cell types in RA and SLE. An increase is also seen in HIV patients which indicates a large lymphocyte turnover, which suggests the viral killing of lymphocytes.
Describe three techniques for quantifying immunoglobulins
Three techniques used to quantify immunoglobulins are Immunoturbidimetric/
Immunonephelometric, labeled techniques such as Radioimmunoassays, Enzyme Immunoassays
and Fluorescent Polarization assays and finally Radial Immunodiffusion techniques.
Sandwich RIA
Is where a antibody is on the surface of a well and the patients serum is added and the antigens attach to the antibody and the rest is washed away and a labeled antibody is the usually the label is I125 and then attaches to the other end of the antibody and then the rest is washed away and the amount of antigen is directly proportional to the amount of antibody present.
Inverse relationships is seen when a two antibody; one that is labeled and the patients antibody compete with one another for binding to the patients antigen such as in
Competitive binding RIA
a Scintillator counter is used to
count antigen-antibody complexes with Sandwich RIA techniques
A standard curve in RID plotes
Antigen concentration as a function of the diameter squared in (mm)^2
what is the function of haptoglobin and when is it increased or decreased
Haptoglobin is increased in binds oxidized hemoglobin in the plasma and is a acute phase reactant.
Haptoglobin is decreased in Intravasular hemolysis ( hemolytic anemia, transfusion reactions, and malaria.)
Haptoglobin is increased in burns, nephrotic syndrome when plasma proteins are lost, RA, tissue necrosis
When vivo hemolysis occurs what happens to haptoglobin levels
decrease
what is the most specific cardiac marker
Troponin cTnl
what are the three subsets of troponin
Troponin C binds calcium
Troponin I is the inhibitory component
Troponin T is the tropomyosin binding component
what method is used to measure troponin
Antibody immunoassay chromagenic which binds troponin and is more cardiac-specific then CKMB which can be falsely elevated in muscle injures due to CK2 elevation
Troponin advantages
it is released from both dead and dying cells and is present for up to 7-10 days as compared to CKMB which is only present for 24-36 hours. It is more specific and can detect even minor heart injuries
what is the first cardiac marker to be detected in myocardial infarction?
Myoglobulin and its peak is 6-8 hours and is only detectable until 24 hours
CHF marker and definition
decreased ablility of the heart to pump blood
marker: BNP or brain natriuretic peptide which cause a increase in cardiac output and a decrease in blood volume and promotes your kidneys to remove water and salt(ANP) a type Natriuretic
interpret this
