Atherosclerosis is one of the leading causes of heart disease and its presence is an important risk factor for events leading to acute myocardial infarction (AMI). In the past, atherosclerosis was described as a cholesterol and lipid storage event. Now we know it is a chronic inflammatory disorder of the arterial vessels with lipid components. Atherosclerosis begins with damage to the cells that line the blood vessels. Some possible causes of this cell injury are bacterial infection, hyperlipidemia, hypertension, glycosylated products of diabetes, cytokines from adipose tissue, or exposure to toxins such as pollution and second-hand smoke. Monocytes and lymphocytes adhere to the injured site; macrophages enter and ingest proteins and, along with modified lipoproteins, create foam cells. An inflammatory milieu results as cytokines and other inflammatory molecules become involved; foam cells and white blood cells begin secreting cytokines and metalloproteinases. Myeloperoxidase is also released by degranulated white blood cells and macrophages. As inflammation and accumulation of these products continues, fatty dots and streaks are formed on the vessel lining and the formation of plaque begins. As the atherosclerotic process continues, involved cells proliferate forming a complex extracellular matrix and a fibrous cap. If development continues, possibly over decades, the plaque formations are distributed throughout various vessels, become calcified or collagenized and make the vessel walls rigid. The risk to patients with significant atherosclerosis is that eventually a narrowing of the artery (stenosis) can cause a reduction in oxygen delivery to tissues and plaque rupture can lead to an acute coronary event.
Even though atherosclerosis is primarily a chronic inflammatory process, lipids are involved in atherosclerotic plaque formation. Lipoproteins are components of the foam cells that eventually develop into plaque if the inflammation in blood vessels continues. Lipids are transported in circulatory system in complexes composed of lipids, phospholipids, and protein. Cholesterol and triglyceride are the primary lipids transported in lipoproteins. There are four major lipoproteins: High-Density Lipoprotein (HDL) Low-Density Lipoprotein (LDL) Very-Low Density Lipoprotein (VLDL) Chylomicron (CM)
In the past, an AMI was primarily diagnosed by evaluating symptoms at patient presentation, ECG measurement, and results of enzyme assays that were considered cardiac enzymes. The enzymes, creatine kinase (CK), lactate dehydrogenase (LD), and aspartate aminotransferase (AST) were assayed several times a day often for several days to observe peak concentration and return to normal level for each enzyme. The first assay result was the baseline level or baseline concentration. Isoenzymes of CK and LD were later added for AMI diagnosis. All three of these enzymes are found in other tissues, making the diagnosis difficult and lengthy. In the 1980s, CK isoenzyme, CK-MB, though not totally cardiac specific, became the benchmark marker for an AMI. None of these enzymes are in any of the current recommendations except for CK-MBCurrent diagnosis, monitoring, and screening relating to heart disease includes measurement of lipids, proteins, enzymes, and other biomolecules. Risk stratification for cardiac and vascular disease is an additional role for measurement of these analytes. The physiological changes in the development of heart disease are better understood and the role of the clinical laboratory is greatly expanded.Today's markers are significant because of their location in the myocyte, the kinetics of their release in myocyte damage, and their rate of clearance from peripheral blood.
Currently, the troponins are the definitive markers of myocyte injury. The troponins are contractile proteins that regulate muscle contractions; they work with calcium ions and another protein, tropomyosin. There are three types of troponin: Troponin C -- Calcium-Binding Component Troponin I -- Inhibitory Component Troponin T -- Tropomyosin-Binding ComponentIsoforms of the troponins are found in cardiac muscle and in slow-twitch and fast-twitch skeletal muscle. Troponin C is found in both cardiac and skeletal muscle in the same form. Troponin I has a specific form found in cardiac muscle, cTnI; cTnT is the cardiac specific form of troponin T.
cTnT is also encoded by a separate gene and after translation, an 11-amino acid chain is added giving this protein its cardiac specificity. Its release characteristics and elevation periods are similar to cTnI. In some AMI patients, a second increase in analyte concentration occurs. It is believed that there are two pools of cTnT, cystolic and structurally bound pools. The first elevation is from the cystolic pool and the second elevation is from the structurally bound pool released later in the necrotic process.
|Use of Troponins|
Both cTnI and cTnT are cardiac specific, rapidly released after injury, remain in circulation for several days, normally in low concentration in serum or plasma, and can be rapidly assayed at relatively low cost.Currently cTnI and cTnT are considered the best markers in diagnosing ACS. Either protein is assayed to detect an AMI or other myocardial injury. These markers are especially helpful when the patient with chest pain and symptoms of an AMI does not have a diagnostic ECG. Cardiac troponin levels are used in risk stratification for a patient with chest pain that is not diagnosed with an AMI at presentation. Elevations of cardiac troponins are especially significant when other markers are normal. These elevations predict higher risk of severe cardiac events in the coming month. In other patients with ACS, troponin elevations identify those who are at risk for cardiac events for up to six months.
|Troponin Measurement and Ranges|
Rapid immunoassays provide concentration levels of cTnI and cTnT that are approximately 96% sensitive and 94% specific for cardiac injury.Each diagnostic company develops their unique antibody against epitopes on the proteins. There is only one assay available for cTnT. However, there are several different antibodies that are used by manufacturers to detect cTnI. Consequently, different assay methods may not correlate well. Standardization is needed for intra-laboratory comparisons. Reference Ranges for an adult: cTnT <0.01 ng/mL No cardiac injurycTnI references may vary with different assay methods, but approximate these values: Equal to or < 0.03 ng/mL -- No detectable cardiac injury 0.04-0.49 ng/mL -- Cardiac muscle injury Equal to or > 0.5 ng/mL-- Myocardial infarction
Myoglobin can also be used as a diagnostic indicator of an AMI. Myoglobin is an oxygen-binding protein in cardiac and skeletal muscle. It is released earlier after muscle injury than cardiac troponins and CK-MB and returns to normal faster than either of these other markers. It rises within 2 - 4 hours after chest pain, peaks in 6 - 12 hours, and is usually normal within 24 - 36 hours.Because of myoglobin's increase after skeletal muscle injury, it lacks the needed specificity for diagnosis of ACS and an AMI. False-positive elevation of myoglobin may also occur in a patient with impaired renal function since myoglobin is cleared through the kidneys.Myoglobin reference ranges for adults when an immunoassay method is used are approximately: Male 17-106 ng/mL Female 1-66 ng/mL Variation in ranges may be seen with different measurement methods.
|BNP Versus NT-ProBNP|
BNP, as an active protein hormone, has a short biological half-life of 20 minutes. NT-ProBNP, inactive fragment, has a half-life of 1-2 hours. Both are increased in CHF and both levels correlate to the severity of condition. In the future these two hormones may be used as prognostic indicators in ACS also; studies have shown that levels predict cardiac mortality and adverse cardiac events in patients with ACS.Because of its longer half-life, there are advantages to measuring NT-ProBNP over BNP. In CHF patients receiving exogenous and synthetic BNP for treatment, BNP levels may be affected while NT-ProBNP will not be affected. NT-ProBNP is thought to be primarily cleared by kidneys and therefore falsely elevated in severe renal disease.
