Test 2
Albumin (info)
synthesized in hepatic parenchymal cells transports fatty acids etc low=nephrotic syndrome, hepatic disease high=dehydration, burns, CHF
The three-dimensional spatial configuration of a single polypeptide chain as determined by disulfide linkages, hydrogen bonds, and van der Waals forces is referred to as the: a. primary structure b. secondary structure c. tertiary structure d. quaternary structure
tertiary structure
Kjeldahl's procedure for total protein is based upon the premise that: a. proteins are negatively charged b. the pKa of proteins is the same c. the N content of proteins is constant d. proteins have similar Tyr and Trp content
the N content of proteins is constant
BUN Methodologies: Nessler's Reaction
the addition of a double iodide compound (2HgI2 + 2KI) results in the formation of a yellow to orange brown compound with NH4+. (classic reaction)
BUN Methodologies: Berthelot's reaction
the ammonium ion is reacted with phenol and hypochlorite in an alkaline medium to form indophenol blue, the chromagen which is measured. (classic reaction)
What is a Quarternary Structure (QS)?
the arrangement of two or more polypeptide chains to form a protein -only proteins with more than one polypeptide chain have QS
What is determined by the Tertiary Structure (TS)?
the chemical and physical properties of the protein
What is Denaturation?
the disruption of the bonds holding the secondary, tertiary, or quaternary structures together
What is electrophoresis?
the migration or separation of charged particles or solutes of a liquid solution in an electrical field. Conventional electrophoresis is tedious and time consuming. Electrophoresis automation and newer electrophoresis techniques have revitalized the utilization of electrophoresis in today's clinical laboratories. Molecular diagnostic analysis using electrophoresis and research in proteomics have also contributed to this revitalization.
Isoelectric focusing is a type of electrophoresis used to phenotype for alpha1-antitrypsin deficiencies. When the protein is electrophoresed, it migrates to: a. the site where the pH is the same as the pI b. the site where the protein's net charge is less than the pI c. the site where the molecular weight of the protein correlates with the pI d. the site where the gel pore size inhibits further migration
the site where the pH is the same as the pI
What is the Tertiary Structure (TS)?
the way in which the chain folds back upon itself to form a 3-dimensional structure -mainly interactions of amino acids with the R-groups of more distant amino acids
Which of the following statements regarding proteins is true? a. total protein and albumin are about 10% higher in ambulatory patients b. plasma total protein is about 15% higher than serum levels c. albumin normally accounts for about 40% of CSF total protein d. transudative serous fluid protein is about two thirds of the serum level
total protein and albumin are about 10% higher in ambulatory patients
The acute-phase reactant proteins include all of the following EXCEPT: a. transferrin b. alpha1-antitrypsin c. haptoglobin d. fibrinogen
transferrin
Which of the proteins listed below migrates in the beta region at pH 8.6? a. haptoglobin b. orosomucoprotein c. antichymotrypsin d. transferrin
transferrin transferrin, beta lipoprotein, C3, and C4 = beta haptoglobin = alpha-2 orosomucoprotein = alpha-1
Postrenal azotemia is found in:
tumors of the bladder obstructing urine flow
Direct relationship between _____ and the glomerular filtration
urea
Renal azotemia is found in:
uremia or nephrotic syndrome
Which of the following serum results correlates best with the rapid cell turnover associated with chemotherpay treatment regimens?
uric acid of 11.0 mg/dL
The plasma ureas concentration is increased by:
urinary stasis or dehydration
Select the correct order of Hb migration on agarose or cellulose acetate at pH 8.6: a. - C F S A + b. - S C A F + c. - C S F A + d. - S F A C +
- C S F A +
What is Uric Acid formed from?
- Exogenous nucleotides: dietary. - Endogenous nucleotides: cells breaking down and being replaced. - Present in plasma as monosodium urate - 70% excreted by kidneys - Remainder is degraded by bacteria in the GI tract.
What is BUN formed from?
- Exogenous protein: protein in the diet. - Endogenous protein: protein from the breakdown of cells in the body
Classes and heavy chains are
- IgM = μ (mu) - IgG = γ (gamma) - IgA = α (alpha) - IgD = δ (delta) - IgE = ε (epsilon)
Total Protein Methodologies: Kjedahl
- Reference Method Principle - measures nitrogen content Acid digestion converts nitrogen in protein to ammonium ion (NH4+) which is measured Difficult to perform—infrequently used. • Considered most accurate
Major Renal Functions: Tubular Reabsorption
-Active (AT) and Passive Transport (PT) -every minutes, 1200 mL of ULTRAFILTRATE is filtered through 2 million nephrons -PCT reabsorbs 80% of fluids and electrolytes filtered through the glomerulus -Active transport substance reabsorbed is combined with a CARRIER PROTEIN; transported against concentration gradient and requires ATP -Sodium: moves by AT in PCT, ascending loop of Henle, and collecting Tubules -PT: requires no energy and moves from high to low concentration
Protein Metabolism
-Digestion of dietary proteins by proteolytic enzymes starts with GI tract -AA are released & absorbed by the Jejunum & transported via portal circulation to AA pools. -Liver & other organs use AA pools to synthesize the body's proteins.
Protein electrophoresis migration
-Direction of migration of proteins in an electrical field determined by surface charge of protein. Protein at pH higher than its isoelectric point (positive charges = negative charges) is negatively charged and migrates toward anode (positive). Albumin smallest MW has largest number of free negative charges and migrates the fastest from point of application.
Major Renal Functions
-Glomerular Filtration -Tubular Reabsorption -Tubular Secretion
What is the function of protein?
-Maintenance of colloidal osmotic pressure and water distribution -Structural: support for the body, tissue, or cell --collagen and keratin (hair and nails) -Transport molecule: Transferrin (Fe3+), Albumin-bilirubin, Thyroid binding globulins, hormones -Enzymes -Peptide hormones, insulin -Coagulation -Hemoglobin -Antibodies
Protein Synthesis
-Most are synthesized by the liver- plasma proteins, coagulation factors, protein hormones & hemoglobin & secreted by the hepatocytes. -Hepatocytes maintain balance between synthesis and degradation-narrow range. -Primary structure determined by the sequence of purine & pyrimidine bases in the DNA molecule. -mRNA carries the template for the initiation and termination codes to begin and end the specific protein molecule.
Transthyretin (TTR; Pre-Albumin)
-TTR binds with thyroxine and triiodothyronine (thyroid hormones) and retinol (vitamin A) and serves as a transport protein. -The main clinical significance of TTR is its role as a sensitive marker of poor nutritional status such as protein-energy malnutrition (PEM).
What the the possible conformations describing the Secondary Structure (SS)?
-alpha helix -Beta pleated sheets -Random coils
Total Protein Methodologies: Biuret
-based on the presence of peptide bonds found in all proteins. -When a solution of protein (serum or plasma) is treated with cupric (Cu+2) divalent ions in a moderately alkaline medium, a violet-colored chelate, which absorbs light at 540 nm, is formed between the cupric ion and carbonyl oxygen and the amide nitrogen atoms of the peptide bond. -Proteins + Biuret Reagent (Cu2+) ----> violet-colored product (540nm) • Sodium potassium tartrate: complexes with cupric ions to prevent their precipitation in alkaline solution. • Copper sulfate: the major reactant providing the Cu+2 ions. • Potassium iodide: an antioxidant that stabilizes the cupric ions. • NaOH: provides the alkaline pH. • Biuret method not sensitive enough for CSF, Urine or transudates. • The Folin-Lowery method for protein synthesis has highest analytical sensitivity. • Most tests done this way
Uric Acid Methodologies: Phosphotungstic Acid
-classic measures the development of a blue color (tungsten blue) as phosphotungstic acid (PTA) is reduced by uric acid in an alkaline medium
Creatinine Methodologies: Jaffe Reaction
-classic reaction between creatinine and picric acid in an alkaline medium yielding a red orange compound, which is creatinine and a picrate ion. ( time consuming, not readily automated) A kinetic Jaffe method was developed to reduce the effect of interfering substances (protein, glucose, uric acid, ascorbic acid, acetone, ketoacids, and medications
Major Renal Functions: Glomerular Filtration
-enhanced by: -pressure in glomerular capillaries due to size difference between afferent and efferent arterioles -semipermeable glomerular basement membrane: allows LMW molecules (albumin, urea, proteins, glucose, and creatinine) to be freely filtered and moved to PCT -basement membrane: negatively charged molecules repelled (large)
Uric Acid Methodologies: Uricase
-enzymatic reaction • Uricase catalyzes the oxidation of uric acid to allantoin measured by: - The decrease in absorbance at 293 nm, which is a peak absorbance for uric acid and one at which allantoin does not absorb. - Peroxidase and a dye (4-aminoantipyrene) is a second modification and the most common automated method. - Interference - Susceptible to bacterial action - keep refrigerated; hemolysis & bilirubin can decrease uric acid in peroxidase methods. 1. Uric Acid + O2 + 2H2O --uricase--> Allantoin + CO2 + H2O2 2. H2O2 + indicator dye --peroxidase--> colored compound + 2H2O OR H2O2 + CH3OH --catalase--> H2CO + 2H2O H2CO + 3C5H8O2 + NH3 ---> 3H2O + colored product
Albumin
-synthesized in the liver and comprises approximately 60% of total serum protein. -maintains plasma colloidal osmotic pressure (COP) -Serves as an endogenous source of amino acids. -Transports and stores a wide variety of ligands • Bilirubin • Long chain fatty acids • Therapeutic Drugs (TDM) • Calcium • Magnesium • Hormones (thyroid, Cortisol)
Isoelectric point (pI)
-the pH at which the amino acid or protein has NO NET CHARGE and the positive charges equal the negative charges -pH greater than pI: protein carries a NEGATIVE CHARGE -pH less than pI: protein carries a POSITIVE CHARGE
Creatinine Reference range for both men and women (put together) mg/dL
0.9-1.2
BUN methodologies 1) Form indophenol blue color 2) Yellow-orange brown compound 3) Most commonly used procedure used for BUN methodologies
1) Berthelot's reaction 2) Nessler's reaction 3) Glutamate dehydrogenase (GLDH)
Creatinine Methodologies 1) Red orange compound (classic) 2) Enzymatic procedure
1) Jaffe reaction 2) Creatininase
Kidneys play a vital role in 1) _____ levels of substances 2) _____ critical components 3) _____ what is not essential
1) Maintaining 2) Retaining 3) Eliminating
Uric Acid methodology 1) Gives nice blue color 2) _____ catalyzes the oxidation of uric acid to allantoin
1) Phosphotungstic acid 2) Uricase
Creatinine levels are affected by 3 main variables
1) Relative muscle mass 2) Creatine turnover 3) Renal function
Tubular secretion two major functions
1) elimination of waste 2) Regulation of acid-base balance
Serum urea nitrogen and serum creatinine determinations are frequently requested together so that thier ratio may be evaluated. What is the range of the normal ratio of urea nitrogen to creatinine?
