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
which is abnormal monoclonal or polyclonal antibodies
Monoclonal or M spike proteins
Polyclonal gammopathy
Is the proliferation of more then on cell line and is due to high exposure to antigens ( infection, neoplastic disorder,
Where is EPO produced
in the kidneys
where is IgA released
from epithelial cells
is Von williebrand factor synthesized by the liver
no
interpret this
Immunofixation electrophoresis
is where six sample wells are applied with samples and then electrophoreses to separate the individual proteins, then antisera for immunoglobulins are applied with a total protein reference sample, then the proteins are stained with coomassie blue.
Order of protein bands and describe protein electrophoresis
-Albumin, alpha 1, alpha 2, beta, gamma+
all major serum protein carry a net negative charge and will migrate toward the cathode. there is a current that is applied and the buffer is alkaline buffer at 8.6pH. The proteins are denatured to allow for visualization by a stain such as coomassie blue or ponceau S)
order of the alpha 1 region
Alpha 1 antitrypsin
alpha 1 acid glycoprotein
alpha 1 lipoprotein
alpha 1 fetoprotein
order of the alpha 2 region
alpha 2 macroglobulin
Haptoglobulin
ceruloplasmin
Beta region
beta q
transferrin
hemopexin
b-lipoprotien
C-4
Beta 2 region
Fibrinogen
C-3
Beta-2-microglobulin
Gamma region
IgG
IgA
IgM
IgD
IgE
C-reactive protein
multiple myeloma pattern
major monoclonal spike in the gamma region
Nephritic syndrome
Decrease in albumin and alpha 1 proteins, with a increase in the alpha 2 portion
Liver disease
albumin is decreased and the beta and gamma region increased in chronic hepatitis and IgM and IgA in billiary or alcoholic cirrhosis.
Obstructive jaundice
A1 is decreased and A2 and beta is increased
what has bridging in the beta and gamma regions
Cirrhosis
Chronic infection
IgG increased but normal beta and albumin is decreased because AA are used up for IgG synthesis
Acute phase reaction
Increase in beta portion
alpha 2( increase haptoglobulin) is increased and alpha 1
albumin is decreased
Malnutrition
decrease in Albumin
What is indicative of multiple sclerosis
Oligoclonal bands(70-90% of patients) are indicative of MS in cases where it is present in the CSF ultra filtrate but not in the serum
Fresh serum containing complement
Will show a double banded increase in B2 region representing of C3 which is the major complement fraction
NPNs
are non nitrogenous proteins that make up the nitrogen portion of the blood not composed of proteins
50% of NPN are creatinine, urea, uric acid, and creatinine.
what does BUN stand for
Blood urea nitrogen is used as a measurement of renal function
Azotemia
Azotemia is a condition characterized by abnormal levels of nitrogen-containing compounds, such as urea, creatinine, various body waste compounds, and other nitrogen-rich compounds in the blood. It is largely related to insufficient filtering of blood by the kidneys.
normal ratio of BUN:creatinine in pre renal azotemia is
greater then 20
normal ratio of BUN to creatinine in azotemia is but increased levels of both are in
azotemia
Post renal azotemia has a increase in 15:1 early but then goes back down to
Normal but BUN is greatly increased
If serum creatinine is elevated then it is most indicative of
Muscle disease
Creatinine In the serum is directly proportional to muscle mass and is increased In males and decreased with age
Prerenal azotemia diseases
Poor perfusion, GI Hemorrhage
Increased protein intake
Renal azotemia diseases
Pyelonephritis
Acute tubular nephrosis
Postrenal disease states
Kidney stones, Tumors
Prostatitis, Kidney blockage
normal ratio of BUN:Creatinine is
10:1-20:1
Normal values for BUN in the urine and serum
50-170mg/L in serum and 7-16g/L in urine
Normal value for Creatinine in males and females
6.4 mg/L in males, and 5.7 – 6.2 mg/L in females
Berthelot reaction
is a colorimetric method for urea
urea——> ammonia and carbonate by urease
Phenol + hypochlorite( color developer) + ammonia + sodium nitroprusside( enzymatic reagent——-> blue green color ( indophenol)
EDTA, fluoride, and citrate are anticoagulants that must be avoided because they inhibit
urease reaction in the berthelot method
The most frequently used method for BUN or urea is
Glutamate dehydrogenase reaction which measures ammonium quantity by detecting NADH—> NAD or a decrease in absorbance at 340nm
Jaffs reaction is used to measure what
What is the reaction temperature and why
What are interfering substances
Creatinine
Jaffe’s reaction is the oldest clinical chemistry method still in use today. This reaction is conducted at 30°C so that the reagent, picrate, does not interact with substances that may give a false positive. Substances that may yield a false positive due to interaction with picrate are glucose, uric acid and ascorbic acid. The reaction
requires a protein-free filtrate and Fuller’s Earth, flordin, to increase specificity of the reaction. Advantages to this reaction are that it is automated and low cost. The disadvantage to this reaction is that Fuller’s Earth needs to be added manually. Pyruvate and oxaloacetate at high concentrations may interfere as well.