Previously, screening for cardiovascular disease (CVD) focused on hyperlipidemia, obesity, and hypertension. However, approximately one half of AMIs occur in healthy men and women with normal or only slightly elevated plasma lipids. With new insights into cardiac disease and the ACS, novel biomarkers such as inflammatory markers, hormones, and other biomolecules indicating myocardial stress are required. Some new screening markers are in use today and many more are in study and evaluation for future use. New screening markers for CVD and ACS are: Highly Sensitive C-Reactive Protein (hs-CRP) Homocysteine Ischemial Modified Albumin (IMA) Myeloperoxidase (MPO)
|High Sensitivity C-Reactive Protein (hs-CRP)|
C-reactive protein (CRP) is an acute-phase protein produced by the liver in response to injury or tissue damage. It has been assayed for many years as a non-specific marker of acute inflammatory diseases, infections, neoplastic diseases, and other conditions where inflammation occurs. It is still assayed in this manner as a marker of inflammation by immunoassay methods that are sensitive to concentrations of 5-20 mg/L. Atherosclerosis is a subclinical chronic inflammatory condition. Highly sensitive measurements of CRP have been developed to detect this protein in lower levels that are sensitive to 0.5-10.0 mg/L. This assay is referred to as high sensitivity C-reactive protein (hs-CRP).
|Future Cardiac Biomarkers|
There are a large number of other molecules being evaluated and studied to determine their use as reliable cardiac biomarkers. Future research will demonstrate if they provide additional helpful information in ACS and if they are helpful in screening for CVD in asymptomatic individuals. Some future markers under study are lipoprotein(a), oxidized LDL, metalloproteinases, lipoprotein-associated phospholipase A2, pregnancy-associated plasma protein A, and placental growth factor. With advances in molecular diagnostics and proteomics, an individual's plasma proteome, their fingerprint of proteins and peptides, may be a biomarker profile of current and future disease.
|Which biomarkers of cardiac disease risk are inflammatory markers?||View Page|
Arneson W, Brickell J, eds.Clinical Chemistry: A Laboratory Perspective. Philadelphia: FA Davis; 2007.Burtis CA, Ashwoood ER, Burns DE, eds. Tietz Fundamentals of Clinical Chemistry. 6th ed. St. Louis, MO: Elsevier Saunders; 2008.Carreiro-Lewandowski E. Update on cardiac markers. Lab Med. 2006;37:597-605.D'Amore PJ. Evolution of C-reactive protein as a cardiac risk factor. Lab Med. 2005;36:234-238.Dotsenko O, Chackathayil J, Lip GY. Cardiac biomarkers:myths, facts and future horizons. Expert Review of Molecular Diagnostics. 2007;7:693-697.Foley, K. BNP: a novel biomarker. Advance for Medical Laboratory Professionals. August 25, 2008:9.Kaplan LA, Pesce AJ, Kazmierczak SC, eds. Clinical Chemistry Theory, Analysis, Correlation. St. Louis, MO: Mosby Elsevier Science; 2007. McDowell J. Reviewing the evidence for BNP, NT-proBNP testing. Clin Lab News. 2006;32:1, 3, 5.Rollins G. cTn and MI - what's the diagnosis? Clin Lab News. 2009;35:1, 3-4.Rollins, G. The BNP debate. Clin Lab News. 2009;35:1,3-4.Schreiber D, Miller SM. Use of cardiac markers in the emergency department. eMedicine. Updated July 2009. Available at: http://emedicine.medscape.com/article/811905-print.Accessed March 25, 2013. Thygesen K, Alpert JS, White HD. Universal definition of myocardial infarction. Circulation 2007;116:2634-2653.
Most S. pneumoniae strains gain penicillin resistance by altering the penicillin-binding proteins in their cell wall. Penicillin molecules that cannot find a penicillin binding site cannot interfere with cell wall synthesis. Several different types of penicillin binding proteins may be involved, explaining the various levels of intermediate resistance that may be encountered with different strains of S. pneumoniae. Because different penicillin binding proteins may be involved, the level of penicillin resistance cannot be predicted by the oxacillin screening test. Infections caused by isolates of S. pneumoniae showing penicillin resistance in the intermediate range may be successfully treated by administering high doses of antibiotic. For this reason, the level of resistance with an accurate minimum inhibitory concentration (MIC) test must be determined for all clinically significant isolates of S. pneumoniae.
|Middle ear damage in cases of S. pneumoniae infections are caused primarily by the: (Choose all that apply)||View Page|
Francois P. Vaudaux P. Foster TJ. Lew DP.: Host-bacteria interactions in foreign body infections. Infection Control & Hospital Epidemiology. 17:514-20, 1996 Persistent staphylococcal infections are a major medical problem, especially when they occur on implanted materials or intravascular catheters. This review describes some of the recently discovered molecular mechanisms of Staphylococcus aureus attachment to host proteins coating biomedical implants. These interactions involve specific surface proteins, called bacterial adhesins, that recognize specific domains of host proteins deposited on indwelling devices, such as fibronectin, fibrinogen, or fibrin. Elucidation of molecular mechanisms of S. aureus adhesion to the different host proteins may lead to the development of specific inhibitors blocking attachment of S. aureus, which may decrease the risk of bacterial colonization of indwelling devices.
|Decreasing the risk of staphylococcal colonization of indwelling catheters in the future may involve:||View Page|
Cunningham MW.: Pathogenesis of group A streptococcal infections. Clinical Microbiology Reviews. 13):470-511, 2000 Group A streptococci are model extracellular gram-positive pathogens responsible for pharyngitis, impetigo, rheumatic fever, and acute glomerulonephritis. A resurgence of invasive streptococcal diseases and rheumatic fever has appeared in outbreaks over the past 10 years, with a predominant M1 serotype as well as others identified with the outbreaks. Emm (M protein) gene sequencing has changed serotyping, and new virulence genes and new virulence regulatory networks have been defined. The emm gene superfamily has expanded to include antiphagocytic molecules and immunoglobulin-binding proteins with common structural features. At least nine superantigens have been characterized, all of which may contribute to toxic streptococcal syndrome. An emerging theme is the dichotomy between skin and throat strains in their epidemiology and genetic makeup. Eleven adhesions have been reported, and surface plasmin-binding proteins have been defined. The strong resistance of the group A streptococcus to phagocytosis is related to factor H and fibrinogen binding by M protein and to disarming complement component C5a by the C5a peptidase. Molecular mimicry appears to play a role in autoimmune mechanisms involved in rheumatic fever, while nephritis strain-associated proteins may lead to immune-mediated acute glomerulonephritis. Vaccine strategies have focused on recombinant M protein and C5a peptidase vaccines, and mucosal vaccine delivery systems are under investigation.
|What are the factors related to the strong resistance of certain strains of group A streptococci to phagocytosis? (Choose all that apply)||View Page|
Rouquette C. Berche P. The pathogenesis of infection by Listeria monocytogenes Microbiologia. 12:245-58, 1996 Listeria monocytogenes is a Gram-positive bacterium responsible for severe infections in human and a large variety of animal species. It is a facultative intracellular pathogen which invades macrophages and most tissue cells of infected hosts where it can proliferate. The molecular basis of this intracellular parasitism has been to a large extent elucidated. The virulence factors, including internalin, listeriolysin O, phospholipases and a bacterial surface protein, ActA, are encoded by chromosomal genes organized in operons. Following internalisation into host cells, the bacteria escape from the phagosomal compartment and enter the cytoplasm. They then spread from cell to cell by a process involving actin polymerisation. In infected hosts, the bacteria cross the intestinal wall at Peyer's patches to invade the mesenteric lymph nodes and the blood. The main target organ is the liver, where the bacteria multiply inside hepatocytes. Early recruitment of polymorphonuclear cells lead to hepatocyte lysis, and thereby bacterial release This causes prolonged septicaemia, particularly in immunocompromised hosts, thus exposing the placenta and brain to infection. The prognosis of listeriosis depends on the severity of meningoencephalitis, due to the elective location of foci of infection in the brain stem (rhombencephalitis). Despite bactericidal antibiotic therapy, the overall mortality is still high (25 to 30%).
