Harr MLS Review Chemistry 5.6 Proteins, Electrophoresis, and Lipids

Select the order of mobility of lipoproteins electrophoresed on cellulose acetate or agarose at pH 8.6.

- Chylomicrons →β→pre-β→α + Although pre-β lipoprotein is lower in density than β lipoprotein, it migrates faster on agarose or cellulose acetate owing to its more negative apoprotein composition. When lipoproteins are separated on polyacrylamide gel, pre-β moves slower than β lipoprotein. Molecular sieving causes migration to correlate with lipoprotein density when PAGE is used.

Select the correct order of Hgb migration on agarose or cellulose acetate at pH 8.6.

- C→S→F→A + Hgb A2 is the slowest of the normal Hgbs, and Hgb A is the fastest. Hgb F migrates just behind Hgb A. Hgb S migrates midway between Hgb A2 and Hgb A. Hgbs C, CHarlem (Georgetown), O, and E migrate with Hgb A2. Hgbs G and DPunjab and Hgb OArab migrate with Hgb S.

Select the correct order of Hgb migration on citrate agar at pH 6.2.

- F→A→S→C + In an acid buffer, the hemoglobins are expected to migrate to the cathode, with hemoglobin A being the slowest because it has the weakest net positive charge. However, Hgb C and Hgb S bind to sulfated pectins in the agar gel, forming a complex that is negatively charged causing them to migrate toward the anode. Hgb C migrates furthest toward the anode, followed by Hgb S. Hgb F migrates furthest toward the cathode. Hgbs A, A2, DPunjab, E, G, and HgbLepore migrate slightly toward the cathode.

What is the most appropriate fasting procedure when a lipid study of triglyceride, total cholesterol, HDL cholesterol, and LDL cholesterol tests are ordered?

12 hours; nothing but water allowed Lipid orders that include triglyceride and LDL cholesterol should always be performed using a plasma or serum specimen collected after a 12-14 hour fast. The patient should be instructed to drink nothing but water during this period. Fasting specimens are preferred for total and HDL cholesterol as well, but nonfasting specimens may be used for initial screening purposes.

Select the reagent needed in the coupling enzyme reaction used to generate a colored product in the cholesterol oxidase method for cholesterol.

4-Aminoantipyrine In the cholesterol oxidase method, cholesterol ester hydrolase converts cholesterol esters to free cholesterol by hydrolyzing the fatty acid from the C3-OH group. Cholesterol oxidase catalyzes the oxidation of free cholesterol at the C3-OH group forming cholest-4-ene-3-one and hydrogen peroxide. The peroxide is used in a peroxidase reaction to oxidize a dye (e.g., 4-aminoantipyrine), which couples to phenol, forming a red quinoneimine complex.

The protein composition of HDL is what percentage by weight?

50% About 50% of the weight of HDL is protein, largely apo A-I and apo A-II. The HDL is about 30% phospholipid and 20% cholesterol by weight. The HDL binds and esterifies free cholesterol from cells and transports it to the liver, where it can be eliminated in the bile.

What is the HDL cholesterol cutpoint recommend by NCEP?

<40 mg/dL The HDL cholesterol cutpoint recommended by NCEP is < 40 mg/dL regardless of sex. A result below 40 mg/dL counts as a risk factor for coronary artery disease. Conversely, if the HDL cholesterol is ≥ 60 mg/dL, then one risk factor is subtracted from the total number. The therapeutic goal for someone with low HDL cholesterol is still reduction of LDL cholesterol (if elevated), weight loss, and increased exercise.

Capillary electrophoresis differs from agarose gel electrophoresis in which respect?

A stationary support is not used Capillary electrophoresis is a rapid automated procedure for separating serum or body fluid proteins. Instead of a stationary support, the proteins migrate based upon their charge/mass ratio inside a small-bore silica capillary tube (20-200 μm). The cations in the buffer are attracted to the negatively charged silicates and migrate to the cathode rapidly when voltage is applied. The electroendosmotic force created moves the proteins toward the cathode and they are detected by an in-line UV photometer that measures their absorbance. High voltage (e.g., 9,000 volts) is used to effect separation of serum proteins in an 8-10 minute run, giving resolution equal to or greater than HR agarose gel electrophoresis.

Which of the following is more commonly associated with a nonmalignant form of monoclonal gammopathy (MGUS)?

Age greater than 60 at the time of monoclonal protein discovery MGUS is the most common cause of monoclonal gammopathy. About 3% of the U.S. population at age 50 and 5% at age 70 have MGUS. The absence of bone lesions and organ damage, plasma cells below 10% of nucleated bone marrow cells, and M-protein below 3.0 g/dL are characteristic of MGUS as opposed to myeloma or other malignant gammopathy. About 50% of persons with MGUS have IgH gene translocations or chromosome 13 deletion associated with multiple myeloma. The risk of transformation of MGUS to malignant disease is about 1% per year.

Which of the following serum protein electrophoresis results suggests an acute inflammatory process?