12/1 and 20/1
What is the value of UREA if the BUN is 60 mg/dL? (urea MW = 60, N=14, C=12) A. 2.8 B. 128 mg/dl C. 28 mg/dl D. 12.8 mg/dl
128 mg/dl
A BUN of 9 mg/dL is obtained by a technologist. What is the urea concentration?
19.3 mg/dL
Of the five immunoglobulin classes, IgG is the most structurally simple, consisting of how many light chains/heavy chains, respectively? A. 5/2 B. 1/1 C. 2/5 D. 2/2
2/2
how many amino acids are coded by DNA?
20
Male Uric Acid Ref Range; Female Uric Acid Ref Range
3.5-7.2 mg/dL; 2.6-6 mg/dL
In a patient with a normal to increased GFR, approximately #% of the BUN is reabsorbed and #% is excreted
40% absorbed; 60% excreted
total protein
6.4-8.3 g/dL elevated=dehydration or infection decreased=malnutrion
Reference range for BUN is #-# mg/dL
7-18
In a dehydrated patient, #% of the BUN is reabsorbed and #% is excreted - as a result, the patient's _____ BUN is increased and the _____ BUN is decreased
70% reabsorbed; 30% excreted; serum BUN is increased; urine BUN is decreased
Portal cirrhosis is a chronic disease of the liver. Observed on an electrophoretic serum protein pattern, a predominant characteristic of this disease is: A) A monoclonal band in the gamma region B) A polyclonal band in the gamma region C) A bridging effect between the beta and gamma bands D) An increase in the alpha2-globulin band E) An increase in the albumin band
A bridging effect between the beta and gamma bands
Amino Acid/Protein Bonds
A molecule of water is split between the carboxyl group of one amino acid and the amino group of another and a covalent bond called a PEPTIDE BOND.
Normal protein electrophoresis (SPE)
A normal serum protein electrophoresis (SPE) has five bands. The % distribution x total protein = g/dL.
Capillary electrophoresis differs from agarose gel electrophoresis in which aspect? a. A stationary support is not used b. An acidic buffer is used c. A low voltage is used d. Electoendosmosis does not occur
A stationary support is not used
Elevations in which of the following are MOST likely to indicate liver disease? A. ALK phosphatase, creatinine, and uric acid B. Amylase, AST, ALT, and glucose C. ALK phosphatase, creatinine, and serum potassium D. AST, ALT and ALK phosphatase
AST, ALT and ALK phosphatase
In a healthy individual, which protein fraction has the greatest concentration in serum? A. Alpha1-globulin B. Beta-globulin C. Gamma-globulin D. Albumin
Albumin
In serum protein electrophoresis, when a barbital buffer of pH 8.6 is employed, hat protein fraction will migrate the fastest toward the anode? A. Albumin B. Alpha1-globulin C.Beta-globulin D. Gamma-globulin
Albumin
Which protein component is responsible for most hypoproteinemias a. Albumin b.Beta globulins c.Alpha globulins d.Gamma globulins
Albumin
f serum proteins were immersed in a buffer of pH 8.6, which protein, albumin (pI = 4.7) or gamma globulin (pI = 7.6) would migrate the farthest by electrophoresis, and toward which electrode? A. Gamma globulin/anode B. Albumin/anode C. Gamma globulin/cathode D. Albumin/cathode
Albumin/anode
Which of the following are non-protein nitrogen compounds? A. Uric acid B. Creatinine C. BUN D. All of the others
All of the others
Why is creatinine sometimes prefered as the test for renal function? A. All of the others B. Creatinine is not reabsorbed by the tubules C. Creatinine is relatively constant and not affected by diet D. Creatinine is not affected by pre-renal conditions
All of the others
In cases of hepatoma, which protein not normally found in adult serum is synthesized by liver cells? A. Alpha1-Acid glycoprotein B. Alpha1-Fetoprotein C. Alpha2-Macroglobulin D. Carcinoembryonic antigen
Alpha1-Fetoprotein
Which of the following proteins is normally produced by the fetus but is found in increased amounts in the amniotic fluid in cases of spina bifida? A. Alpha1-Antitrypsin B. Alpha1-Acid glycoprotein C. Alpha1-Fetoprotein D. Alpha2-Macroglobulin
Alpha1-Fetoprotein
The acute phase reactant protein that also inhibits proteolysis and has the highest concentration within its globulin fraction is: A) C-reactive protein B) Haptoglobin C) Alpha2-macroglobulin D) Alpha1-antichymotrypsin E) Alpha1-antitrypsin
Alpha1-antitrypsin
A protein that is normally produced by the fetus but is found in increased amounts in the amniotic fluid in cases of spina bifida is: A) Alpha1-antitrypsin B) Alpha1-acid glycoprotein C) Alpha1-fetoprotein D) Alpha2-macroglobulin E) Hemopexin
Alpha1-fetoprotein
Proteins may become denatured when subjected to mechanical agitation, heat, or extreme chemical treatment. How are proteins affected by denaturation? A. Alteration in primary structure B. Alteration in secondary structure C. Alteration in tertiary structure D. Increase in solubility
Alteration in tertiary structure
what do amino acids contain?
Amino Group (NH2) Carboxyl Group (COOH) Hydrogen R Group (radical or side chain)
Azotemia refers to: A) An increase in creatinine in urine B) A decrease in BUN in blood C) An increase in urea in blood D) An increase in creatinine in blood
An increase in urea in blood
Using a buffer with a higher pH than the isoelectric point will cause protein to migrate toward which electrode? a. Neither anode or cathode b. Cathode c. Anode
Anode
Which statement is correct for a protein With an isoElectric point (pI) of 8.2 IN A Ph OF 7.0 A. At pH 7.0, this protein will migrate to the cathode B. At pH 7.0, this protein will have an equal number of negatively and positively charged groups C. At pH 7.0. this protein will have a net positive charge. D. At pH 7.0, this protein will not move in an electrical field.
At pH 7.0, this protein will migrate to the cathode
Protein Electrophoresis at pH of 8.6
At pH 8.6 the order of migration for the five major bands: Albumin, Alpha 1, alpha 2, beta and gamma. These proteins are negatively charged and migrate toward the anode Albumin is the fastest migrating protein toward the anode. It has the highest net negative charge, therefore the lowest pI. Gamma globulins (IgG) have the highest pI.
An increase blood urea and other NPN compounds
Azotemia
BUN Clinical Significance include (2 disorders)
Azotemia and Uremia
The pI of a protein is 9.2. This protein is placed in an electrical field where a buffer sets the pH at 10.0. Select the correct statement regarding the electrophoretic migration of this protein. A. This protein will have a net positive charge. B. This protein will have a net negative charge. C. This protein will migrate toward the anode, the positively charged electrode. D. This protein will migrate to the cathode, the negatively charged electrode
B and C This protein will have a net negative charge. This protein will migrate toward the anode, the positively charged electrode.
There are several different types of media that can be used in electrophoresis. Most methods today use a gel, cellulose acetate, agarose, or polyacrylamide gel. Which one of the following statements is true regarding these gels? a. The compounds used to make cellulose acetate gels are carcinogenic. B. Polyacrylamide gels are formed by polymerizing and crosslinking acrylamides. C. Polyacrylamide gels have the greatest resolution or separation of solutes D. Cellulose acetate and agarose gels are the more commonly used gels
B and D Polyacrylamide gels are formed by polymerizing and crosslinking acrylamides. Cellulose acetate and agarose gels are the more commonly used gels
Which one of the following will slow down the migration of solutes in electrophoresis? increasing the temperature A.Increasing the voltage B. Increasing the size & shape of molecule C. Increasing the pH of the buffer D. Increasing the pore size of the medium. E. Increasing the viscosity of the medium.
B and E Increasing the size & shape of molecule Increasing the viscosity of the medium.
A high protein diet increases...