|Chemical Urinalysis Reagent Strips|
A chemical urinaylsis reagent strip, also called a dipstick, for screening urine is a narrow band of paper which has been saturated with chemical indicators for specific substances or properties. Depending on the product being used, chemical urinalysis reagent strips may include test indicators for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocyte esterase. The results obtained from urine screening using chemical urinalysis strips can indicate the patient's carbohydrate metabolism status, kidney and liver function, urinary tract infection, and acid-base balance. Most chemical urinalysis reagent strips can be read visually and do not require instrumentation for automatic reading, though many laboratories utilize instruments for this purpose. When performing chemical urinalysis reagent strip analysis, the directions must be performed exactly. Accurate timing is paramount in order to achieve appropriate and optimal results. In addition, the reagent strips must be stored properly in their containers with the lid tightly closed to maintain reagent reactivity. It is always essential to utilize well-mixed urine which has been collected within 2 hours of analysis.Always read the package insert for your particular brand of chemical urinalysis reagent strip, as each manufacturer may have slightly different instructions and interpretations.
|Urine pH: Acidic and Alkaline|
Urine pH results must be evaluated in conjunction with a patient's medical condition and clinical history. Factors to be considered include:Respiratory and metabolic statusRenal functionCrystal or calculi formationDietThe table below summarizes dietary and medical conditions as well as preanalytic and analytic errors that may affect urine pH:ConditionAcid pHAlkaline pHHigh meat dietXVegetarian dietXRespiratory/metabolic acidosisXRespiratory/metabolic alkalosisXHypochloridemiaXHigh concentration of urine glucoseXBacterial infection caused by urease-producing bacteriaXProlonged storage of specimen at room temperature, allowing multiplication of urease-producing bacteriaX (above 8.0)Improper procedural technique; excess urine left on reagent strip, allowing acid buffer in protein pad to run over into adjacent pH pad (refers to some reagent strip configurations)XKidney failureXUrinary tract infectionsXVomitingXDiabetic ketoacidosis XDiarrheaXStarvationX
|Protein Error of Indicators|
Testing for protein in the urine is based on the phenomenon called the "Protein Error of Indicators" (ability of protein to alter the color of some acid-base indicators without altering the pH). In a solution void of protein, tetrabromphenol blue, buffered at a pH of 3, is yellow. However, in the presence of protein, particularly albumin, the color changes to green, then blue, depending upon the concentration. This method is more sensitive to albumin than to globulin, detecting as little as 5 mg albumin/dL urine. Bence Jones protein and mucoprotein are examples of globulin components that are sometimes present in urine, but are not distinguishable by the chemical reagent strip method for urine protein. False positive results can occur when testing for urine protein. A urine specimen that has remained at room temperature for an extended period of time may produce a false-positive protein result on a reagent strip. A false positive may also occur in the presence of bacterial contamination, alkaline medication, quaternary ammonium compounds such as disinfectants or drugs, and with skin cleansers containing chlorhexidine.
|Follow-up Testing for Positive Urine Protein|
A 24-hour urine protein may be ordered if a large amount of protein is detected with the dipstick method or if protein persists in the urine. A 24-hour urine protein may also be ordered if the physician suspects the release into the urine of protein other than albumin.
|Confirmatory Testing for Urine Protein|
Semiquantitative tests are used in some laboratories to confirm the presence of protein in a specimen when the result is positive on the urine chemical reagent strip. Tests that are used for confirmation include: sulfosalicylic acid (SSA), heat and acetic acid, nitric acid ring test, and Roberts' Ring Test. Any one of these procedures may be used for confirmation of the presence of protein. NOTE: A protein result on a urine chemical reagent strip that is greater than a trace may be an indication of proteinuria.
|Clinical Significance of Urine Protein|
The presence of an increased amount of protein in a urine specimen is often the first indicator of renal disease. Proteinuria may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the proteinuria. Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dL. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.
|Clinical Significance of Urine Protein (continued)|
Individuals with diabetes mellitus may excrete small amounts of albumin in the urine (microalbumin) which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. Both type I and type II diabetes mellitus are leading causes of renal failure. Microvascular damage caused by excessive renal exposure to glucose can lead to diabetic nephropathy. By the time the urine protein level reaches the 30 mg/dL level that is necessary for detection by routine reagent strips, damage to the kidneys may have already occurred. Reagent strips are available that use a dye-binding technique rather than the traditional protein-error of indicators principle. These strips are more sensitive and specific for albumin, detecting levels as low as 8 mg/dL.Women in the last month of pregnancy may develop proteinuria as the first sign of impending eclampsia. Eclampsia is the gravest form of toxemia of pregnancy. The presence of protein in this situation must be evaluated by the physician in conjunction with other clinical symptoms.Benign transient proteinuria may be the result of: exposure to cold, strenuous exercise, dehydration, and/or high fever. Benign transient proteinuria may also occur during the acute phase of a severe illness. Patients over the age of 60 have a greater chance of having protein in their urine. Occult malignancies and glomerulonephritis, that occur more frequently in the elderly, may be signaled by the presence of proteinuria. Orthostatic proteinuria is a condition seen most often in young adults. The condition may be caused by pressure on the renal nerve. When this condition is suspected, two urine specimens are tested. One specimen is collected upon arising in the morning, and the second is collected several hours later. When this condition is present, the first morning specimen, after the patient has been in a supine position, will be negative for protein. The second specimen, taken after the patient has been upright for several hours, would be positive for protein.
|The presence of increased levels of protein in the urine may be an early indicator of which of the following conditions?||View Page|
|Examples of conditions resulting in benign proteinuria include: (Choose ALL correct answers)||View Page|
|A copper reduction method (e.g. Clinitest® or Benedict's) is performed on pediatric specimens in order to check for the presence of:||View Page|
|False Positive and Negative Results|
False Positives:A false positive result for blood on the urine chemical reagent strip can occur when oxidizing contaminants, such as hypochlorite (bleach), remain in collection bottles after cleaning. Contamination of the urine with provodine-iodine, a strong oxidizing agent, used in surgical procedures can also result in a false positive reaction. Microbial peroxide found in association with urinary tract infections may also cause false-positive results. Capoten® (Captopril) can cause decreased reactivity.The muscle tissue form of hemoglobin, myoglobin is a well-known cause of false-positive reactions on the blood portion of the reagent strip. When tissue hemoglobin is present, the urine specimen has a clear red appearance. Patients suffering from muscle-wasting disorders or muscular destruction due to trauma, prolonged coma, or convulsions or individuals engaging in extensive exertion may have myoglobin in their urine. Specific tests for myoglobin, such as immunodiffusion techniques or protein electrophoresis, are needed to confirm the presence of this substance in a urine specimen. Levels of ascorbic acid normally found in urine do not interfere with this test. False Negatives:False negative results may occur in some analysis methods when the concentration of ascorbic acid is greater than 5 mg/dL. The sensitivity of the blood portion of the test strip is decreased in specimens with a high specific gravity and increased protein. High levels of nitrites may delay the reaction, causing a false negative to be reported. If the pH of a urine sample is below 5, hemolysis of red cells as part of the test reaction is inhibited which results in a false negative reaction. An improperly mixed specimen may test negative if the red blood cells are in the sediment.