Albumin: Decreased α1: Increased α2: Increased Β: Normal γ: Normal Acute inflammation is characterized by increased production of acute phase proteins. These include α1-antitrypsin, α1-acid glycoprotein, α1-antichymotrypsin, and haptoglobin. Albumin is slightly decreased. γ- and β-fractions are normal.

What do "direct" or homogenous methods for LDL cholesterol assay have in common?

All use a detergent to facilitate selective reactivity with reagent enzymes The direct LDL cholesterol assays are all detergent based methods. One commonly used method employs a polyanionic detergent to release cholesterol from HDL, chylomicrons, and VLDL. The detergent binds to LDL and blocks its reaction with the esterase and oxidase enzymes in the reagent. Cholesterol oxidase oxidizes the non-LDL cholesterol, forming H2O2, and peroxidase catalyzes the oxidation of an electron donor by the H2O2,which does not result in color formation. A second nonionic detergent and chromogen is added. The second detergent removes the first from the LDL, allowing it to react with the enzymes. The resulting H2O2 reacts with the chromogen, forming a colored product.

Which of the following mechanisms accounts for the elevated plasma level of β lipoproteins seen in familial hypercholesterolemia (formerly type II hyperlipoproteinemia)?

ApoB-100 receptor defect The production of excess insulin leads to hypertriglyceridemia and is one mechanism responsible for familial endogenous hypertriglyceridemia. ApoC-II is an activator of lipoprotein lipase, and a homozygous deficiency results in high plasma chylomicrons and VLDL. ApoE3 deficiency is synonymous with inheritance of two apo-E2 alleles that lead to β dyslipoproteinemia. Familial hypercholesterolemia is inherited as an autosomal dominant trait. The classical form results from one of many mutations affecting the LDL receptor that cause it to have a lower affinity for LDL. A related hypercholesterolemia common in people of European ancestry results from a mutation of the apo-B100 gene that causes LDL to have a lower affinity for the LDL receptor. Together, they make familial hypercholesterolemia the most common inherited hyperlipoproteinemia with a frequency over 1:500.

Which apoprotein is inversely related to risk of coronary heart disease?

Apoprotein A-I Apoprotein A-I and apo A-II are the principal apoproteins of HDL, and low apo A-I has a high correlation with atherosclerosis. Conversely, apo-B100 is the principal apoprotein of LDL, and an elevated level is a major risk factor in developing coronary heart disease. Apoprotein assays are not recommended as screening tests because they are not as well standardized as LDL cholesterol assays. However, apo-B100 assay is more sensitive than LDL cholesterol in predicting coronary artery disease risk. Apo-B100 may be abnormal in persons with increased small dense LDL. Small dense LDL is more atherogenic than large LDL molecules. In addition, persons with hyperapobetalipoproteinemia overproduce apo-B100 without having significantly elevated LDL cholesterol.

Which of the following is associated with Tangier disease?

Apoprotein A-I deficiency Deficiency of apo A-I is seen in Tangier disease, a familial hypocholesterolemia. Heterozygotes have about half of the normal level of HDL (familial hypoalphalipoproteinemia) and homozygotes have almost no detectable HDL. Tangier disease is caused by a mutation of the ATP-binding cassette gene. The deficient gene prevents apo A-I from binding lipids, and it is rapidly catabolized. Abetalipoproteinemia results from defective hepatic transport of apo-B100, and is also inherited as an autosomal recessive condition. LDL is absent, and the condition is associated with hemolytic anemia and central nervous system damage.

Which of the following dyes is the most specific for measurement of albumin?

Bromcresol purple (BCP) Tetrabromphenol blue and tetrabromosulfophthalein are dyes that change pKa in the presence of protein. Although they have greater affinity for albumin than globulins, they are not sufficiently specific to apply to measurement of serum albumin. BCG and BCP are anionic dyes that undergo a spectral shift when they bind albumin at acid pH. BCP is more specific for albumin than BCG. Reaction of both dyes with globulins requires a longer incubation time than with albumin, and reaction times are kept at 30 seconds or less to increase specificity. Both dyes are free of interference from bilirubin. However, BCG is the method used most often. One reason for this is that renal dialysis patients produce an organic acid that competes with BCP for the binding site on albumin, causing a falsely low result.

Which method is considered the candidate reference method for triglyceride measurement?

CDC modification of van Handel and Zilversmit Enzymatic methods for triglyceride measurement are widely used because they eliminate the need for extraction and saponification. However, they are subject to positive interference from endogenous glycerol and variations in the efficiency of lipase, which can result in under- or overestimation of triglycerides. The most accurate method for triglyceride assay is the nonenzymatic method based upon reaction of formaldehyde with chromotropic acid. In this method, extraction with silicic acid and chloroform separates triglycerides from lipoproteins, phospholipids, and glycerol. Saponification with alcoholic potassium hydroxide (KOH) produces glycerol, which is oxidized to formaldehyde by periodate. The formaldehyde reacts with chromotropic acid to form a pink product.

Which statement regarding IFE is true?