BUN
Major nitrogen containing metabolic product of protein catabolism in humans
BUN
Many compounds in human serum contain nitrogen. Some of these substances are protein in nature, whereas others are nonprotein nitrogen compounds. What is the compound that is comprised 16% nitrogen? A) Creatinine B) Uric acid C) BUN D) Ammonia
BUN
Nonprotein Nitrogen Compounds (6)
BUN Amino acids Uric acid Creatinine Creatine Ammonia
Kidney Function Test includes
BUN and Creatinine
Which of the statements below about serum urea is true? A) BUN is elevated in prerenal as well as renal disease B) Urea is not reabsrobed by renal tubules C) Alpha-1 globulin D) Levels are independent of diet
BUN is elevated in prerenal as well as renal disease
What is the most common electrophoretic pattern seen in liver cirrhosis? a. Increased alpha 1 b. Beta-gamma bridge c. Increase albumin d. M spike (M: monoclonal protein)
Beta-gamma bridge
Which of the following is a low-weight protein that is found on the cell surfaces nucleated cells? A. C-reactive protein B. Beta2-Microglobulin C. Ceruloplasmin D. Alpha2-Macroglobulin
Beta2-Microglobulin
Common clinical laboratory methods for the measurement of serum albumin are based on the properties of albumin as a/an: A. Glycoprotein B. Homogeneous protein C. Binding protein D. Enzyme E. Antibody
Binding protein
Which term describes a congenital disorder that is characterized by a split in the albumin band when serum is subjected to electrophoresis? A. Analbuminemia B. Anodic albuminemia C. Prealbuminemia D. Bisalbuminemia
Bisalbuminemia
What is the common name for the Total Protein reagent? a. Biuret b. Jaffe c. Kjeldahl d. Dye Binding
Biuret
Which total protein method requires copper sulfate, potassium iodide in sodium hydroxide, and potassium sodium tartrate in its reagent system? a. Biuret b. Ultraviolet absorption c. Kjeldahl d. Folin-Ciocalteu
Biuret
measurement of total protein
Biuret = violet color complex gross hemolysis (elevated results) lipemic sera (elevated results) Dye binding method turbidimetric/nephelometer
The best specimen for early newborn screening is a A.) Timed urine specimen B.) Blood specimen C.) First morning blood specimen D.) Fecal specimen
Blood specimen
Which of the following is an anionic dye that binds selectively with albumin? A. Amido black B. Ponceau S C. Bromcresol green D. Coomassie brilliant blue
Bromcresol green
Urea is produced from: A) Oxidation of pyrimidines B) The breakdown of complex carbohydrates C) Oxidation of purines D) Catabolism of proteins and amino acids
Catabolism of proteins and amino acids
Uric acid is derived from the A) Oxidation of proteins B) Catabolism of purines C) Oxidation of pyrimidines D) Reduction of catecholamines
Catabolism of purines
Which of the following is a copper transport protein that migrates as an alpha2-globulin? A. Ceruloplasmin B. Haptoglobin C. Transferrin D. Fibrinogen
Ceruloplasmin
Which serum protein should be measured in patient suspected of having Wilson's disease? a. Alpha-1 antitrypsin b. Hemopexin c. Ceruloplasmin d. Haptoglobin
Ceruloplasmin
Principle of Electrophoresis
Charged particles under the influence of a liquid media placed in an electric field will migrate to the electrode of the opposite charge. •Positive ions (cations) will migrate to the cathode, the negative electrode. •Negative ions (anions) will migrate to the anode, the positive electrode.
The abnormal metabolism of several of amino acids has been linked with disorders classified as inborn errors of metabolism. What techniques is used to differentiate among several different amino acids? A.) Chromatography B.) Microbiological analysis C.) Enzyme immunoassay D.) Electrophoresis
Chromatography
What determines the concentration of bands on a protein electrophoresis?
Concentration of bands determined by densitometry (Scans)
Which of the following is a cause of pre-renal azotemia? A. Addison disease B. Congestive heart failure C. Cystitis D. Nephrolithotasis
Congestive heart failure
Which of the following reagents can be used to measure protein in cerebrospinal fluid? A. Biuret B. Coomassie brilliant blue C. Ponceau S D. Bromcresol green
Coomassie brilliant blue
It is difficult to actually measure glomerular filtration rate. What test can be used to estimate it? A. Creatine B. Creatinine C. Creatinine Clearance D. Urea clearance
Creatinine
Primarily an index of renal function; Measures the GFR
Creatinine
Which of the following formulas is the correct expression for creatinine clearance? A) Creatinine Clearance = U/P X V X 1.73/A B) Creatinine Clearance = P/V X U X 1.73/A C) Creatinine Clearance = U/PX U X A/1.73 D) Creatinine Clearance = U/V X { X 1.73/A
Creatinine Clearance = U/P X V X 1.73/A
BUN is a better indicator of glomerular function (kidney function), why?
Creatinine has a lot more variables that can alter the results that have nothing to do with kidney disease...
Peptide bonds of proteins bind with _________ ions of biuret reagent to produce a colored product. A. Ferrous B. Cuprous C. Ferric D. Cupric
Cupric
What substance may be measured as an alternative for creatinine for evaluating GFR? A) Cystatin C B) Potassium C) Plasma urea D) Uric acid
Cystatin C
What is the common electrophoretic pattern seen in nephrotic syndrome? a. Decreased albumin & increase alpha 2 b. Increased albumin & increase alpha 1 c. Increased albumin & increase alpha 2 d. Beta bridging
Decreased albumin & increase alpha 2
In what condition would an increased level of serum albumin be expected? A. Malnutrition B. Acute inflammation C. Dehydration D. Renal disease
Dehydration
Increased serum albumin concentrations are seen in which of the following conditions? A. Nephrotic syndrome B. Acute hepatitis C. Dehydration D. Chronic inflammation
Dehydration
What physiological condition causes most cases of hyperproteinemia? a. Salt retention b. Burns c. Multiple myeloma d. Dehydration
Dehydration
Creatinine is formed from the: A. Dehydration of creatinine B. Oxidation of protein C. Metabolism of purines D. Deamination of dibasic amino acids
Dehydration of creatinine
In which of the following disorders would the maternal serum level of alpha1-fetoprotein not be elevated? A. Neural tube defect B. Spina bifida C. Fetal distress D. Down syndrome
Down syndrome
Bence Jones proteinuria is a condition characterized by the urinary excretion of what type of light chain? A. Kappa light chains B. Lambda light chains C. Both kappa and lambda light chains D. Either kappa or lambda light chains
Either kappa or lambda light chains
Basic Metabolic Profile (BMP)
Electrolytes (Na, K, Cl, CO2) Glucose, Calcium, BUN, Creatinine
Serum protein electrophoresis is routinely performed on the serum obtained from a clotted blood specimen. If a plasma specimen is substituted for serum, how will the electrophoresis be affected? A. Electrophoresis cannot be performed because the anticoagulant will retard the mobilities of the protein fractions. B. Electrophoresis cannot be performed because the anticoagulant will cause migration of the protein fractions in the direction of the cathode C. Electrophoresis will show an extra fraction in the beta-gamma region D. Electrophoresis will show an extra fraction in the prealbumin area
Electrophoresis will show an extra fraction in the beta-gamma region
Major Renal Functions: Tubular Secretion
Elimination of wastes Regulation of Acid-Base balance: secretes 90% of hydrogen ions
Uric acid formed both from
Exogenous nucleotides and Endogenous nucleotides
True or False Of the three types of gels discussed, agarose gels are stronger, thermostable, and transparent
False
True or False Proteins in a buffer with the pH set at 8.6 will become anions and move to the positively charged electrode.
False
Urine screening tests for PKU utilize: A.) Microbial inhibition B.) Nitroso-napthol C.) Dinitrophenylhydrazine D.) Ferric chloride
Ferric chloride
The physician is concerned that a pregnant patient may be at risk for delivering prematurely. What would be the best biochemical marker to measure to assess the situation? A. Inhibin A B. Alpha1-Fetoprotein C. Fetal fibronectin D. Human chorionic gonadotropin
Fetal fibronectin
Which of the following protein methods has the highest analytical sensitivity? a. Refractometry b. Folin-Lowry c. Turbidimetry d. direct UV absorption
Folin-Lowry
When performing protein electrophoresis, which way do the electrons flow? a. Depends upon the pH of the buffer b. From the anode c. From the cathode d. Depends upon the charges on the proteins
From the cathode
Of the following protein fractions, the one that is NOT produced in the liver is: A) Albumin B) Alpha1 globulin C) Alpha2 globulin D) Beta globulin E) Gamma globulin
Gamma globulin
There are five immunoglobulin classes: IgG, IgA, IgM, IgD, and IgE. With which globulin fraction do these immunoglobulins migrate electrophoretically? A. Alpha1-globulins B. Alpha2-globlins C. Beta1-globulins D. Gamma-globulins
Gamma-globulins
Pressure in the glomerular capillaries is high because of the difference in size between the afferent and efferent arterioles
Glomerular filtration
Which of the following vacutainer tubes can NOT be used for BUN? A. Red B. Grey C. SST D. Lavender
Grey
A glycoprotein that binds with hemoglobin to facilitate the removal of hemoglobin by the reticuloendothelial system is: A) Haptoglobin B) Ceruloplasmin C) Transferrin D) Hemopexin E) C-reactive protein
Haptoglobin
Which of the following abnormal Hbs migrates to the same position as Hb S on agarose or cellulose acetate at pH 8.6? a. Hb C b. Hb D Punjab c. Hb O d. Hb E
Hb D Punjab
Which of the following conditions is associated beta-gamma bridging? a. Hepatic cirrhosis b. Rheumatoid arthritis c. Multiple myeloma d. Malignancy
Hepatic cirrhosis
Which of the following is not characteristic of multiple myeloma? A. Monoclonal band in the gamma region B. Hypercalcemia C. Hyperalbuminemia D. Hyperglobulinemia
Hyperalbuminemia
Which disorder is not associated with an elevated protein level in cerebrospinal fluid? A. Bacterial meningitis B. Multiple sclerosis C. Cerebral infarction D. Hyperthyroidism
Hyperthyroidism
A serum UA concentration of >7 mg/dL
Hyperuricemia
Increased dietary intake, over production of uric acid, under excretion of uric acid, specific enzyme defects cause what?
Hyperuricemia
Which immunoglobulin class, characterized by its possession of a seccretory component, is found in saliva, tears and body secretions? A. IgA B. IgD C. IgG D. IgM
IgA
Which immunoglobulin class is able to cross the placenta from the mother to the fetus? A. IgA B. IgD C. IgE D. IgG
IgG
Which of the following plasma proteins is not manufactured by the liver? a. IgG b. Haptoglobin c. Fibrinogen d. Albumin
IgG
Which of the proteins listed below has the highest pI? a. albumin b. transferrin c. ceruloplasmin d. IgG
IgG
What technique is used to quantify specific immunoglobulin classes? A. Immunononephelometry B. Serum protein electrophoresis C. Isoelectric focusing D. Immunoelectrophoresis
Immunononephelometry
Hyperuricemia
Increased Uric Acid • Causes of hyperuricemia: - Increased dietary intake - Overproduction of uric acid - Under excretion of uric acid - Specific enzyme defects > 7.0 mg/dL Men > 6.0 mg/dL Female
What is hypoalbuminemia?