|False Positive and Negative Urine Leukocyte Esterase|
False Positives:A false positive result may occur in the presence of strong oxidizing agents in the collection container. In random urine specimens from women, a positive result for leukocyte esterase may be due to a source external to the urinary tract. Other urine sediment findings such as bacteria, squamous or renal epithelial cells, lymphocytes or red blood cells do not contain esterases, and would not produce a positive leukocyte esterase test. False Negatives:False negative results may occur in the presence of significant levels of protein or glucose and in urines with high specific gravity which can crenate the white blood cells, leaving them unable to release esterases. Some drugs such as Cephalexin (Kelfex®), Cephalothin Keflin®), Tetracycline, or high concentrations of oxalic acid may also cause falsely decrease leukocyte esterase test results.
|Clinical Significance of Leukocyte Esterase in Urine|
Using the esterase test in conjunction with pH, protein and nitrite provides a combination of tests which can screen for the presence of bacterial infections in the urinary system.
|Advantages and Limitations of the Chemical Reagent Strip Method for Specific Gravity|
Specific gravity (SG) measured with the chemical reagent strip method correlates well with gravimetric measurement, and, unlike the gravimetric or refractometer methods, does not need to be corrected for glucose or protein. Cloudy/turbid urines do not need to be clarified before measuring specific gravity with the reagent strip method. It is the recommended method for determining specific gravity if a urine specimen contains x-ray contrast media or plasma expanders. Alkaline urine can affect the indicator system and lower the specific gravity result on the reagent pad. If the result is being read visually, it is recommended that .005 be added to the specific gravity result when the pH is alkaline. Most chemical reagent strip readers, however, will automatically adjust the specific gravity reading for pH. A specific gravity reading higher than the chemical reagent strip range would need to be measured by another method, and may require dilution.
|False-positive tests for protein on a urine reagent strip may be caused by: (Choose ALL of the correct answers)||View Page|
|The reagent strip method for protein primarily tests for which type of protein?||View Page|
|Which of the following reagent strip tests is based on the Ehrlich- aldehyde reaction.||View Page|
|Sulfosalicylic acid can be used to confirm the result of which of the following tests included on a urine reagent strip?||View Page|
The urine specimen should be freshly voided. Urine is an ideal medium for the proliferation of bacteria due to the large amount of urea present. These bacteria metabolize urea, producing ammonia that causes the urine pH to become more alkaline. If there is a delay before performance of the test, the sample should be refrigerated.
|Protein Error of Indicators|
Testing for protein is based on the phenomenon called the "Protein Error of Indicators" (ability of protein to alter the color of some acid-base indicators without altering the pH). In a solution void of protein, tetrabromphenol blue, buffered at a pH of 3, is yellow. However, in the presence of protein (albumin), the color changes to green, then blue, depending upon the concentration. This method is more sensitive to albumin than to globulin, detecting as little as 5 mg albumin/dL urine. Bence Jones protein and mucoprotein are examples of globulin components that are sometimes present in urine, but are not distinguishable by the dipstick method for protein.
|False Positive Protein Results|
A urine specimen that has remained at room temperature for an extended period of time may produce a false-positive protein result on a reagent strip. A false positive may also occur in the presence of bacterial contamination, alkaline medication, or quaternary ammonium compounds such as disinfectants or drugs, and with skin cleansers containing chlorhexidine.
|Follow-up Testing of Urine Dipstick Protein Results|
A 24-hour urine protein may be ordered if a large amount of protein is detected with the dipstick method or if protein persists in the urine. A 24-hour urine protein may also be ordered if the physician suspects the release into the urine of protein other than albumin.
|Confirmatory Testing for Protein|
Semiquantitative tests are used in some laboratories to confirm the presence of protein in the specimen when the result is positive on the urine dipstick. Tests that are used for confirmation include: sulfosalicylic acid (SSA); heat and acetic acid; nitric acid ring test; and Roberts' Ring Test. Any one of these procedures may be used for confirmation of the presence of protein. A protein dipstick result that is greater than a trace may be an indication of proteinuria.
The presence of protein in a urine specimen can have serious implications. It may signal severe kidney damage, be a warning of impending kidney involvement, or be transient and unrelated to the renal system. Further quantitative testing of urine for protein may be needed to determine the significance of the proteinuria.
|Clinical Significance cont'd|
Proteinuria related to kidney impairment may be due to glomerular membrane damage caused by toxic agents, immune complexes found in lupus erythematosus, or streptococcal glomerulonephritis. The amount of protein present in urine samples from patients with glomerular damage usually ranges from 10-40 mg/dl. If the urinary protein is due to a disorder that affects tubular reabsorption, the urine protein quantities will be much greater. In patients with multiple myeloma, proteinuria is due to the excretion of the Bence Jones protein. This low molecular weight protein produced by a malignant clone of plasma cells circulates in the blood and is filtered in the kidneys in quantities exceeding the tubular capacity. This excess protein is excreted in the urine.
|Clinical Significance cont'd|
Individuals with diabetes mellitus may excrete small amounts of protein in the urine which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. Women in the last month of pregnancy may develop proteinuria as the first sign of impending eclampsia. Eclampsia is the gravest form of toxemia of pregnancy. The presence of protein in this situation must be evaluated by the physician in conjunction with other clinical symptoms.Benign transient proteinuria may be the result of: exposure to cold, strenuous exercise, dehydration, and/or high fever. Benign transient proteinuria may also occur during the acute phase of a severe illness.
|Clinical Significance cont'd|
Patients over the age of 60 have a greater chance of having protein in their urine. Occult malignancies and glomerulonephritis, that occur more frequently in the elderly, may be signaled by the presence of proteinuria. Orthostatic proteinuria is a condition seen most often in young adults. The condition may be caused by pressure on the renal nerve. When this condition is suspected, two urine specimens are tested. One specimen is collected upon arising in the morning, and the second is collected several hours later. When this condition is present, the first morning specimen, after the patient has been in a supine position, will be negative for protein. The second specimen, taken after the patient has been upright for several hours, would be positive for protein.
|The ability of proteins to alter the color of tetrabromphenol blue without altering the pH is an application of:||View Page|
|A copper reduction method (e.g. Clinitest® or Benedict's) is performed on pediatric specimens in order to check for the presence of:||View Page|
|False Positive Results|
A false positive result for blood on the reagent strip can occur when oxidizing contaminants, such as hypochlorite (bleach), remain in collection bottles after cleaning. Contamination of the urine with provodine-iodine, a strong oxidizing agent, used in surgical procedures can result in a false positive reaction. Microbial peroxide found in association with urinary tract infections may also cause false-positive results. Capoten® (Captopril) can cause decreased reactivity. The muscle tissue form of hemoglobin, myoglobin is a well-known cause of false-positive reactions on the blood portion of the reagent strip. When tissue hemoglobin is present, the urine specimen has a clear red appearance. Patients suffering from muscle-wasting disorders or muscular destruction due to trauma, prolonged coma, or convulsions or individuals engaging in extensive exertion may have myoglobin in their urine. Specific tests for myoglobin, such as immunodiffusion techniques or protein electrophoresis, are needed to confirm the presence of this substance in a urine specimen. Levels of ascorbic acid normally found in urine do not interfere with this test.
|False Negative Results|
False negative results may occur with some methods when the concentration of ascorbic acid is greater than 5 mg/dL. The sensitivity of the blood portion of the test strip is decreased in specimens with a high specific gravity and increased protein. High levels of nitrites may delay the reaction, causing a false negative to be reported. If the pH of a urine sample is below 5, hemolysis of red cells as part of the test reaction is inhibited which results in a false negative reaction. An improperly mixed specimen may test negative if the red blood cells are in the sediment.