CSF should be concentrated 50- to 100-fold before performing IFE Any monoclonal precipitin band formed when heavy- or light-chain-specific antiserum reacts with a sample should also be found in the same position when sample is fixed with sulfosalicylic acid or reacted with polyvalent antihuman Ig. The normal free κ:λ ratio can vary between 0.26 and 1.65. In a monoclonal gammopathy, this ratio always heavily favors the light chain type of M protein. A diagnosis of multiple sclerosis is usually confirmed by demonstration of oligoclonal banding in the CSF, which is not present in the serum. CSF is usually concentrated 50-100 times to increase sensitivity.

Which serum protein should be measured in a patient suspected of having Wilson's disease?

Ceruloplasmin α-1 antitrypsin, haptoglobin, and ceruloplasmin are acute phase proteins and will be increased in inflammatory diseases. Ceruloplasmin is an α-2 globulin that binds the majority of the serum copper. Levels are low in almost all patients with Wilson's disease, an autosomal recessive disorder caused by accumulation of copper in liver, brain, kidney, and other tissues. Low ceruloplasmin may occur in patients with nephrosis, malnutrition, and hepatobiliary disease. Therefore, the diagnosis of Wilson's disease is made by demonstrating decreased plasma ceruloplasmin, increased urinary copper, and the presence of Kayser-Fleischer rings (brown deposits at the edge of the cornea).

In double immunodiffusion reactions, the precipitin band is:

Concave to the protein of greatest molecular weight In double immunodiffusion (Ouchterlony), the molecules of lower molecular weight move fastest through the gel, causing a visible precipitin arc when antigen and antibody approach equivalence. At equivalence the precipitin arc remains stationary. If the concentration of antisera is constant, the distance of the precipitin arc from the antigen well is proportional to antigen concentration.

What is the purpose of the saponification step used in the Abell-Kendall method for cholesterol measurement?

Convert cholesterol esters to free cholesterol The Abell-Kendall method is the reference method for cholesterol assay because differences in esterase activity and interference in the peroxidase step are potential sources of error in enzymatic assays. Saponification is performed to hydrolyze the fatty acid esters of cholesterol, forming free cholesterol. This is required because the reagents react more intensely with cholesterol esters than with free cholesterol. Saponification is followed by extraction of cholesterol in petroleum ether to separate it from proteins and interfering substances. The extract is reacted with sulfuric acid, acetic anhydride, and acetic acid (Liebermann-Burchard reagent), which oxidizes the cholesterol and forms a colored product.

Upon which principle is the biuret method based?

Coordinate bonds between Cu+2 and carbonyl and imine groups of peptide bonds. Biuret is a compound with two carbonyl groups and three amino groups and forms coordinate bonds with Cu+2 in the same manner as does protein. Therefore, proteins and peptides are both measured in the biuret reaction. The biuret reagent consists of an alkaline solution of copper II sulfate. Tartrate salts are added to keep the copper in solution and prevent turbidity. Potassium iodide prevents autoreduction of Cu+2.

Hyperalbuminemia is caused by:

Dehydration syndromes A high serum albumin level is caused only by dehydration or administration of albumin. Liver disease, burns, gastroenteropathy, nephrosis, starvation, and malignancy cause hypoalbuminemia.

At pH 8.6, the cathodal movement of γ globulins is caused by:

Electroendosmosis Agarose and cellulose acetate contain fixed anions (e.g., acetate) that attract counterions when hydrated with buffer. When voltage is applied the cations migrate to the cathode, creating an osmotic force that draws H2O with them. This force, called electroendosmosis, opposes protein migration toward the anode and may cause some γ-globulins to be displaced toward the cathode.

Which of the following protein methods has the highest analytical sensitivity?

Folin-Lowry The Folin-Lowry (Lowry's) method uses both biuret reagent and phosphotungstic and molybdic acids to oxidize the aromatic side groups on proteins. The acids oxidize the phenolic rings of tyrosine and tryptophan. These, in turn, reduce the Cu+2 in the biuret reagent, increasing sensitivity about 100-fold.

Which of the following enzymes is common to all enzymatic methods for triglyceride measurement?

Glycerol kinase All enzymatic triglyceride methods require lipase to hydrolyze triglycerides, and glycerol kinase to phosphorylate glycerol, forming glycerol-3-phosphate. The most common method couples glycerol kinase with glycerol phosphate oxidase and peroxidase. 1. Triglyceride + H2O in the presence of Lipase goes to glycerol + fatty acids 2. Glycerol + ATP in the presence of GK goes to glycerol-3-phosphate + ADP 3. Glycerol-3-phosphate+O2 in the presence of GPO goes to dihydroxyacetone phosphate + H2O2 H2O2 + phenol + 4-aminophenazone in the presence of Px goes to quinoneimine dye + H2O GK = glycerol kinase; GPO = glycerol phosphate oxidase; Px = peroxidase

A patient with hemolytic-uremic syndrome associated with septicemia has a haptoglobin level that is normal, although the plasma free hemoglobin is elevated and hemoglobinuria is present. Which test would be more appropriate than haptoglobin to measure this patient's hemolytic episode?