Increased catabolism: tissue damage and inflammation Impaired or decreased synthesis -Primary: liver disease -Secondary: diminished protein intake, malnutrition, malabsorption Increased loss of protein -nephrotic syndrome -chronic glomerulonephritis -diabetes mellitus/diabetic neuropathy -extensive burns -acute viral gastroenteritis
Muscle wasting (starvation, GI hemorrhage, stress, steroids, uncontrolled DM, high fever all caused by what?
Increased protein catabolism (pre renal azotemia)
Creatinine is considered the substance of choice to measure endogenous renal clearance because: A. Plasma levels are highly dependent on diet B. The rate of formation is independent of body size C. It is completely filtered by the glomeruli D. Clearance is the same for both men and women
It is completely filtered by the glomeruli
Which test is commonly used for creatinine? A. Hydrolysis B. Nesslerization C. Jaffe Reaction- Kinetic D. Biuret reaction
Jaffe Reaction- Kinetic
Total Protein Methodologies
Kjeldahl Biuret Acid-Sulfosalicylic Acid
Protein Electrophoresis: Monoclonal increase
Left to Right Albumin: 2nd highest peak, lower than normal, 30% Alpha 1: 3.9% Alpha 2: 9.5% Beta: 6.0%--- 1.9% Gamma: highest peak, higher than normal, 48.7%
Protein Electrophoresis: Cirrhosis
Left to Right Albumin: highest peak Alpha 1: Alpha 2: Beta: higher than normal Gamma: higher than normal Bridge between beta and gamma
Protein Electrophoresis: Nephrotic Syndrome
Left to Right Albumin: highest peak Alpha 1: Alpha 2: 2nd highest peak, higher than normal Beta: 3rd highest peak, higher than normal Gamma:
Protein Electrophoresis: Inflammation
Left to Right Albumin: highest peak Alpha 1: increased, 3rd highest peak Alpha 2: increased, 2nd highest peak Beta: increased, 4th highest peak Gamma:
Protein Electrophoresis Image: Reference Pattern
Left to Right Albumin: highest peak at 62% alpha 1: 4% alpha 2: 8.6% beta: 12.5% gamma: 12.9%
Protein Electrophoresis: alpha1-Antitrypsin Deficiency
Left to Right Albumin: highest peak, higher than normal Alpha 1: none, 0% Alpha 2: slightly elevated Beta: slightly elevated Gamma: slightly elevated
Which of the conditions below is usually associated with an acute inflammatory patterns? a. MI b. malignancy c. RA d. hepatitis
MI
Which of the following disorders is not associated with the phenylalanine-tyrosine pathway? A.) Tyrosinemia B.) Alkaptonuria C.) Albinism D.) Maple syrup urine disease
Maple syrup urine disease
All of the following disorders can be detected by newborn screening except: A.) Tyrosyluria B.) Maple Syrup Urine Disease C.) Melanuria D.) Galactosemia
Melanuria
Identification of which of the following is useful in early stages of glomerular dysfunction? A. Microalbuminuria B. Ketonuria C. Hematuria D. Urinary light chains
Microalbuminuria
All states require newborn screening for PKU for early: A.) Modification of diet B.) Administration of antibiotics C.) Detection of diabetes D.) Initiation of gene therapy
Modification of diet
What is the most characteristic electrophoretic pattern seen in multiple myeloma? a. Increased alpha 2 globulin b. Decreased albumin c. Monoclonal protein spike d. Increased beta globulin
Monoclonal protein spike
High serum total protein but low albumin is usually seen in: a. Glomerulonephritis b. Multiple myeloma c. Hepatic cirrhosis d. Nephrotic syndrome
Multiple myeloma
monoclonal immunoglobulins
Multiple myeloma
Nonprotein Nitrogen (NPN)
NPN refers to products of catabolism of proteins and nucleic acids, which contain nitrogen but are not part of a protein molecule. • 75% of NPN (Nonprotein Nitrogen) is eventually secreted. • Most of the protein is converted to Urea during breakdown of proteins Direct relationship between Urea & GFR-glomerular filtration rate. • Urea freely filtered in normal functioning kidneys • Patient with normal to increased GFR- 40% BUN reabsorbed & 60% excreted - therefore a well hydrated patient excretes more BUN—resulting in lower serum/plasma BUN • Dehydrated patients 70% BUN is reasbsorbed & 30% excreted—Resulting in an increased BUN -plasma/serum & a decreased urine BUN. • BUN dependent on three variables: Urea concentration, Glomerular filtration rate and level of hydration.
Proteins, carbohydrates and lipids are the three major biochemical compounds of human metabolism. What is the element that distinguishes proteins from carbohydrate and lipid compounds? A. Carbon B. Hydrogen C. Oxygen D. Nitrogen
Nitrogen
Comprises the products of catabolism (breakdown) of proteins and nucleic acids, which contain nitrogen but are not part of a protein molecule
Nonprotein Nitrogen (NPN)
Clinical Significance of Albumin
Normal Range: 3.4-5.0 g/dL**** -Largest Plasma protein (52-62%) -Regulator of Collodial Oncotic pressure -Transports protein due to ease of binding with blood components. -Causes for Increased Values: · No Clinical Significance (above 5 is not typically significant) · Hemoconcentration · Dehydration -Causes of Decreased Values: (when to worry) · Decreased Synthesis · Malabsorption/ Malnutrition · Severe Burns
When quantifying serum total proteins, upon what is the intensity of the color produced in the biuret reaction dependent? A. Molecular weight of the protein B. Acididty of the medium C. Number of peptide bonds D. Nitrogen content of the protein
Number of peptide bonds
Which of the following stains is used for lipoprotein electrophoresis? a. Oil Red O b. Coomassie Brilliant Blue c. Amido Black d. Ponceau S
Oil Red O
Which of the following statement regarding paraproteins is true? a. The Bence-Jones protein heat test is confirmatory for monoclonal light chains b. Light chains found in urine are always derived from monoclonal proteins c. Oligoclonal banding is seen in the CSF of about 90% of multiple sclerosis cases
Oligoclonal banding is seen in the CSF of about 90% of multiple sclerosis cases
The term below that is related to the ability of plasma proteins to maintain plasma water volume is: A) Isotonic equilibrium B) Crystalloid osmotic pressure C) Oncotic pressure D) Hydrostatic pressure
Oncotic pressure
Creatinine is formed from: A) Metabolism of purines B) Oxidation of creatine in skeletal muscle C) Deamination of basic amino acids D) Oxidation of proteins in the blood
Oxidation of creatine in skeletal muscle
Requires no energy; characterized by movement of a substance from an area of high concentration to one of lower concentration
Passive transport
For the Total Protein reagent to work, what type of bond must exist a. Electrostatic bonds b. Peptide bonds c. Hydrogen bonds d. Van der Waal forces
Peptide bonds
Clinical significance of A/G ratio
Plasma proteins - albumin and globulins · Liver produces albumin, alpha -1, alpha-2 and beta globulins · RE system produces gamma globulin (antibodies secreted by plasma cells) Prealbumin · Appears as a faint band on serum electrophoresis · Used clinically to assess nutritional status
Protein Electrophoresis: Specimen Collection and Handling
Plasma samples mistaken as serum will cause a false positive result due to fibrinogen between the gamma beta fractions.
Which of the following electrophoretic media separates proteins primarily on the basis of molecular size? A. Agarose B. Polyacrylamide gel C. Cellulose acetate D. Salt fractionation
Polyacrylamide gel
If you have blockage of a ureter, it would cause: A. Pre-renal azotemia B. Renal azotemia C. Post-renal azotemia
Post-renal azotemia
Tumors of the bladder or prostate gland, prostatic hypertrophy, gynecologic tumors, nephrolithiasis, severe infections all caused by what?
Postrenal azotemia (Maybe something wrong with ureters, bladder, etc.)
Which of the following would NOT be a possible cause for an increased uric acid? A. Gout B. Pregnancy C. Leukemia D. Inheritance
Pregnancy
Azotemia is classified into 3 categories
Prerenal (blood going into kidney) Renal Postrenal (After urine leaves the kidney)
Gout; Idiopathic cause..
Primary Hyperuricemia
Inborn error of metabolism; predominant in men 30-50 years old. Symptoms: arthritis, nephropathy, nephrolithiasis; attacks can increase from alcohol, high-protein diets, stress
Primary gout
Which of the following disorders is not associated with an elevation of serum alpha1-fetoprotein? A. Testicular germ cell tumors B. Prostatic carcinoma C. Pancreatic carcinoma D. Gastric carcinoma
Prostatic carcinoma
Protein electrophoresis Bands
Protein: Relative (%) : g/dL Albumin (anode- fastest: 53-65%: 3.5-5.0 g/dL a1-globulin: 2-5%: 0.1-0.3 g/dL a2-globulin: 7-13%: 0.6-1.0 g/dL B-globulin: 8-14%: 0.7-1.1 g/dL Gamma-globulin (cathode-slowest): 12-20%: 0.8-1.6 g/dL
What is the basis for the Kjeidahl technique for the determination of serum total protein? A. Quantification of peptide bonds B. Determination of the refractive index of total proteins C. Ulttraviolet light absorption by aromatic rings at 280 nm D. Quantification of the nitrogen content of protein
Quantification of the nitrogen content of protein
Amino Acid formula
RCH(NH2)COOH -R differentiates amino acids
Uric Acid (UA)
Reference Range: 3.5-7.2 mg/dL • UA is the major product of purine (adenine and guanine) catabolism in man and higher primates. (seen a lot in patients with high cell turnover (cancer) and/or gout) • It is produced in the liver from xanthine by the action of the enzyme xanthine oxidase.