False negative results may occur in the presence of significant levels of protein or glucose and in urines with high specific gravity which may crenate the white blood cells causing them to be come unable to release esterases. Some drugs such as Cephalexin (Kelfex®), Cephalothin Keflin®) or high concentrations of oxalic acid may also cause decreased test results. Tetracycline may cause decreased activity, and high levels of the drug may cause a false negative reaction. Large amounts of ascorbate may cause false negative results.
Using the esterase test in conjunction with pH, protein and nitrite provides a combination of tests which can screen for the presence of bacterial infection.
|False-negative results on reagent strips for leukocytes may occur when the specimen contains: (Choose ALL of the correct answers)||View Page|
|Advantages and Limitations of the Reagent Strip Method for Specific Gravity|
Specific gravity (SG) measured with the reagent strip method correlates well with gravimetric measurement, and, unlike the gravimetric or refractometer methods, does not need to be corrected for glucose or protein. Cloudy/turbid urines do not need to be clarified before measuring SG with the reagent strip method. It is the recommended method for determining SG if a urine specimen contains x-ray contrast media or plasma expanders. Alkaline urine can affect the indicator system and lower the SG result on the reagent pad. If the result is being read visually, it is recommended that .005 be added to the SG result when the pH is alkaline. Most dipstick readers, however, will automatically adjust the SG reading for pH. A SG reading higher than the reagent strip range would need to be measured by another method, and may require dilution.
|Which of the following tests confirms the presence of Bence-Jones proteinuria:||View Page|
|Which of the following methods is not a quantitative method for the determination of
|Which of the following thyroid function assays is affected least by pregnancy:||View Page|
|Which of the following conditions will not produce a characteristic protein electrophoresis pattern:||View Page|
|Elevation in CSF total protein may be seen in all of the following conditions except:||View Page|
|Which of the following is found in plasma but absent in serum:||View Page|
|The migration rate of proteins on cellulose acetate is primarily the result of:||View Page|
|In a normal CSF the protein concentration as compared to that in the serum is generally:||View Page|
|Which of the following methods would be used to confirm the presence of Bence-Jones
protein in the urine:||View Page|
|Label this lipoprotein electrophoresis scan:
Ch = Cholesterol,
Tr = Triglycerides,
Pr = Protein,
Ph = Phospholipid.||View Page|
|Chylomicrons are primarily composed of:||View Page|
|This SPE scan most likely represents which of the following disease states:||View Page|
|This serum protein electrophoresis scan most likely represents which condition?||View Page|
|Lipemia in a serum sample is most likely caused by an increase in serum levels of:||View Page|
|Which band on the following serum protein electrophoresis scan is not made up of a mixture of proteins:||View Page|
|Which of the following tests would be used in the assessment of glomerular filtration:||View Page|
|Bence-Jones proteinuria can be seen in all of the following conditions except:||View Page|
|In a patient with acute glomerulonephritis you would expect to find all but the following
in the urine except:||View Page|
|Which of the following methods may be employed to definitively identify Bence-Jones proteins:||View Page|
|Which of the following would be the most appropriate method to confirm a positive
protein from a urine dipstick:||View Page|
|Diseases Associated with Proteinuria|
Normal urine contains very little protein, usually less than 10 mg/dL. The majority of the protein that is found in normal urine is albumin. The presence of an increased amount of protein in the urine (proteinuria) can be an indicator of renal disease. The two mechanisms that can lead to proteinuria are glomerular damage or a defect in the reabsorption process of the tubules in the nephron. The concentration of protein in the urine is not necessarily indicative of the severity of renal disease. Severe proteinuria (greater than 3.5 g/day) is characteristically seen in patients with glomerulonephritis, lupus nephritis, lipoid nephrosis, and severe venous congestion of the kidney. Moderate proteinuria (0.5-3.5g/day) is seen in nephrosclerosis, multiple myeloma, diabetes nephropathy, malignant hypertension, and pyelonephritis with hypertension. Mild proteinuria (less than 0.5 g/day) may be seen with polycystic kidneys, chronic pyelonephritis, benign orthostatic proteinuria, and some renal tubular diseases. Transient proteinuria can also be due to physiologic conditions such as stress, exercise, cold exposure, and fever, in the absence of renal disease.
|Screening and Secondary Tests for Protein|
The reagent strip protein method is based on the principle of "protein-error-of-indicators." It produces a visible colorimetric reaction that is capable of detecting most instances of proteinuria. Falsely elevated results can occur if the urine sample is visibly bloody.At one time, sulfosalicylic acid (SSA) was used to confirm all positive protein reagent strip results. However, this no longer routinely done. SSA is a precipitation method that reacts with all forms of protein. False-positives can occur. Any substance that is precipitated by acid will produce false-positive SSA results, including radiographic dyes, cephalosporins, penicillins, and sulfonamides. SSA may be used as a secondary protein detection method if the urine is highly colored so that the colorimetric reaction is masked on the reagent strip.
|Sulfosalicylic Acid Test (Exton's Modification)|
There are several acids which can be used to precipitate proteins - sulfosalicylic, trichloroacetic, nitric, and acetic acids. Sulfosalicylic acid (SSA) is the most frequently used acid test because it does not require the use of heat. Exton's reagent is 5% sulfosalicylic acid in a solution of sodium sulfate. Exton (1925) found that adding sodium sulfate to the SSA causes a more uniform precipitate to be formed. To perform the SSA procedure mix equal parts of patient urine and the reagent. Rate the amount of turbidity according to the following scale:
|Persons with type 1 or type 2 diabetes should periodically have their urine monitored for which of the following to avert renal complications related to diabetes?||View Page|
|The screening test most commonly used to detect Bence-Jones protein in urine is:||View Page|
|Which of the following conditions may require the use of the sulfosalicylic acid (SSA) test to detect protein as part of a routine urinalysis?||View Page|
|Albumin is the main serum protein found in normal urine.||View Page|
|A routine reagent strip test for protein is based on the principle of:||View Page|
|Heat and Acid Test for Urinary Protein||View Page|
Normally, small amounts of conjugated bilirubin, regurgitate back from the bile duct and enter the blood stream, so small amounts of conjugated bilirubin can be found in the plasma. Since conjugated bilirubin is not bound to protein, it is easily filtered through the glomerulus and excreted in the urine whenever the plasma level is increased. Normally, no detectable amount of bilirubin (sometimes referred to as "bile") is found in the urine.
Serum and plasma are the most common clinical specimens used for electrophoresis applications. Urine and cerebrospinal fluids (CSF) are also suitable. Other body fluids such as pleural fluid and pericardial fluid are analyzed less frequently. 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.
|After reviewing the information on specimen samples for electrophoresis, select the one correct statement.||View Page|
|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.Types of electrophoresis that will be discussed are:Routine electrophoresisHigh resolution electrophoresisPolyacrylamide gel electrophoresisCapillary electrophoresisIsoelectric focusingImmunochemical electrophoresisTwo-dimensional electrophoresisPulsed field electrophoresis
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 and carboxyl groups are amphoteric. Nucleic acids (deoxyribonucleic acid or DNA and ribonucleic acid or RNA) are charged and thus are amphoteric.
|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. An amphoteric molecule such as a protein in a pH below its pI will have a net positive charge. Conversely, if the pH is above the pI, a protein will have a net negative charge.
|Which statement is correct for a protein with an isolectric point (pI) of 7.0?||View Page|
|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.||View Page|
|Buffers and pH|
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.
|Proteins in a buffer with the pH set at 8.6 will become anions and move to the positively charged electrode.||View Page|
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.