Hemopexin Hemopexin is a small β globulin that binds to free heme. Haptoglobin is an α-2 globulin that binds to free hemoglobin and disappears from the serum when intravascular hemolysis produces more than 3 grams of free plasma hemoglobin. However, haptoglobin is an acute phase protein, and hepatic production and release are increased in response to acute infections. The normal serum haptoglobin is most likely the result of increased synthesis and would not accurately estimate the hemolytic episode in this patient.

Which of the following conditions is associated with "β-γ bridging"?

Hepatic cirrhosis Hepatic cirrhosis produces a polyclonal gammopathy associated with a high IgA level. This obliterates the valley between β and γ zones. Malignancy and rheumatoid arthritis produce polyclonal gammopathies classified as chronic inflammatory or delayed response patterns. Multiple myeloma produces a zone of restricted mobility usually in the γ, but sometimes in the β- or α2-region.

Which of the following abnormal types of Hgb migrates to the same position as Hgb S on agarose or cellulose acetate at pH 8.6?

Hgb DPunjab Hgb D Punjab migrates with Hgb S on cellulose acetate or agarose at pH 8.6-9.2. Hgb C, E, OArab, and CHarlem migrate to the same position as Hgb A2 on cellulose acetate or agarose at pH 8.6-9.2. Hgb S may be differentiated from Hgb D Punjab using citrate (acid) agar at pH 6.2. Using this technique, Hgb S migrates further toward the anode than Hgb D Punjab.

Which Hgb separates from Hgb S on citrate (acid) agar, but not agarose or cellulose acetate?

Hgb DPunjab Hgbs OArab, E, and CHarlem migrate to the same position as Hgbs A2 and C on agarose × or cellulose acetate) at pH 8.6. Hgb DPunjab migrates to the same position as Hgb S on agarose, but moves with Hgb A on citrate agar. Agarose is a purified form of agar; it lacks the sulfated pectins required to separate Hgbs DPunjab and G from Hgb S, and Hgbs E, CHarlem, and OArab from Hgb C. Hgb CHarlem is a sickling Hgb and it migrates to the same position as Hgb S on citrate (acid) agar.

Which Hgb is a β-δ chain hybrid and migrates to the same position as Hgb S at pH 8.6?

HgbLepore HgbLepore results from translocation of β and δ globin genes, resulting in a polypeptide chain that migrates midway between Hgb A2 and Hgb A. The chain is transcribed more slowly than the β polypeptide chain, causing the quantity of HgbLepore to be less than 15%. HgbLepore is suspected when Hgb migrating in the "S" zone comprises less than 20% of the total Hgb. In Hgb S trait, the AS phenotype produces 20%-40% Hgb S.

Quantitative determination of Hgb A2 and Hgb F are best performed by:

High-performance liquid chromatography Hgb A2 and Hgb F are often quantitated to diagnose persons with thalassemia. The method of choice is HPLC using cation exchange chromatography. Hemoglobins are eluted from the column in order of increasing positive charge using a sodium phosphate buffer to produce a gradient of increasing ionic strength. Hemoglobin F elutes from the column earlier than Hgb A2 because it is less positively charged.

Which of the following is one advantage of high-resolution (HR) agarose electrophoresis over lower-current electrophoresis?

High-resolution procedures detect monoclonal and oligoclonal bands at a lower concentration HR agarose procedures use higher current and a cooling device to resolve 12 or more bands. Advantages include phenotyping of α1-antitrypsin (detection of Z and S variants), detection of β2 microglobulin in urine indicating tubular proteinuria (often associated with drug-induced nephrosis), and greater sensitivity detecting monoclonal gammopathies, immune complexes, and oligoclonal bands in CSF associated with multiple sclerosis. Its disadvantage is that densitometric scans of HR gels usually underestimate albumin.

Cholesterol esterase is used in enzymatic assays to:

Hydrolyze fatty acids bound to the third carbon atom of cholesterol Approximately two-thirds of the serum cholesterol has a fatty acid esterified to the hydroxyl group of the third carbon atom of the cholesterol molecule. Cholesterol esterase hydrolyzes fatty acids and is required because cholesterol oxidase cannot utilize esterified cholesterol as a substrate.

Which of the following conditions is most consistently associated with secondary hypercholesterolemia?

Hypothyroidism The conditions listed are very commonly encountered causes of secondary hyperlipoproteinemia. Oral contraceptives, pregnancy, and estrogens may cause secondary hypertriglyceridemia owing to increased VLDL and endogenous triglycerides. Hypothyroidism and obstructive hepatobiliary diseases are usually associated with secondary hypercholesterolemia owing to high LDL. Diabetes mellitus and chronic pancreatitis may produce hypertriglyceridemia, chylomicronemia, or mixed hyperlipidemia.

In familial β dyslipoproteinemia (formerly type III hyperlipoproteinemia), which lipoprotein accumulates?