Urea/Blood Urea Nitrogen (BUN)
Reference Range: 7-18 mg/dL • BUN: major nitrogen-containing metabolic product of protein catabolism in humans. (breakdown of protein)
Albumin: C-reactive protein- Complement
Reference Value: Molecular Mass: Function: immune response resulting in cell lysis
Albumin: a1-Globulin- a1-fetoprotein
Reference Value: Molecular Mass: 70,000 D Function: antenatal diagnosis of neural tube defects
Albumin: B-Globulin- B-microglobulin
Reference Value: 0.0001-0.0002 g/dL Molecular Mass: 11,800 D Function: Cellular membrane of nucleated cells, especially lymphocytes; renal tubular function test
Transthyretin (prealbumin)
Reference Value: 0.01-0.04 g/dL Molecular Mass: 55,000 D Function: Marker of poor nutritional status; transport protein- hormones and retinol (vitamin A)
Albumin: a2-Globulin- Ceruloplasmin
Reference Value: 0.02-0.60 g/dL Molecular Mass: 133,000 D Function: Copper metalloprotein; Peroxidase activity; oxidant-antioxidant
Albumin: a1-Globulin- a1-antichymotrypsin
Reference Value: 0.03-0.06 g/dL Molecular Mass: 68,000 D Function: Proteinase inhibitor
Albumin: a2-Globulin- haptoglobin
Reference Value: 0.03-0.20 g/dL Molecular Mass: 85,000 - 1 million D Function: acute phase reactant; binds free hemoglobin
Albumin: Gamma Globulin- IgM
Reference Value: 0.03-0.36 g/dL Molecular Mass: 970,000 D Function: Antibody
Albumin: a1-Globulin- a1-acid glycoprotein (orosmucoid)
Reference Value: 0.04-0.14 g/dL Molecular Mass: 44,000 D Function: Acute phase reactant
Albumin: B-Globulin- Hemopexin
Reference Value: 0.05-0.10 g/dL Molecular Mass: 60,000 D Function: Binds heme
Albumin: a1-Globulin- a1-antitrypsin
Reference Value: 0.09-0.19 g/dL Molecular Mass: 52,000 D Function: Acute phase reactant protease inhibitor; associated with early onset emphysema, juvenile hepatic cirrhosis
Albumin: a2-Globulin- a2-macroglobulin
Reference Value: 0.13-0.30 g/dL Molecular Mass: 725,000 D Function: Protease inhibitor (trypsin, pepsin, and plasmin)
Albumin: B-Globulin- Transferrin
Reference Value: 0.20-0.36 Molecular Mass: 78,000 D Function: Transports iron (Fe3+)
Albumin: C-reactive protein- Fibrinogen
Reference Value: 0.20-0.45 g/dL Molecular Mass: 341,000 D Function: coagulation factor
Albumin: B-Globulin- B-lipoproteins
Reference Value: 0.25-0.36 g/dL Molecular Mass: 3,000,000 D Function: Transports lipids, especially cholesterol
Albumin: Gamma globulins- IgG
Reference Value: 0.7-1.6 g/dL Molecular Mass: 144,00-150,000 D Function: Antibody, increased in immune reactions
Albumin: Gamma Globulin- IgA
Reference Value: 0.90-0.410 g/dL Molecular Mass: around 160,000 D Function: Antibody
Albumin: C-reactive protein
Reference Value: 00.1 g/dL Molecular Mass: 118,000 D Function: Acute phase reactant; indicator of risk of myocardial infarction
Albumin characteristics
Reference Value: 3.5-5.5 g/dL Molecular Mass: 66,300 D Function: maintain plasma colloidal pressure; transport ligands
The rate at which the kidneys remove a substance from the plasma or blood (What is the patient cleaning in a 24 hour period)
Renal Clearance
Clearance Tests (3)
Renal Clearance, Creatinine Clearance, eGFR
Uremia, Acute kidney injury, Glomerulonephritis, Nephrotic syndrome, and acute renal failure all caused by what?
Renal azotemia
What condition is closely related to an increase in ammonia? A. Cushing disease B. Addison disease C. Pre-eclampsia D. Reyes syndrome
Reyes syndrome
What is the clinical utility of testing for serum prealbumin (transthyretin)? a. Levels correlate with glomerular injury in patients with diabetes mellitus b. High levels are indicator of acute inflammation c. Low levels are associated with increased free cortisol d. Serial low levels indicate compromised nutritional status
Serial low levels indicate compromised nutritional status
Monitoring of _______ in patients on chemotherapy due to leukemia, lymphoma or multiple myeloma is necessary to avoid nephrotoxicity. A) Serum creatinine B) Urine creatinine C) BUN D) Serum uric acid E) Urea in urine
Serum uric acid
Electrophoresis: Irregular, distorted, or atypical bands
Short Migration Pattern: Contaminated or aged buffer Diffuse Bands: Markedly wet gels, poor sample application Streaks perpendicular to bands: tearing/poking get in sample application Weak Bands: not enough sample or stain Other causes of irregular and distorted bands: hemolyzed sample, bent or dirty applicator, air bubbles in sample application, too much sample, wick flow, too much heat or drying
Protein Electrophoresis Stains
Stains include Amino Black, Ponceau S and Coomassie Brilliant Blue
Protein electrophoresis support media
Support media include cellulose acetate, agarose gel, and starch gel. Loss of contact between buffer chamber and the medium will stop the migration across the gel. Movement of proteins is dependent upon the presence of a salt bridge that allows current to flow & transport ion to the electrodes across the gel. If salt bridge is not present, proteins will not migrate
Urine from a newborn with maple syrup urine disease will have a significant: A.) Pale Color B.) Yellow precipitate C.) Milky appearance D.) Sweet odor
Sweet odor
The biuret method for total serum protein is used for serum but not for protein in urine or cerebrospinal fluid (CSF) because: A. Ammonium ions in CSF interfere with the reaction. B. The method is not sufficiently sensitive for use with urine or CSF. C. Protein is not the major solute present in urine. D. The color of CSF interferes with a reading at 550 nm.
The method is not sufficiently sensitive for use with urine or CSF.
Mobility or Rate of Migration: Electrophoresis
The mobility or rate of migration of ions in electrophoresis is dependent upon the following factors: •Net charge of the molecules •Size and shape of the molecules •Support medium properties •Strength of the electrical field •Ionic strength of the buffer •Temperature
What is hyperalbuminemia?
This condition is only caued by dehydration or administration of albumin.
Which of the following proteins is indicative of a patients nutritional status? A) Hemopexin B) Transthyretin C) Ceruloplasmin D) Haptoglobin
Transthyretin
Passage of substances from the peritubular capillaries into the tubular filtrate
Tubular secretion
many compounds in human serum contain nitrogen. Some of these substances are protein in nature, whereas other are nonprotein nitrogen compounds. What is the compound that compromises the majority of the nonprotein-nitrogen fraction in serum?
Urea
What is BUN dependent on?
Urea concentration, Glomerular filtration rate and level of hydration.
An increased urea/BUN
Uremia
The major product of purine catabolism in man and higher primates
Uric Acid
In gout, what analyte deposits in joints and other tissues? A. Uric acid B. Urea C. Creatinine D. Calcium
Uric acid
Urine vs Serum Electrophoresis
Urine protein electrophoresis same as serum except it must be concentrated before application
Urease is...
a BUN methodology
Nutritional assessment with poor protein-caloric status is associated with: a. a decreased level of prealbumin b. a low level of gamma globulins c. an elevated ceruloplasmin concentration d. an increased level of alpha1-fetoprotein
a decreased level of prealbumin
Substance to be reabsorbed must be combined with carrier protein (uses ATP)
active transport
The following pattern of serum protein electrophoresis is obtained: albumin: ↓ alpha 1 and 2 -globulins: ↑ gamma globulins: normal This pattern is characteristic of which condition? a. acute inflammation (primary response) b. cirrhosis c. nephrotic syndrome d. gammopathy
acute inflammation (primary response)
The order of blood flow through the nephron is:
afferent arteriole, efferent arteriole, peritubular capillaries, vasa recta
The plasma protein mainly responsible for maintaining the colloidal osmotic pressure in vivo is: a. albumin b. hemoglobin c. fibrinogen d. alpha2-macroglobulin
albumin
which of the following is the serum protein fraction most likely to be decreased in nephrotic syndrome?
albumin
Hb F concentration is usually measured by: a. alkali denaturation of Hb A and Hb A2 b. specific Hb F peroxidase reaction c. turbidimetric assay of Hb F after precipitation w/ ammonium sulfate d. gas chromatography
alkali denaturation of Hb A and Hb A2
The measurement of creatinine is based on the formation of a yellow-red color when creatinine reacts with:
alkaline picrate
what would be a protease inhibitor- and acute phase reactor
alpha 1 antitrypsin
a principal fetal protein
alpha 1 fetoprotein
The electrophoretic pattern shown in a densitometric tracing showing marked alpha-1 globulin decrease, slightly decreased albumin, and slight increase in alpha-2 most likely indicates: a. alpha-1 antitrypsin deficiency b. infection c. nephrosis d. systemic sclerosis
alpha-1 antitrypsin deficiency
A peptide bond is: a. amino group and carboxyl group bonded to the alpha carbon b. a double carbon ring c. a tertiary ring of amino group and carboxyl group bonded to the alpha carbon d. two amino groups bonded to the alpha carbon
amino group and carboxyl group bonded to the alpha carbon
Urea may be determined after reaction with urease by measuring ________ in an indicator reaction.
ammonia
What is an amino acid called when both are ionized?
ampholyte or dipolar ion
Structure of Amino Acids
amphoteric-containing two ionizable sites: a proton-accepting group (NH2) proton-donating group (COOH)
When a protein is dissolved in a buffer solution, the pH of which is more alkaline than the pI, and an electric current is passed through the solution, the protein will act as: a. an anion and migrate to the anode b. a cation and migrate to the cathode c. an anion an migrate to the cathode d. an uncharged particle
an anion and migrate to the anode
When does Denaturation occur?
as a result of: -heat (most common) -changes in pH -mechanical forces -exposure to chemicals (solvents, detergents, metals) -exposure to ultraviolet light
haptoglobin
binds to Hb irreversibly increase=nephrotic syndrom deceased= ext hemolysis, estrogens, liver disease
transthyretin (prealbumin, TTR)
binds/transports thyroid hormones indicative of protein status decrease= inflammation, cancer, cirrhosis, nutrition status
Which of the following dyes is the most specific for measurement of albumin? a. bromcresol green b. bromcresol purple c. tetrabromosulfophthalein d. tetrabromphenol blue
bromcresol purple
What do all proteins contain?
carbon, hydrogen, oxygen, and nitrogen -some have sulfer
Uric acid is derived from the:
catabolism of purines
BUN Methodologies: Diacetyl or Fearon Reaction
colorimetric reaction based on the condensation of diacetyl with urea to form the chromogen diazine.