Jaffs reaction
Creatinine + picrate → Janovski complex (red) (alkaline pH)
This kinetic reaction is measured spectrophotometrically at 520 nm.
Creatinine clearance
C = UV/P and you need V in Ml/min
U = Urine creatinine (mg/dL)
V = urine Volume (mL/min)
P = plasma creatinine (mg/dL)
C=UV/P*1.73/SA where SA is the Surface area
SA= square root of [ (Height (cm or in) x Weight (kg or lb)) / 3600 (cmkg) or 3131 (inlb) ]
Cancer associated with a-fetoprotien is
liver and germ cell tumors of the testies and ovaries
what marker is associated with adenocarcinoma of the colon
Carcinoembryonic ag or CEA
what organism is grown to detect PKU in the Guthrie test
Bacillus subtilis will overcome inhibition by thienylalanine and flourish
Ninhydrin test
will separate AA by ion exchange chromatography and then the ninhydrin will react with alpha protein by producing a colored compound
PKU can be prevented by
following a strict diet that excludes phenylalanine before 1 year to prevent intellectual disability.
PKU deficiency is
Phenylalanine hydroxylase which catalyzes the conversion of phenylalanine to tyrosine
cryoglobulins are
IgG that precipitate at low temperatures and are measured by precipitate centrifugation
CA125
ovarian and endometrial carcinoma; evaluated with RIA/EIA
PSA
prostate specific antigen is measured specifically for screening prostate cancer; high levels are not indicative but levels >4ng/L are significant; measured with immunoassay
CA-15-3
carbohydrate antigen-15-3 is a glycoprotein antigen found in high serum levels of breast cancer patients; although it is not diagnostic, it is a good monitor for patient response to chemotherapy and post surgery.
abnormal uric acid could result in
1.) kidney failure or stones
2.) gravel in an infants diaper
3.) neurological problems
4.) gout
Alkaline picrate is used to measure what
Creatinine + picrate—-> orange color in an alkaline medium
520nm
IgG lambda in
Multiple myeloma
BCG measures albumin _____________
BCG is ________ ph
G as in
Directly
Acidic pH
G as in glue
What decreases interferences with the Jaffs reaction
Koalin
Uses a protien free filtrate
Fuller earth
Hepatic cirrhosis has blank on electrophoresis pattern
Gamma-beta bridge
What are the two subunits of LDH
LD1 has 4 _______
LD 5 has 4 ______
M and H as in muscle and heart
LD1 has 4Hs
LD5 has 4Ms
LD1 and LD2 are found in the
Heart and RBCs
LD3 is found in the
Renal cortex and kidney
LD4 and 5 are found in
Skeletal muscle and liver
Lactate to pyruvate occurs at what pH
7.8 and makes NADH
What is measured by glass ph electrode
Na2+ and bicarbonate
In ISE what method requires a dilution
Indirect method for ISE and Flame emission
Normal range for CSF protien is
14-45mg/dl
Osmolality can be used with what samples
Serum and urine