IDL IDLs have roughly equal amounts of cholesterol and triglyceride. The IDL has a density of about 1.006-1.020, causing it to float on the 1.063 density potassium bromide solution used to recover LDL by ultracentrifugation. IDL has faster electrophoretic mobility on agarose than beta lipoprotein. These observations gave rise to the terms "floating beta" and "broad beta," respectively. Familial dysbetalipoproteinemia is in part caused by a polymorphism of apoE (apo-E2) that has poor affinity for the apo-E receptor on hepatocytes. Not all persons with the homozygous polymorphism develop the disease; thus, other factors are necessary for the accumulation of IDL.

Which of the following proteins has the highest pI?

IgG Albumin is the fastest migrating protein toward the anode at pH 8.6 followed by α1-, α2-, β-, and γ-globulins. Because albumin is fastest, it has the greatest net negative charge and lowest pI (about 4.6). γ-Globulins are predominantly immunoglobulins and have the highest pI (about 7.2).

Electrophoretic movement of proteins toward the anode will decrease by increasing the:

Ionic strength of the buffer Electrophoresis is the migration of charged molecules in an electric field. Increasing the strength of the field by increasing voltage (or current) increases migration. However, increasing ionic strength decreases the migration of proteins. Counterions (cations) in the buffer move with the proteins, reducing their electromagnetic attraction for the anode.

Lipoprotein (a), or Lp(a), is significant when elevated in serum because it:

Is an independent risk factor for atherosclerosis Lp(a) is a complex of apo-B100 and protein (a) formed by a disulfide bridge. The complex is structurally similar to plasminogen and is thought to promote coronary heart disease by interfering with the normal fibrinolytic process. Lp(a) is measured by immunoassay; however, the measurement will vary depending on the type of antibodies used and their epitope specificity.

Treatment recommendations for patients with coronary heart disease are based upon measurement of which analyte?

LDL cholesterol NECP has identified LDL cholesterol as the target of therapy for reducing the risk of heart attack because lowering LDL cholesterol has proven to be an effective intervention. The greater the risk of coronary heart disease, the lower the cutpoint for intervention. For persons at high risk (a 10-year risk of heart attack > 20%) the cutpoint is ≥ 100 mg/dL for initiation of statin therapy. For highest-risk persons (those that have acute coronary syndrome, and multiple or uncontrolled risk factors) the treatment goal is LDL cholesterol below 70 mg/dL.

What is the lipid testing protocol for adults recommended by the National Cholesterol Education Program (NCEP) to evaluate risk for atherosclerosis beginning at age 20?

Lipid profile, fasting, every 5 years Because LDL cholesterol, HDL cholesterol, VLDL cholesterol, and triglycerides are all risk factors for coronary artery disease, NCEP recommends a fasting lipid profile to include triglycerides, total cholesterol, HDL cholesterol, and LDL cholesterol be performed every 5 years beginning at age 20. However, because LDL cholesterol is the target of treatment, therapeutic goals are based on the LDL cholesterol. New guidelines recommend an LDL cholesterol goal below 70 mg/dL for the highest-risk persons.

Which of the following conditions will prevent any migration of proteins across an electrophoretic support medium such as agarose?

Loss of contact between a buffer chamber and the medium Movement of proteins is dependent upon the presence of a salt bridge that allows current to flow via transport of ions to the electrodes across the support medium. If the salt bridge is not intact, there will be no migration, even if voltage is maintained across the electrodes. For agarose and cellulose acetate, heat causes evaporation of solvent from the buffer. This increases the ionic strength, causing current to rise during the run. Excessive heat can damage the support medium and denature proteins. Power = E (voltage) × I (current) × t (time); since E = I × R (resistance), heat is proportional to the square of current (P = I2 × R × t). Constant current or power mode is used for long runs to prevent heat damage.

Which of the following conditions is most commonly associated with an elevated level of total protein?

Malignancy Malignant disease is usually associated with increased immunoglobulin and acute-phase protein production. However, nutrients required for protein synthesis are consumed, causing reduced hepatic albumin production. Glomerular damage causes albumin and other low molecular weight proteins to be lost through the kidneys. Liver failure and starvation result in decreased protein synthesis.

Which test is the most useful way to evaluate the response to treatment for multiple myeloma?

Measurement of serum-free light chains Unlike electrophoresis methods, serum free light chain assays are quantitative and an increase in free light chain production with an abnormal kappa:lambda ratio occurs earliest in recurrence of myeloma. Light chains have a shorter plasma half life than intact Ig and therefore, the reduction in free light chain concentration is an earlier indicator of treatment effect than measurement of intact Ig. It is not subject to the variation in 24-hour urinary light chain measurement caused by sample collection error and abnormal renal function. A 50% or more reduction in serum free light chain concentration is considered a partial response to treatment. A full response is indicated by reduction to within normal limits and a return of the kappa:lambda ratio to normal. An abnormal free light chain ratio has a 3.5-fold higher risk of progression to myeloma in persons with monoclonal gammopathy of undetermined significance (MGUS).

Which type of dietary fatty acid is not associated with an increase in serum LDL cholesterol production?