Quantitative determination of Hb A2 is best performed by: a. column chromatography b. alkali denaturation c. electrophoresis d. direct bichromatic spectrophotometry
column chromatography
What are proteins?
complex polymers of α-amino acids that are produced by living cells in all forms of life
Prerenal azotemia is caused by:
congestive heart failure
ceruloplasmin
contains serum copper, involved in plasma redox reactions (oxidant/antioxidant)
The term "biuret reaction" refers to: a. the reaction of phenolic groups w/ CuSO4 b. coordinate bonds between Cu2+ and carboxyl and amino groups of biuret c. the protein error of indicator effect producing color when dyes bind protein d. the reaction of phosphomolybdic acid w/ protein
coordinate bonds between Cu2+ and carboxyl and amino groups of biuret
what enzyme measures creatinine?
creatine kinase
Waste product derived from creatine and creatine _____ is ?
creatine phosphate; creatinine
Which of the following serum protein electrophoresis results suggests an acute inflammatory response? a. decreased albumin, increased alpha-1, decreased alpha-2, normal beta, normal gamma b. normal albumin, increased alpha-1, normal alpha-2, increased beta, increased gamma c. decreased albumin, increased alpha-1, increased alpha-2, normal beta, normal gamma d. increased albumin, increased alpha-1, increased alpha-2, increased beta, increased gamma
decreased albumin, increased alpha-1, increased alpha-2, normal beta, normal gamma
Prerenal azotemia causes decreased _____ and increased _____.
decreased renal blood flow increased protein catabolism
What causes CHF, Dehydration, Shock?
decreased renal blood flow (pre renal azotemia)
High serum total protein with high levels of both albumin and globulins is usually seen in: a. glomerulonephritis b. waldenstrom's macroglobulinemia c. dehydration d. cirrhosis
dehydration
hyperalbuminemia is caused by: a. dehydration syndromes b. liver disease c. burns d. gastroenteropathy
dehydration syndromes
Albumin Method
dye binding-spectral shift in absorbance maximum of dye. Binding of albumin with anionic dyes BCP or BCG + Albumin -----> Colored Complex (bromocresol Purple) (bromocresol Green) or HABA-2-4 (Hydroxyazobenzene -benzoic acid) ***BCP most specific for the measurement of albumin ***BCG - penicillin binds at same site as the dye causing falsely low results Sample type: Serum (preferred) - Plasma (heparin & fibrinogen falsely increase
Use of an estimating or prediction equation to estimate glomerular filtration rate from the serum creatinine level in patients with chronic renal disease and those at risk for CKD (chronic kidney disease)
eGFR
At pH 8.6, the cathodal movement of gamma-globulins is caused by: a. electroendosmosis b. wick flow c. a net positive charge d. cathodal sample application
electroendosmosis
globulins
elevated=chronic infection, liver disease, MM low= nephrosis
A/G ratio
elevated=hypothyroidism decrease= liver dysfunction lab method= bromcresol green/purple
Creatinine Methodologies: Creatininase (Creatinine amidohydrolase)
enzymatic reaction creatininase followed by creatinase, sarcosine oxidase, and peroxidase Creatinine + H2O --creatininase--> Creatine Creatine + H2O --creatinase--> Sarcosine + Urea Sarcosine + O2 + H2O --sarcosine oxidase--> formaldehyde + glycine + H2O2 Indicator (reduced) + H2O2 --peroxidase--> indicator (oxidized) + 2H2O
BUN is formed from... (2)
exogenous and endogenous protein
what band could you see in plasma electrophoresis?
fibrinogen
he protein electrophoretic pattern of plasma, as compared with serum, reveals a: a. fibrinogen peak between the beta and gamma globulins b. broad increase in the gamma globulins c. fibrinogen peak with the alpha2-globulins d. decreased albumin peak
fibrinogen peak between the beta and gamma globulins
C-reactive protein
first to elevate with inflammation most sensitive to MI, stress, trauma, infection (6hrs)
creatinine is a good indicator of glomerular filtration rate for 3 reasons
freely filtered through water; not reabsorbed by the tubules released into plasma at a constant rate
Creatinine clearance is used to estimate the:
glomerular filtration rate
3 Major renal functions
glomerular filtration, tubular absorption, tubular secretion
John doe is admitted to the hospital for a complete workup. His chemistry screening profile results are: BUN 12 mg/dL creatinine 1.5 mg/dL Uric acid 60g.dL
gout
Hemoplexin
heme binding plasma glycoprotein, second line of defense against hemoglobin mediated damage, intravascular hemolysis
A patient w/ HUS associated w/ septicemia has a normal haptoglobin level although plasma free Hb is elevated and hemoglobinuria is present. Which test would be more appropriate than haptoglobin to measure this patient's hemolytic episode? a. hemopexin b. alpha-1-antitypsin c. CRP d. transferrin
hemopexin
Which of the following is one advantage of high-resolution (HR) agarose electrophoresis over lower current electrophoresis? a. high-resolution procedures detect monoclonal and oligoclonal bands at lower concentration b. smaller sample volume is used c. results obtained more rapidly d. more samples can be applied to the support medium
high-resolution procedures detect monoclonal and oligoclonal bands at lower concentration
In a patient with nephrotic syndrome, the total protein levels in urine would be: a. normal b. lower than normal c. higher than normal d. similar to levels of CSF total protein levelse. lower albumin levels and higher levels of IgG
higher than normal
What is indicated by the sequence of amino acids in the polypeptide chain?
identity and specific order of the amino acids
Review the following serum test results: Creatine 2.5 mg/dl Cholesterol: 200 mg Glucose: 110 mg/dl Urea (BUN): 40 mg/dl Uric acid: 6.9 mg/dL
impaired renal function
What are aminoacidopathies?
inherited errors of metabolism
What determines the Secondary Structure (SS) of an amino acid?
interaction of adjacent amino acids
Electrophoretic movement of proteins toward the anode will be made to decrease by increasing the: a. buffer pH b. ionic strength of the buffer c. current d. voltage
ionic strength of the buffer
Alpha 2 macroglobulin
large, major plasma proteinase inhibitor increased in nephrotic syndrome decreased acute pancreatitis
Beta/ gamma bridge
liver dysfunctionmonoclonal gammpathies (malignant) polyclonal (reactive or inflammatory)
Creatinine is synthesized in the... from 3 amino acids
liver; arginine, glycine, and methionine
Which of the conditions below will prevent any migration of proteins across an electrophoretic support medium such as agarose? a. using too high a voltage b. excessive current during procedure c. loss of contact between a buffer chamber and the medium d. evaporation of solvent from the surface of the medium
loss of contact between a buffer chamber and the medium
Role of the kidneys
maintaining levels of many substances in the human body, retaining critical components and eliminating what is not essential Ø Removal of wastes Ø Toxic & surplus products Ø Regulation of acid-base levels & electrolytes Ø Hormone regulation
alpha 1 fetoprotein
major protein in fetal serum elevated maternal serum= open neural tube decreased =trisomy
Which of the following conditions is most commonly associated w/ an elevated total protein? a. glomerular disease b. starvation c. liver failure d. malignancy
malignancy
complement proteins
mediators of inflammationdestroy/ remove infectious agents
An immunofixation protein electrophoresis is performed on serum from a patient with the most common form of multiple myeloma. The resulting pattern revealed: a. monoclonal bands of the IgM type b. oligoclonal bands c. beta-gamma bridging d. monoclonal bands of the IgG type
monoclonal bands of the IgG type
In a well-hydrated patient, _____ BUN is excreted, resulting in _____ serum BUM
more excreted; lower serum
BUN Methodologies: glutamate dehydrogenase (GLDH)
most commonly used The disappearance of NADPH is measured as a decrease in absorbance as NADPH is oxidized to NAD+. (Enzymatic) urea + 2H2O --(urease)--> 2NH4+ + CO3^2- NH4+ + 2-oxoglutarate --GLDH--> Glutamate + H2O NADH + H+ ---> NAD+ GLDH = glutamate dehydrogenase
In which of the following conditions is a normal level of myoglobin expected? a. multiple myeloma b. acute myocardial infarction c. renal failure d. crushing trauma from a car accident
multiple myeloma
Distinct oligoclonal bands in the gamma zone on CSF protein electrophoresis are diagnostic of: a. multiple myeloma b. multiple sclerosis c. waldenstrom's macroglobulinemia d. myoglobinemia
multiple sclerosis
Constancy of endogenous creatinine production is proportional to _____ _____ of an individual and it is released into the body fluids at a constant rate
muscle mass
At pH 8.6, proteins are ____ charged and migrate toward the ____. a. negatively; anode b. positively; cathode c. positively; anode d. negatively; cathode
negatively; anode
Each kidney is composed of more than a million urinary units. Each of these is called a:
nephron
APP (acute phase reaction)
non specific response to inflammation or tissue damage causes a positive APP
The creatine clearance formulas the term 1.73 is used to:
normalize clearance making it independent of muscle mass (size)
Water and urea are always reabsorbed through _____ transport
passive
Which of the following factors is most likely to cause a falsely low result when using the bromcresol green dye-binding assay for albumin? a. penicillin b. incubation time > 120 seconds c. bilirubin d. lipemia
penicillin
immunoglobulins
plasma cells that recognize foreign and initiate mechanism to destroy
all of the statements below regarding creatinine are true except:
plasma levels are highly dependent on diet
transferrin (TRF)
plasma transport protein for iron hemochromatosis (heriditary) TRF normal but saturation increased decrease= neph syndrome, inflammation, cancer
Which support medium can be used to determine the MW of a protein? a. cellulose acetate b. polyacrylamide gel c. agar gel d. agarose gel
polyacrylamide gel
Which statement about the biuret reaction for total protein is true? a. it is sensitive to protein levels below 0.1 mg/dL b. it is suitable for urine, exudates, and transudates c. polypeptides and compounds w/ repeating imine groups react d. hemolysis will not interfere
polypeptides and compounds w/ repeating imine groups react
A hight BUN: creatinine ratio with a significantly elevated creatinine is usually seen in:
postrenal azotemia
indicator of nutrition
prealbumin
Substances removed from the blood by tubular secretion include primarily:
protein bound substances, hydrogen, and potassium
Total Protein Methodologies: Acid-Sulfosalicylic Acid
protein precipitates -Visual method
alpha 1 antittrypsin (AAT)
proteinase inhibitorinhibit leukocyte elastase (lungs)pulmonary emphysema
CSF
proteins w/ increased permeability to BBB MS- oligoclonal bands
Wilson disease
rare autosomal rec trait where ceruloplasmin levels reduced, excessive accumulation of Cu in liver
Renal function tests are included in chemistry screen profiles to test for (3)
renal disease, water balance, and acid-base disorders
Select the primary reagent used in the Jaffe method for creatinine A) saturated picric acid and NaOh B) Sodium nitroprusside and phenol C) Phosphotungstic acid D) Alkaline copper II sulfate
saturated picric acid and NaOh
Malignancy, acute or chronic renal disease or renal failure, glycogen storage disease, high purine diet, ethanol abuse, toxemia of pregnancy cause...