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.
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.A manual agarose gel electrophoresis of eight serum samples is pictured below. After electrophoresis, the gel was stained with Ponceau S.
|High Resolution Electrophoresis (HRE)|
High resolution electrophoresis (HRE) is routine electrophoresis using a high voltage. Serum samples separated with HRE may yield approximately fifteen distinct protein bands. Other HRE applications are the separation of CSF proteins for the diagnosis of multiple sclerosis and light chains in urine for early detection of lymphoproliferative disorders such as multiple myeloma. Both of these specimen separations require more resolution of proteins than routine protein electrophoresis can provide. Increasing the voltage will increase heat generated. To prevent denaturation of proteins, drying out of gels and other system components, a cooling system is included in HRE instrumentation.
|Polyacrylamide Electrophoresis (PAGE)|
More separations are also achieved with layers of polyacrylamide gels each with a different pore size. The gels can be horizontal or vertical slabs or incorporated into vertical cylinders or rods. Varying the pore size in each layer is significant especially if very small pore sizes are created. DNA of 100 base pairs (bp) or less can be separated.Common applications of PAGE are separation of proteins and nucleic acids. Polyacrylamide gels are also used as the medium in several other types of electrophoresis described in this section.
|Denaturing Polyacrylamide Gels|
Denaturing chemicals can be added to the acrylamides during formation of polyacrylamide gels. These additives keep the solutes or molecules in a denatured state during separation. Urea denatures double-stranded DNA to single-stranded DNA. A detergent, sodium dodecyl sulfate (SDS), denatures proteins. Adding SDS with heat denatures proteins to small, similar shaped particles and coats each so that protein structures are not reformed. SDS is usually added to the gel and the protein sample. Then the mixture of protein coated fragments moves through polyacrylamide gel pores with speed similar to a mixture of DNA fragments.
|IEF Advantages and Applications|
IEF's greatest advantage is its high resolution, resulting in greater separation of solutes. IEF of serum proteins results in many more bands; these bands are sharper because each pH region is very narrow. Performing IEF is easier because the placement of sample application is not important. The sample and ampholytes can be mixed before application; the ampholytes will migrate, create the gradient, and then the proteins separate and migrate.Some isoenzymes and variant hemoglobins in prenatal screening are separated with IEF. Detection of oligoclonal bands in gamma-globulin is a newer use of IEF. IEF is commonly used as one of the separations in two-dimensional electrophoresis.
|CE Advantages and Applications|
CE is very rapid, efficient, easily automated, computerized, and requires only a microvolume of sample. Again a cooling system is included in instrumentation to prevent problems associated with the heat generated as discussed previously. The resolving power of CE is another important advantage.Proteins can be separated with CE along with inorganic ions, amino acids, drugs, vitamins, carbohydrates, oligonucleotides, and DNA fragments.
Two-dimensional electrophresis is separating the same sample with two distinct separation techniques or two different electrophoresis separations. The separated bands from one electrophoresis are resolved more with the second electrophoresis. IEF followed by PAGE or AGE is the most frequent two-dimensional electrophoresis. The gel from the IEF capillary is removed and placed across the PAGE or AGE gel slab at right angles for the second electrophoresis. If PAGE is used for the second electrophoresis, it is often PAGE with SDS.Two-dimensional electrophoresis can also be a single sample run on either agarose or polyacrylamide gels. The gel is then turned 90 degrees and the same type electrophoresis is run on the separated solutes to separate each band from the first run into more bands.The image below shows a two-dimensional electrophoresis separation of proteins which is IEF followed by PAGE with SDS. The proteins were first separated by IEF on a very narrow gel strip. This strip was then positioned at top of a polyacrylamide gel with SDS for the second electrophoresis. The IEF gel is the very narrow strip on top and remainder of the image is the many separated proteins on the PAGE with SDS.
|Two-Dimensional Electrophoresis Advantages and Applications|
Because of the two separation processes, more information and separated solutes can be gained from a sample. The use of two-dimensional electrophoresis is specialized and most applications are in research fields. It is used to study families of proteins in the field of proteomics and protein content in different types of cells. It is also used extensively in genetics to study differences in diseases, gene mutations, and bacterial DNA. In an effort to find ways to detect malignancies earlier, two-dimensional electrophoresis is also used to study tumor cells.
There are several immunochemical electrophoresis methods used to investigate protein antigens and antibodies in serum. Two methods will be discussed: Immunofixation electrophoresis (IFE) Electroimmunoassay 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.
In electroimmunoassay electrophoresis, the antiserum is mixed in the gel during preparation. In the electrophoresis of the serum sample, the voltage drives the sample antigen into the antiserum creating a precipitin line in the shape of a rocket. This line is proportional to the concentration of the antigen, which is the protein to be detected. Each gel contains several serum samples, one antibody suspended in the gel, and standards of known concentration of antigen. Quantitation of the unknown antigen is derived from the height of the sample rockets compared to the height of the standard rockets. Electroimmunoassay electrophoresis is often referred to as rocket electrophoresis.
|Currently there has been a revitalization in the clinical usage of electrophoresis. Previously, methods were primarily used to separate proteins in blood and other body fluids. From the following statements, select the statements that correctly describe newer applications of electrophoresis.||View Page|
|Sodium dodecyl sulfate is added to polyacrylamide gels to denature the proteins in the sample and enhance their separation.||View Page|
|Stains and Dyes|
Substance Stain or DyeCommentsProteinsPonceau SCoomassie Brilliant BlueSilverSpecific for ProteinsSilver is a biohazardLipoproteinsSudan Black BOil Red O-EnzymesEnzyme substrate and achromagen or fluorescent dyeReaction catalyzed by enzyme and color or fluorescence detectedHemoglobinNot neededIs intensely coloredNucleic Acids (DNA/RNA)Ethidium Bromide (EtBr)SyBr GreenSilver EtBr is CarcinogenicSyBr Green is new - Introduced in 1995Silver is a biohazard
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.An integrator or microprocessor evaluates the area under each peak and reports each as a percent of the total sample. If the electrophoresis is for separation of serum proteins, the concentration of each band is derived from this percent and the total protein concentration. If the electrophoresis is for separation of enzymes, the enzyme activity of each band is derived from this percent and the total enzyme activity. 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. The densitometer scan from this electrophoresis shows five bands with two peaks in the beta band. Recall the order of protein fractions from left to right is: Albumin, alpha 1, alpha 2, beta, and gamma.