Monounsaturated cis fatty acids Polyunsaturated and cis monounsaturated fatty acids are not associated with increased production of LDL cholesterol. On the other hand, saturated and trans monounsaturated fatty acids are both associated with increased LDL. Cis fatty acids are those in which the H atoms belonging to the double-bonded carbons are on the same side of the molecule. Ω-9 (n-9) fatty acids are those with a double bond located 9 carbons from the terminal methyl group. Ω Fatty acids are associated with increased cholesterol, if the hydrogens attached to the double-bonded carbons are in the trans position.

High serum total protein but low albumin is usually seen in:

Multiple myeloma In multiple myeloma, synthesis of large quantities of monoclonal immunoglobulin by plasma cells often results in decreased synthesis of albumin. In glomerulonephritis and nephrotic syndrome, both total protein and albumin are low owing to loss of proteins through the glomeruli. In hepatic cirrhosis, decreased hepatic production of protein results in low total protein and albumin.

Which of the following conditions is usually associated with an acute inflammatory pattern?

Myocardial infarction (MI) MI produces a pattern of acute inflammation usually associated with tissue injury. This pattern results from production of acute phase proteins including α1-antitrypsin, α1-antichymotrypsin, and haptoglobin. It is also seen in early infection, pregnancy, and early nephritis. Malignancy, rheumatoid arthritis, and hepatitis are associated with a chronic inflammatory pattern. This differs from the acute pattern by the addition of a polyclonal gammopathy.

At pH 8.6, proteins are _________ charged and migrate toward the _________.

Negatively, anode Proteins are amphoteric owing to ionization of acidic and basic side chains of amino acids. When the pH of the solution equals the isoelectric point ( pI ), the protein will have no net charge and is insoluble. When the pH of the solution is above the pI, the protein will have a net negative charge. Anions migrate toward the anode (positive electrode).

Which of the following diseases is caused by a deficiency of sphingomyelinase?

Niemann-Pick disease The diseases mentioned result from inborn errors of lipid metabolism (lipidoses) caused by deficiency of an enzyme needed for lipid degradation. Specific lipids accumulate in the lysosomes. Niemann-Pick disease results from a deficiency of sphingomyelinase; Gaucher disease from β-glucocerebrosidase; Fabry disease (sex linked) from α-galactosidase A; and Tay-Sachs from N acetylglucosaminidase A.

Which of the following stains is used for lipoprotein electrophoresis?

Oil Red O Oil Red O and Sudan Black B stain neutral fats and are used to stain lipoproteins as well as fat in urine or stool. The other stains are used for proteins. Coomassie Brilliant Blue is more sensitive than Ponceau S or Amido Black, and all three stains have slightly greater affinity for albumin than globulins. In addition, silver nitrate may be used to stain CSF proteins because it has greater sensitivity than the other stains.

Which of the following statements regarding paraproteins is true?

Oligoclonal banding is seen in the CSF of greater than 90% of multiple sclerosis cases The α heavy chain is more acidic than γ or μ chains, giving IgA a greater net negative charge at alkaline pH. The IgA precipitin band is anodal to the IgG or IgM band. In hepatic cirrhosis, the β-γ bridging observed on serum protein electrophoresis results from increased IgA. Light chains in the form of Fab fragments are often found in increased amounts in the urine of patients with polyclonal gammopathies, especially from patients with an autoimmune disease. These can cause a positive Bence-Jones test and will produce a polyclonal (spread-out) appearance on IFE gels.

SITUATION: A lipemic specimen collected from an adult after a 12-hour fast was assayed for total cholesterol, triglycerides, and HDL cholesterol using a direct HDL method. Following are the results: Total cholesterol = 220 mg/dL HDL cholesterol = 40 mg/dL Triglyceride = 420 mg/dL The physician requests an LDL cholesterol assay after receiving the results. How should the LDL cholesterol be determined?

Perform a direct LDL cholesterol assay An accurate LDL cholesterol can be reported, if the direct (detergent) method for LDL cholesterol is employed. These methods are not subject to interference by triglycerides at a concentration below 700 mg/dL.

Which support medium can be used to determine the molecular weight of a protein?

Polyacrylamide gel Polyacrylamide gels separate by molecular sieving as well as charge. Sodium dodecyl sulfate (SDS) is a nonionic detergent that binds to proteins, neutralizing their charge. Polyacrylamide gel electrophoresis (PAGE) after treating with SDS separates proteins on the basis of molecular size. The smaller proteins become trapped in the pores of the gel and migrate more slowly.

Which statement about the biuret reaction for total protein is true?

Polypeptides and compounds with repeating imine groups react The biuret reaction is not sensitive to protein levels below 0.1 g/dL and, therefore, is not sensitive enough for assays of total protein in CSF, urine, or transudates. Slight hemolysis does not cause falsely high results, if the absorbance of the Cu+2-protein complexes is measured bichromatically. However, frankly hemolyzed samples contain sufficient globin to cause positive interference. The reagent reacts with peptides containing at least two peptide bonds, but due to the high concentration of proteins in plasma relative to peptides present this reactivity causes insignificant bias.

Which enzyme deficiency is most commonly associated with familial hypertriglyceridemia associated with fasting plasma cholomicrons (formerly type I hyperlipoproteinemia)?