secondary hyperuricemia
Plasma concentration of urea is significantly elevated by all of the following except:
severe liver disease
The amount of creatinine in urine is directly related to:
severity of renal disease
renal calculi (kidney stones)
• 67% calcium oxalate with or without phosphate -calcium oxalate crystals can be any in any pH • 12% magnesium ammonium phosphate • 8% calcium phosphate • 8% urate (uric acid) • 1-2% cystine • 2-3% mixtures
Acute Attacks of Gout can be Precipitated by
• Alcohol • High-protein diets • Stress • Acute infection • Surgery • Certain medications
Clinical Significance of BUN
• Azotemia: an increased blood urea and other NPN compounds. • Uremia: an increased urea/BUN. • Azotemia is classified into three categories: prerenal, renal, and postrenal.
BUN Reference Ranges
• BUN is 7-18 mg/dL. • A high protein diet increases BUN. • BUN < 8-10 mg/dL probably result of overhydration • BUN 50-150mg/dL- impaired Glomerular Filtration Rate (GFR) • BUN 150-250mg/dL - Severe renal impairment
Creatinine Clinical significance
• CR is primarily an index of renal function and measures the glomerular filtration rate (GFR). • Increased serum CR is present when the formation or excretion of urine is impaired due to prerenal, renal, or postrenal causes. • CR levels in urine can be used to detect if a urine has been diluted, e.g., in drug testing. • CR and BUN levels can be used to identify a fluid as urine.
Creatinine (Cr)
• Cr is synthesized in the liver from three amino acids: arginine, glycine, and methionine. • It is a waste product derived from creatine and creatine phosphate. • The constancy of endogenous CR production is proportional to the muscle mass of the individual and it is released into the body fluids at a constant rate. Cr is readily filtered by the glomeruli and does not undergo any significant tubular reabsorption
Creatinine Clearance
• Creatinine is a very good indicator of glomerular filtration rate for three reasons. - It is freely filtered by the glomeruli. - It is not reabsorbed by the tubules to any significant extent. - It is released into the plasma at a constant rate, resulting in constant plasma levels over 24 hours.
What is Protein Electrophoresis?
• Electrophoresis is the migration of charged solutes or particles in a liquid medium under the influence of an electrical field.
Inulin
• Endogenous, naturally occurring polysaccharide found in artichokes - gold standard for measuring glomerular filtration rate. • Inject inulin & collect urines at appropriate times—exogenous to people Disadvantage - invasive procedure—must inject inulin
A/G ratio (albumin/globulin)
• Globulin concentration can be calculated by subtracting the albumin from total protein. - Globulin = Total Protein (g/dL) - Albumin (g/dL) • The A/G ratio can then be determined by dividing the albumin concentration by the calculated globulin.
Hyperproteinemia
• Hyperproteinemia is associated with a positive nitrogen balance; dietary nitrogen intake is greater than the excretion or loss of nitrogen, which occurs mainly in the urine.
Immunoglobulin A (IgA)
• IgA has a molecular weight of 160,000 and comprises 10-15% of Ig. • Secretory IgA, a dimer, is found in secretions including tears, sweat, saliva, and milk as well as gastrointestinal and bronchial secretions.
Immunoglobulin E (IgE)
• IgE has a molecular weight of 180,000 and concentrations are very low (0.3 μg/mg). • IgE are produced in allergic reactions, urticaria, hay fever, and asthma.
Immunoglobulin G (IgG)
• IgG has a molecular weight of 150,000 and is the most abundant Ig in serum (70-75% of Igs). • IgG antibodies are produced in response to the antigens of most bacteria and viruses.
Immunoglobulin M (IgM)
• IgM is the largest Ig with a molecular weight of 900,000 and accounts for 5-10% of total Igs. • IgM is the first produced during an immune response (primary response).
Primary Gout
• Inborn error of metabolism • Predominantly in men 30 to 50 years of age • 7 times more common in men than women • Symptoms - Arthritis (pain, inflammation of the joints) -see uric crystals - Nephropathy - Nephrolithiasis
What factors influence the formation of kidney stones?
• Increase in concentration of chemical salts as a result of dehydration or increase in salts in the diet, e.g., vegetarians ingest more oxalates • Change in urinary pH (more alkaline pH results in less solubility of Ca and oxalate) • Urinary stasis -flow of urine has stopped; something making it "back up" • Presence of a foreign body
Protein:Creatinine Ration
• Proteinuria - determined with 24 hour collection- Several sources of error - mainly collection incomplete. • Ratio- best performed on 4hour or random sample- has strong correlation with 24 hour results.
Hypoproteinemia
• Relative hypoproteinemia: an increase in plasma water volume or hemodilution that results in a decrease in the concentration of all proteins.
What are the three main variables of creatinine levels?
• Relative muscle mass- low levels not significant—serum & urine excretion function of muscle mass • Creatine turnover - High levels unique to kidney Renal function
Renal Function tests
• Renal function tests, e.g., BUN (blood, urea, nitrogen) and creatinine, and electrolytes are included in chemistry screening profiles to test for renal disease, water balance, and acid-base disorders.
Cystatin C
• Single chain- low molecular weight protein synthesized in nucleated cells. • Cysteine protein inhibitor. • Small size & high isoelectric point - pI = 9.2 - enables it to be freely filtered by glomerulus & catabolized in the PCT • Cystatin C produced at a constant rate & serum concentrations are not affected by muscle mass, diet, race, age or gender. • Superior to CRCL tests for detecting renal disease—especially mild to moderate impairment. • Expensive
Nephrotic Syndrome
• Symptoms: - Massive proteinuria (>3 g/day) - lose a lot protein - Albuminuria (>1.5 g/day) - Pitting edema - fingers sink into skin and leave a mark/indentation due to edema (accumulation of fluid) - Hyperlipidemia - Hypoalbuminemia
eGFR
• The estimating or prediction equation eGFR estimates glomerular filtration rate (GFR) from the serum creatinine level in patients with chronic renal disease and those at risk for CKD, e.g., diabetes, hypertension, cardiovascular disease, and family history of kidney disease.
BUN: Creatinine (BUN:Cr) Ratio:
• The normal ratio is between 12:0 to 20:1. • In renal disease, BUN and Cr are both elevated proportionally; the ratio will fall within the normal range. • A high ratio >20:1 to 30:1 with a high BUN and a normal or only slightly elevated Cr is associated with prerenal azotemia. • High ratios with an elevated Cr suggest postrenal obstruction (azotemia) or prerenal azotemia in addition to renal disease. • Decreased ratios less common—seen in renal dialysis patients because BUN is more dialyzable than creatinine. Other conditions Acute Tubular Necrosis, Low-protein diets, starvation, severe diarrhea, vomiting, SIADH & sever liver disease.
Renal Clearance
• The rate at which the kidneys remove a substance from the plasma or blood. • A quantitative expression of the rate at which a substance is excreted by the kidneys in relation to the concentration of the same substance in the plasma usually expressed as mL cleared per minute.
Total protein reference range
• The serum protein in healthy, ambulatory adults in 6.0-8.3 g/dL. • A physiological decrease of approximately 0.5 g/dL occurs in bed-ridden patients.
Electrophoresis: Densitometry
•After electrophoresis, a stained gel is passed through the optical system of a densitometer to create an electrophoregram, a visual diagram or graph of the separated bands. • A densitometer is a special spectrophotometer that measures light transmitted through a solid sample such as a cleared or transparent but stained gel. Using the optical density measurements, the densitometer represents the bands as peaks. •These peaks compose the graph or electrophoregram and are printed on a recorder chart or computer display. •Absorbance and/or fluorescence can be measured with densitometry. •The densitometer scan below depicts the separated bands from a serum sample electrophoresis. The SPIFE 3000, Helena Laboratories, electrophoresis splits the beta zone into two fractions for easier detection of small beta-migrating monoclonal gammopathies.
Electrophoresis: Types of Support Media: Agarose Gel
•Agarose gels are chemically purified forms of agar, a polysaccharide extracted from seaweed. The gel pores allow for separation of proteins based on their individual charge and mass. Agarose gel will naturally clear after drying the separated proteins. •Common clinical uses of agarose gel electrophoresis (AGE) are separations of plasma proteins, hemoglobin variants, lipoproteins, and isoenzymes. The gels come prepackaged with a plastic template to lay over gel for sample application or slots etched in the gel for these samples.