Minute-size fractions achieved in two-dimensional electrophoresis, IEF and PAGE with SDS, and bands from electrophoresis of nucleic acids are detected differently than protein electrophoresis fractions. Labeled polypeptide probes are used to detect these proteins; labeled single-stranded nucleic acid fragments are used for the detection of nucleic acids. Each probe is made with a label designed to generate a detectable signal. The label is bound to a probe and a system is created such that the signal is visualized when the probe is bound to the target.The most common labels are radioactive isotopes and fluorescence dyes. Chemiluminescence and color or ultraviolet absorbance are also used.
|Which statements below are correct descriptions of visualization and detection methods used in electrophoresis?||View Page|
With a pH 8.0-9.0 used for protein electrophoresis, proteins take on a negative charge, that is a negative ion cloud forms. As the negative ion cloud migrates to the anode, the proteins are pulled to the anode. Several gels used routinely for protein electrophoresis attract positive ions from the buffer and form a positive ion cloud. This ion cloud moves in the opposite direction to the cathode. This phenomenon is called electroendosmosis or endosmosis.The tension created by these oppositely moving ion clouds can affect the movement of sample macromolecules. The migration of some proteins can be slowed, some proteins can become immobile, and other proteins are pushed toward the cathode. Many protein electrophoresis methods take advantage of this tension and use it to achieve better separation of protein bands. The gamma globulin band in serum, urine, and other body fluids will separate more sharply by being pushed to the cathode and will appear behind the point of sample application.
|Which statements below are associated with electroendosmosis?||View Page|
|Wick flow is caused by movement of the buffer ions into the medium when there has been a loss of moisture in the medium due to heat generation.||View Page|
|Resurgence of Electrophoresis|
Traditionally, most clinical laboratory electrophoresis utilizes methods that separate and identify proteins in serum, urine, CSF, and some other body fluids. Most studies are designed to detect serum protein abnormalities and gathering more information about gammopathies.In recent years, there has been a resurgence in electrophoresis use and methods. Development of automated methods has enhanced this. The evolution of numerous molecular diagnostic investigations and research in proteomics have also augmented electrophoresis.Applications of two-dimensional electrophoresis discussed the use of electrophoresis in proteomics. Electrophoresis and molecular diagnostics, blotting techniques, and current uses of CE in molecular diagnostics will be discussed now.
Blotting techniques were developed to discriminate fragments of nucleic acids. These techniques involve several processes; electrophoresis is one of the processes and is used to separate fragments of DNA and RNA. In Southern blotting (named after Edward Southern) restriction enzymes cut fragments of DNA are separated by AGE or PAGE, transferred to a membrane or blot, and visualized by hybridization with labeled probes.Northern blotting (not named after an inventor but by analogy to Southern blotting) separates RNA. RNA molecules are shorter and have defined lengths; cutting by restriction enzymes is not required. Denaturing conditions are required because of RNA secondary structures. After membrane blotting, the separated types of RNA are visualized with staining or labeled probes.Western blotting (again not named after an inventor but by analogy to Southern blotting) does not separate nucleic acids; it separates proteins in a mixture. The proteins are usually separated with PAGE, transferred to the membrane and visualized with a labeled antibody against the proteins of interest.
|Transport of Lipophilic Substances|
Many lipophilic substances, including fat-soluble vitamins, cholesterol, and triglycerides are essential for life. The body needs to be able to absorb and transport these substances. However, lipophilic substances are not water-soluble, and, since blood is aqueous, this presents a challenge. The body addresses this need by using 'carriers' which can bind or sequester lipophilic molecules to aqueous 'vehicles' and thus transport them through the aqueous environment of the blood. Small lipid-soluble hormone molecules like estrogen, testosterone or cortisone are carried through the blood by binding to carrier proteins. Cholesterol and triglycerides are carried through the body in small spherical particles which trap the lipophilic molecules in their centers. These particles have an outer shell that is polar on the surface so that the particles are soluble in the blood but they have a lipophilic core which can hold fat-soluble molecules.
Lipoproteins differ in size and density as well as in their content (what they tend to carry). They also can differ in their origination (where they are made). Another significant difference between lipoprotein molecules is the proteins they have on their surfaces. These proteins, known as apolipoproteins, are the major identifying characteristics of a lipoprotein. There are many different apolipoproteins and we are continually learning more about them. Apolipoproteins have multiple roles. One role is that these amphipathic (detergent-like) proteins increase the overall solubility of the lipid particle, helping it to dissolve in the aqueous environment of the blood. Apolipoproteins can also function as enzyme co-factors (receptor ligands) and facilitate the transfer of their lipid cargo to specific cells. Thus, the apoliproteins are the smart or working-end of the lipoprotein particle. The apolipoproteins dictate where the particles will dock and where they can bind, and in so doing the apolipoproteins regulate lipid metabolism in the body.
|What are apolipoproteins?||View Page|
Lipoprotein (a) is a modified version of LDL containing a unique protein, apolipoprotein (a). It was discovered in 1963 and is well-associated with vascular disease. Do not confuse apolipoprotein (a) with apolipoprotein A that is found on high density lipoprotein particles. Lipoprotein (a) is abbreviated as Lp(a). Lp(a) is an LDL particle whose ApoB molecule has formed a disulfide bond with another protein called Apo(a), see figure. Apo(a) is a protein very similar in structure to plasminogen. Numerous retrospective case control studies and prospective studies have shown Lp(a) to be an independent risk factor for vascular disease. This means that Lp(a) levels alone (not in conjunction with LDL, or patient risk factors) can predict cardiovascular risk. Lp(a) has been called the most atherogenic lipoprotein. Serum concentrations of Lp(a) are related to genetic factors; drugs and diet changes do not typically lower Lp(a) as they do LDL.
One of the problems with Lp(a) measurement is that the Apo(a) protein has a variable mass. It can have a molecular weight ranging from 275,000 to 800,000 daltons. This is due to variable amounts of repeating regions of the protein. Immunoassay antibodies which recognize these regions will thus give more signal for larger Apo(a) molecules compared to smaller Apo(a) molecules. This is not ideal since again, we would prefer to quantify the number of particles and Lp(a) containing large Apo(a) molecules will produce more signal, skewing the count. One assay system that tries to correct for this is the Lp(a) Cholesterol Electrophoresis Assay sold by Helena Laboratories. This assay uses electrophoresis followed by cholesterol staining and densitometry to calculate the concentration of cholesterol in Lp(a). Although this method still does not enumerate particles, it does appear to have less heterogeneity.Lp(a) is an acute phase reactant. This means that Lp(a) levels will rise in the context of general inflammation. Thus, Lp(a) should not be measured when there is extensive inflammation, such as immediately following a cardiovascular event. Concentrations of Lp(a) above 30 mg/dL are associated with increased cardiovascular risk. The risk of having a cardiovascular event increases 2 to 3 fold if Lp(a) cholesterol is > 30 mg/dL. Fifteen to 20% of the Caucasian population have Lp(a) levels >30 mg/dL. Africans, or people of Aftican descent, generally have levels higher than Caucasians and Asians, however, results must be evaluated in conjunction with clinical history.
|High Sensitivity-C-Reactive Protein|
C-reactive protein (CRP) is a very sensitive acute phase reactant. Serum CRP levels increase following a variety of pro-inflammatory events such as infection, tissue necrosis, trauma, surgery and even malignancy. CRP levels can increase quickly and dramatically (often 100 fold) during inflammation. CRP can activate compliment, bind Fc receptors and can function as an opsonin, enhancing phagocytosis with certain infections. Measurement of CRP is not new, it has been on clinical laboratory testing menus for decades. However, a newer version of the CRP test is now in use to assess cardiovascular risk.High sensitivity-CRP (hs-CRP) assays have been developed that are more sensitive to the more subtle changes that can occur during chronic vascular inflammation. (Recall that atherosclerosis is an inflammatory process.) By measuring hsCRP we can get a glimpse at vascular function. CRP has been shown to be an independent risk factor for atherosclerotic disease and cardiac death. A 2002 prospective study of more than 27,000 patients showed that the CRP concentration is a stronger predictor of cardiovascular events than the LDL-cholesterol level.