Post-heparin-activated lipoprotein lipase deficiency Deficiency of capillary endothelial lipase is the most common cause of fasting chylomicronemia. This lipase is also known as post-heparin- activated lipase and apo C-II-activated lipase. β Glucocerebrosidase deficiency results in accumulation of glucocerebrosides and is the cause of Gaucher's disease. ApoC-II deficiency results in decreased activity of peripheral and hepatic lipases and is associated with hypertriglyceridemia. Apo-B deficiency resulting from a point mutation in the apo-B gene, is responsible for hypobetalipoproteinemia, and is inherited as an autosomal dominant trait. LDL levels are about half normal in heterozygotes, and this reduces their risk of coronary artery disease.

Which statement best describes immunofixation electrophoresis (IEF)?

Proteins are separated by electrophoresis followed by overlay of monospecific anti-immunoglobulins Immunofixation electrophoresis (IFE) is used to identify monoclonal bands in serum or urine. Electrophoresis is performed on the serum or urine sample in the same manner as for protein electrophoresis, except that six lanes are used for the same sample. After the proteins are separated, a different monospecific antiserum is applied across the surface of each lane. After incubating, the gel is washed and blotted to remove uncomplexed proteins and salts. The immune complexes that remain are stained. Monoclonal bands will be seen only in those lanes where the monoclonal immunoglobulins were recognized by the corresponding antiserum.

What is the clinical utility of testing for serum prealbumin?

Serial low levels indicate compromised nutritional status Prealbumin (also called transthyretin) is a small protein with a half-life of only 2 days. Serum levels fall rapidly in patients with deficient protein nutrition. As a result, prealbumin is used to detect malnutrition and to measure the patient's response to dietary supplementation. The cutpoint used to identify nutritional deficiency in elderly patients is usually 11 mg/dL. Prealbumin is usually measured by immunonephelometry.

Which test is the most sensitive in detecting early monoclonal gammopathies?

Serum-free light chain immunoassay Immunonephelometric free light chain assays can detect monoclonal protein production before the mass is sufficient to cause a monoclonal spike on protein electrophoresis or capillary electrophoresis, but will be positive only in cases where monoclonal light chain production occurs. Therefore, measurement of free light chains is recommended along with protein electrophoresis when testing for myeloma. Free light chains are normally present in serum because L chains are made at a faster rate than H chains. However, in cases where free L chains are the result of monoclonal plasma cell proliferation, the kappa:lambda ratio will be abnormal in addition to one of the L chain types being elevated.

Which of the following reagents is used in the direct HDL cholesterol method?

Sulfated cyclodextrin The direct HDL cholesterol method most commonly employed uses cholesterol esterase and oxidase enzymes conjugated to polyethylene glycol. In the presence of sulfated cyclodextrin, the enzymes do not react with non-HDL cholesterol molecules. Anti-apoA-I binds to HDL and is not used in HDL assays.

Kjeldahl's procedure for total protein is based upon the premise that:

The nitrogen content of proteins is constant Kjeldahl's method measures the nitrogen content of proteins as ammonium ion by back titration following oxidation of proteins by sulfuric acid and heat. It assumes that proteins average 16% nitrogen by weight. Protein in grams per deciliter is calculated by multiplying protein nitrogen by 6.25. The Kjeldahl method is a reference method for total protein that is used to assign a protein assay value to calibrators.

Which of the following factors is most likely to cause a falsely low result when using the BCG dye-binding assay for albumin?

The presence of penicillin BCG and BCP are not significantly affected by bilirubin or hemolysis, although negative interference caused by free Hgb has been reported with some BCG methods. Lipemic samples may cause positive interference, which can be eliminated by serum blanking. Incubation times as long as 2 minutes result in positive interference from globulins, which react with the dye. Penicillin and some other anionic drugs bind to albumin at the same site as the dye, causing falsely low results.

Which of the following statements is correct?

There are several subfractions of both HDL and LDL There are 7 subfractions of LDL and 10 subfractions of HDL. These are grouped into subclasses defined by their molecular sizes. In general, the small, dense LDL subclasses contain more oxidized LDL and are more atherogenic than the larger LDL molecules. The larger HDL subfractions comprising the HDL-3 subclass are associated with a lower risk of coronary artery disease.

An EDTA blood sample is collected from a nonfasting person for a CBC. The physician collected the sample from the femoral vein because venipuncture from the arm was unsuccessful. He called the lab 15 minutes after the sample arrived and requested a lipid study including triglyceride, total cholesterol, HDL cholesterol, and LDL cholesterol. Which test results should be used to evaluate the patient's risk for coronary artery disease?

Total cholesterol and HDL cholesterol NCEP recommends a 12-hour fasting sample when screening persons for risk of coronary artery disease. However, if a fasting sample is unavailable, NCEP recommends performing the total cholesterol and HDL cholesterol because these tests are least affected by recent ingestion of food. If the total cholesterol is ≥ 200 mg/dL or the HDL cholesterol is < 40 mg/dL, then testing for LDL cholesterol and triglycerides should be performed when a fasting sample can be obtained. An EDTA plasma sample is acceptable for most enzymatic cholesterol and triglyceride assays.