Types of Electrophoresis: Immunofixation electrophoresis
•An agarose gel electrophoresis first separates the proteins in a serum sample. Antiserum against the protein of interest is spread directly on the gel. The protein of interest precipitates in the gel matrix. After a wash step to remove other proteins, the precipitated protein is stained. This method is qualitative and is used to identify proteins found in multiple myeloma. •Below is the immunofixation electrophoresis gel from a serum sample analyzed on SPIFE 3000, Helena Laboratories. After electrophoresis, the precipitated proteins are stained with Acid Violet, a stain developed and used by Helena Laboratories. •The SP lane represents a routine serum protein electrophoresis of this specimen. On the next three protein separations, antiserum against IgG, IgA, and IgM were applied to the G, A, M lanes respectively. Antiserum to kappa light chain was added to the next protein separation and antiserum to lambda light chain to the last protein separation. •When testing for monoclonal gammopathy both serum and urine must be tested. •CSF Samples must be concentrated 50-100 fold before performing IFE
Electrophoresis: Amphoteric
•An amphoteric molecule has the ability to be negatively or positively charged. Changing the pH using buffers will alter the charge and magnitude of the charge. •A molecule with this amphoteric ability is sometimes referred to as an ampholyte or even by the older term, zwitterion. •Proteins with their ionizable amino(NH2) and carboxyl (COOH) groups are amphoteric. Nucleic acids (deoxyribonucleic acid or DNA and ribonucleic acid or RNA) are charged and thus are amphoteric.
High performance chromatography (HPLC)
•Aqueous or organic solutions are pumped through columns under high pressure, which allows high resolution with fast and accurate quantitation. •Both qualitative and quantitative results.
Electrophoresis: Automation
•Automated systems for protein electrophoresis are available for large volumes of samples for electrophoresis. An automated system is capable of separating 10-100 samples simultaneously. •There are several different automated systems and the number of process steps that are automated varies. Automated steps may include reagent addition, sample application, electrophoresis separation, staining, and detection.
Electrophoresis: Types of Support Media
•For electrophoretic separation of solutes, the sample of solutes is placed on a gel or membrane in contact with buffer for separation. Common gels are cellulose acetate, agarose, and polyacrylamide gels. •These gels are formed into sheets, slabs, or inserted into columns or tubes. The gel can be positioned horizontally or vertically.
Electrophoresis Equipment
•In addition to the specimen sample, support medium and buffer for electrophoresis, a power supply, positive and negative electrodes, chamber, and identification or detection method are needed. •The power supply is a source of constant voltage or current that provides energy to the electrodes. This drives the movement of the ions in the medium and results in the movement and separation of the molecules or solutes in the specimen. Control of current or voltage comes with the power source in order to make adjustments. •The chamber is divided into two sections or has two reservoirs for the buffer and one electrode is placed in each. The support medium is laid over the chamber in such a way that it connects the two reservoirs. A lid or cover is placed over the chamber during electrophoresis.
Rate of Migration in Electrophoresis: strength of electrical field/Temperature
•Increased heat increases the rate of migration. Increasing the strength of the electrical field by increasing voltage and increasing the temperature used for the electrophoresis both increase the mobility and rate of migration. •an increase in temperature that can possibly denature the sample and alter the characteristics of the support medium.
Isoelectric point (pI)
•Isoelectric point (pI) is the PH where the net charge of a molecule is zero. At its pI, a molecule will not move in an electrical field because it does not have a charge. •The ph of an electrophoretic gel is determined by the buffer used. IF the pH of the buffer used is: •pH below its pI will have a net positive charge & migrate to the negative pole of the gel (Cathode). •PH above the pI, a protein will have a net negative charge & migrates to the positive pole of the gel (anode).
Electrophoresis: Types of Support Media: Polyacrlamide gels
•Polyacrylamide electrophoresis (PAGE) is performed on a gel formed by polymerizing and cross-linking acrylamides. These gels are stronger than agarose gels and also thermostable and transparent. The matrix created by cross-linking the polymer chains is more regular and the pore sizes are more uniform in an individual gel. The pore size can be changed by changing the concentrations of the acrylamides used. •In addition to separating fragments by charge and mass, PAGE also separates solutes by molecular size. When using PAGE, the gel allows more fractions of smaller size to be detected than the traditional agarose gel methods. •Care is required in polyacrylamide gel preparation and use because acrylamides are carcinogenic.
Chromatography
•Principle: Separates mixture into individual components on basis of specific differences in physical characteristics. •Types: •Liquid-liquid-chromatography •Ion Exchange chromatography •Gas-liquid chromatography •Thin Layer chromatography High performance chromatography
Types of Electrophoresis: Routine Electrophoresis
•Routine electrophoresis is a generic term for the traditional clinical laboratory electrophoresis performed on a rectangle-shaped slab gel. Routine electrophoresis is mostly used for separation of proteins and has some use in separating nucleic acids. Generally several patient specimens and control(s) can be placed on one gel and solutes separated in one run. This type of electrophoresis is sometimes called zone electrophoresis. •A serum sample with normal plasma proteins yields five zones or bands of separated proteins: albumin, alpha-1-globulins, alpha-2-globulins, beta-globulins, and gamma-globulins. Proteins in CSF and urine proteins are also separated with routine electrophoresis. Using whole blood treated with a reagent to lyse red blood cells, variant and glycosylated hemoglobins can be detected. With different visualization methods, isoenzymes and lipoproteins in a serum sample can be identified. •Albumin is the fastest migrating protein toward the anode at ph 8.6 with the greatest net negative charge and lowest PI (4.6). followed by alpha-1. Alpha-2, Beta, Gamma globulins are predominately immunoglobulins and have highest PI about 7.2 and greatest net positive charge.
Electrophoresis: Visualization and Detection Methods
•Separated bands or zones can be visualized with stains and dyes. • Densitometry can be used to detect and usually quantitate stained separated fragments. •Some electrophoresis methods use labeled probes to detect presence of unknowns in samples. •Serum-free light chain immunoassays are most sensitive for detecting monoclonal gammopathies. Immunonephelometric free light chain assays can detect monoclonal protein production before the mass is sufficient to cause a monoclonal spike on SPE, but will only be positive when monoclonal light chain production occurs.
Liquid-liquid chromatography
•Separation based on differences in solubility between two liquid phases: •Separation of molecules is based on their relative distribution between two different phases. Separation takes place on a stationary or nonmoving phase under the influence of a mobile or moving phase. • Molecules are separated based on their solubility and their interaction with these two phases. •Sample mixture with a mobile phase -often liquid or gas, while the stationary phase is a medium like a aluminum silicate gel and resin particles. •Sample applied to the column & sample travels via gravitational force or pressure from a liquid or gas. •Sample will travel at different rates & elute from the column at different times. •Separated components can be detected by using different solvents.
Ion exchange chromatography
•Separation depends on molecular weight, size and charge of the ions or molecules. Based on the interaction of the analyte charges with an oppositely charged group bound to the chromatographic support. •Ions with the greater charge densities are held most strongly on ion exchange material. •Retention can be varied by varying pH or ionic strength.
Thin Layer chromatography (TLC)
•Separation depends on rate of diffusion and solubility of the substance in the solvents as the components migrate through media. •Used to identify drugs, lipids, carbohydrates and amino acids. •TLC used a thin layer of slica gel, alumina gel, poly acrylamide or starch gel attached to a glass plate as stationary phase. The mobile phase is a liquid solvent. The fractions in the sample are generally quite soluble in the solvent and move with it up the stationary phase by capillary action. •Rf = Distance moved (constituent) Distance moved (solvent)
Gas-liquid chromatography (GLC or GC) Gas Chromatography
•Separation depends on sample volatility and rate of diffusion into liquid layer (partition coefficient or inert gas (mobile phase) •Retention Time: Used to identify volatiles (methanol, isopropyl, alcohol), drugs, organic acids in urine and catecholamines. •Best method for blood alcohol •Confirmatory testing-GC with mass spectrophotometry (MS) required for regulated drugs of abuse.
Electrophoresis: Buffers and Specimens
•Serum and plasma are the most common clinical specimens used for electrophoresis applications. Urine and cerebrospinal fluids (CSF). •SPE - Serum Protein Electrophoresis- Must use Serum!- Plasma is not used because fibrinogen will produce a band resembling a small monoclonal protein in the beta region. •Some specimens require pretreatment before electrophoresis. Low concentrations of proteins normally in urine and CSF are concentrated in order to have enough proteins for detectable separations. Some body fluids require removal of pigments, salts, and other compounds that interfere with electrophoresis or the detection of separated solutes. •In molecular diagnostic testing of DNA and RNA, the nucleic acids must first be isolated from the specimen and then purified before separation with electrophoresis.
Rate of Migration in Electrophoresis: Ionic Strength
•The ionic strength of the buffer affects the thickness of the ionic cloud, the rate of migration, and the sharpness of the separated solutes. In electrophoresis, a cloud of ions forms over the medium and is composed of buffer ions, sample ions and other nonbuffer ions. Increasing the buffer ionic strength increases the buffer ions in the cloud and slows the movement of solutes and creates sharper bands. However, this also increases heat production
Rate of Migration in Electrophoresis: Net charge
•The net charge of a molecule is the most important factor affecting the mobility of that molecule. The greater the net charge, the greater the mobility or the more quickly the molecule migrates. The net charge of a particular compound depends upon the buffer and the resultant pH set by that buffer.
Rate of Migration in Electrophoresis: Size and Shape
•The size and shape of a molecule also influence the rate of migration in that the larger the size, the slower the molecule will move in electrophoresis.
Electrophoresis: Role of Buffers
•The two important purposes of the buffer are to create the pH and to conduct the current. The buffer ions will carry the current during electrophoresis. •The pH set by the buffer determines the net charge on the solutes. The pH ionizes these solutes and the resulting net charge determines which electrode the solutes migrate toward. Besides setting the pH, the buffer also maintains the pH throughout the electrophoresis of the sample. •The isoelectric point of most proteins is between pH 4.0 and 7.5. In pH 8-9, proteins will take on a negative charge and migrate to the anode. Most protein electrophoresis is performed at pH 8.6. •Buffers most commonly used are barbital or tris-boric acid-EDTA buffers. They fix the pH at 8.6, leading to sharper bands and good separations.
Rate of Migration in Electrophoresis: Support Media
•The viscosity and the pore size in the support media or gels used for electrophoresis influence the rate of migration. Increased viscosity slows the migration increasing pore size speeds up the migration.
Types of Electrophoresis
•There are numerous applications of electrophoresis. Routine protein electrophoresis performed in clinical laboratories is the oldest method and therefore the most frequently used method. With the advent of molecular diagnostics, several other electrophoresis methods have become very important, highly automated, and have several important applications.