A person has a fasting triglyceride level of 240 mg/dL. The physician wishes to know the patient's non-HDL cholesterol level. What cholesterol fractions should be measured?

Total cholesterol and HDL cholesterol When the HDL cholesterol is subtracted from the total cholesterol, the result is called the non-HDL cholesterol. This result, the sum of LDL cholesterol and VLDL cholesterol, represents the fraction with atherogenic remnant lipoproteins as well as LDL cholesterol. People who have a fasting triglyceride ≥ 200 mg/dL are at increased risk for coronary artery disease owning to atherogenic VLDL remnants, and the treatment goal is to have a non-HDL cholesterol no more than 30 mg/dL greater than the LDL cholesterol.

Which of the following statements regarding proteins is true?

Total protein and albumin are about 10% higher in ambulatory patients Water pools in the vascular bed in nonambulatory patients, lowering the total protein, albumin, hematocrit, and calcium. Plasma levels of total protein are 0.2-0.4 g/dL higher than serum (about 5%) owing to fibrinogen. Cerebrospinal fluid albumin levels are normally 10-30 mg/dL, which is approximately two-thirds of the CSF total protein. Transudates have a total protein below 3.0 g/dL and less than 50% of the serum total protein.

Which of the following proteins migrates in the β region at pH 8.6?

Transferrin Transferrin, β lipoprotein, C3, and C4 are the dominant proteins in the β-globulin region. Haptoglobin and α2- macroglobulin are the principal proteins in the α2-fraction. α1-Antitrypsin, α1-lipoprotein, and α1-acid glycoprotein (orosomucoprotein) make up most of the α1-fraction. Immunoglobulins dominate the γ region. Plasma is not used for protein electrophoresis because fibrinogen will produce a band resembling a small monoclonal protein in the beta region.

Which of the following methods for HDL cholesterol is the reference method?

Ultracentrifugation Ultracentrifugation of plasma in a potassium bromide solution with a density of 1.063 is used to separate HDL from LDL and VLDL. The HDL fraction is transferred from the bottom of the tube and assayed for cholesterol content by the Abell-Kendall method. The remaining three methods rely upon selective precipitation of lipoproteins containing apoprotein B using a polyanionic solution. All of these methods are subject to interference by very high triglycerides and vary somewhat in specificity depending on the efficiency of precipitation.

Select the lipoprotein fraction that carries most of the endogenous triglycerides.

VLDL The VLDL is formed in the liver largely from chylomicron remnants and hepatic-derived triglycerides. Therefore, the VLDL transports the majority of endogenous triglycerides, while the triglycerides of chylomicrons are derived entirely from dietary absorption.

Following ultracentrifugation of plasma, which fraction correlates with pre-β lipoprotein?

Very low-density lipoprotein (VLDL) The VLDL (very low-density lipoprotein) migrates in the pre-β zone. The VLDL is about 50% triglyceride, whereas LDL is only 10% triglyceride by weight. LDL is formed from VLDL in the circulation. The process is initiated by apoC-II on VLDL activating peripheral lipoprotein lipase. Hydrolysis of triglycerides and transfer of apoproteins from VLDL to HDL result in formation of IDL. Larger IDLs are returned to the liver as remnant lipoproteins. Further hydrolysis of triglycerides, transfer cholesterol esters from HDL, and transfer of apoproteins to HDL convert IDL to LDL.

Which of the following statements regarding the identification of monoclonal proteins by IFE is true?

When testing for a monoclonal gammopathy, both serum and urine must be examined Quantitation of IgG, IgA, IgM, or IgD indicates the concentration of each class of immunoglobulin but does not distinguish monoclonal from polyclonal gammopathies. Monoclonal characteristics are determined by demonstrating restricted electrophoretic mobility, indicating that all immunoglobulins in the band are of the same amino acid sequence. Monoclonal light chains can be demonstrated in about 60% of monoclonal gammopathies. In up to 25% of multiple myeloma patients, a heavy chain gene deletion results in production of monoclonal light chains only. Because these are filtered by the glomerulus, the procedure must be performed on urine as well as serum. Some patients with a monoclonal protein fail to develop malignant plasma cell proliferation. This state is called a monoclonal gammopathy of undetermined significance (MGUS). Within 10-15 years, 15%-20% of persons with MGUS develop some form of lymphoproliferative disease.

The electrophoretic pattern shown in the following densitometric tracing most likely indicates:

α1-Antitrypsin deficiency This pattern shows a marked decrease in the α1-globulin (slightly less than one-fifth of the expected peak area). Staining of the α1-globulin fraction is predominately determined by the α1-antitrypsin level. A value of less than 20% of normal (0.2-0.4 g/dL) is usually caused by homozygous α -antitrypsin deficiency. There is a slight decrease in albumin and increase in the α2-fraction. Patients with α1-antitrypsin deficiency often display elevations in the α2-globulin and γ-globulin fraction because the condition is associated with chronic emphysema and hepatic cirrhosis.