|The hs-CRP Test|
The traditional CRP test uses immunoassay methods that are sensitive to concentrations of 5-20 mg/L. The hs-CRP test, with its increased sensitivity, is able to detect C-reactive protein in lower levels, 0.5-10.0 mg/L. As with most risk markers, the results of hs-CRP testing are generally interpreted on a relative scale; the higher the value, the higher the risk of a future cardiovascular event.The American Heart Association and Centers for Disease Control and Prevention has defined risk groups with hs-CRP as follows: Low risk: < 1.0 mg/L Average risk: 1.0 to 3.0 mg/L High risk: > 3.0 mg/L It is important to note that hs-CRP assays are measuring the same protein as traditional CRP assays. Thus, in patients with active inflammation (such as chronic, active arthritis; lupus; infection; etc.) hs-CRP values would be expected to be high and would not necessarily implicate cardiovascular risk. If values greater than 10 mg/L are seen in repeated measurements, a non-cardiovascular cause should be considered. Taking anti-inflammatory drugs (NSAIDs, aspirin, etc.) or the statin-class of cholesterol-lowering drugs may reduce CRP levels in patients. This is not an artifact, but is thought to be an effect of treating the underlying inflammatory process.
|Which of the following is FALSE concerning CRP or hs-CRP?||View Page|
Atherosclerosis. U.S. Department of Health & Human Services National Institutes of Health. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis_WhatIs.html Accessed March 25, 2013.Daniels LB, Barrett-Connor E, Sarno M, Laughlin GA,Bettencourt R, Wolfert RL. Lipoprotein-associated phospholipase A2 (Lp-PLA2) independently predicts incident coronary heart disease (CHD) in an apparently healthy older population: The Rancho Bernardo study. J Am Coll Cardiol. 2008;51:913-919.Executive Summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001; 285:2486-2497. Frostegard, J, Wu R, Lemne C, Thulin T, Witztum JL and de Faire U. Circulating oxidized low-density lipoprotein is increased in hypertension, Clin Sci 2003; 105, 615.Garza CA, Montoir VM, McConnell JP, et al. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc. 2007;82(2):159-165.Interpretive Handbook, (MC0440rev0407) Mayo Clinic, RochesterMN;2007. Maksimowicz-McKinnon K, Bhatt DL, Calabrese LH: Recent advances in vascular inflammation: C-reactive protein and other inflammatory biomarkers. Curr Opin Rheumatol. 2004;16:18-24.Mora S, Szklo M, Otvos JD, et al. LDL particle subclasses, LDL particle size, and carotid atherosclerosis in the multi-ethnic study of atherosclerosis. Atherosclerosis. 2007;192:211-217.NACB Laboratory Medicine Practice Guidelines. Emerging biomarkers of cardiovascular disease and stroke. NationalAcademy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006.PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed March 25, 2013.Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: BurtisCA, Ashwood ER. BrunsDE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Saunders: 2006; chap. 26.Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347:1557-1565.Sniderman AD. Differential response of cholesterol and particle measures of atherogenic lipoproteins to LDL-lowering therapy: Implications for clinical practice. J Clin Lipidol 2008;2:36-42.Tsimikas, S, Brilakis ES, Miller ER, et al. Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease, N Engl J Med: 2005;353:46.Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol. 2003; 41: 360.Tsimikas, S, Lau HK, Han KR, et al. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and long-term immunologic responses to oxidized low-density lipoprotein. Circulation. 2004;109, 3164.Tsimikas S, Witztum JL, Miller ER, Sasiela WJ, et al. High-dose atorvastatin reduces total plasma levels of oxidized phospholipids and immune complexes present on apolipoprotein B-100 in patients with acute coronary syndromes in the MIRACL trial, Circulation: 2004;110, 1406. Walldius G, Jungner I, Holme I, et al. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet. 2001;358:2026-2033.Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet. 2004;364:937-952.
|Oxidized LDL Tests|
There are currently two antibodies that have been developed that target oxidized lipids; the 4E6 antibody and EO6 antibody. The 4E6 antibody is directed against an oxidized epitope on the ApoB-100 protein on LDL. The EO6 antibody recognizes oxidized phospholipids and the assay measures the content of these oxidized phospholipids on lipid particles that contain ApoB-100.
Because hereditary hemochromatosis (HH) is a disease of iron overload, a review of the basic principles of iron metabolism is helpful in understanding its pathophysiology. Iron is needed by all body cells and is crucial for oxygen transport, oxidative metabolism, and cell growth and proliferation. To serve these functions, iron must be bound to protein. Iron is potentially harmful when ionized or complexed to inorganic compounds. Iron must be present in amounts sufficient to carry out these normal functions, but not in excessive amounts which may be toxic.Two types of iron-containing compounds are normally found in the body: compounds that serve in metabolic or enzymatic functions and storage compounds. Hemoglobin, myoglobin, cytochromes and other proteins are involved in oxygen transport and utilization. Iron in hemoglobin comprises about 67% of total body iron, thus erythrocytes are rich in iron. Approximately 27% of iron is found in storage compounds. Myoglobin, other tissue iron, and transport iron comprise the remaining 6% of total body iron. (2)
|What is a mobilizable, water-soluble form of storage iron that is bound to protein?||View Page|
|Regulation of Iron Equilibrium|
Regulation of iron equilibrium occurs mainly through the process of absorption. Iron is absorbed through the mucosal cells lining the duodenum. A variety of proteins are involved in this process. Hepcidin, an antimicrobial protein primarily produced in the liver, has been recently found to be a major (negative) regulator of dietary iron absorption by disrupting cellular iron transport in the intestine. Decreased levels of hepcidin are related to increased iron absorption into the bloodstream. Hepcidin is increased in response to iron overload and inflammation. (4)Additional proteins involved in iron metabolism include transferrin (Tf), transferrin receptor (TfR), ferroportin, HFE protein, hemojuvelin, and others. Their roles in iron absorption are complex and in some instances incompletely understood.Factors affecting iron absorption include: Tissue stores, e.g., decreased stored iron is associated with a decrease in hepcidin and increase in iron absorption. Rate of hematopoietic activity, e.g., an increased rate of erythropoiesis is associated with a decrease in hepcidin and an increase in iron absorption. Oxygen concentration in tissues, e.g., hypoxia decreases hepcidin and increases iron absorption, thereby promoting increased erythopoiesis. Dietary intake, including form of iron ingested, e.g., heme iron is more readily absorbed than non-heme forms of iron. Condition of GI tract mucosal cells Intraluminal factors, e.g. intestinal motility
|Which of the following is NOT considered to be an important protein regulator of iron metabolism?||View Page|
Once absorbed through the mucosal cells of the duodenum, iron is bound to a carrier plasma protein, transferrin (Tf), for movement to sites of utilization. Almost all iron in plasma is bound to Tf, and most Tf-bound iron is carried to the bone marrow to be incorporated into developing erythrocytes. Transferrin is normally about 20% to 40% saturated with iron. (5)Transferrin releases iron to specific transferrin receptors (TfRs) for movement into cells. Transferrin receptors are found on all cells, but are found in relatively high concentration in erythroid precursors, hepatocytes, and placental cells. When the capacity of plasma Tf to bind iron is exceeded, i.e., transferrin saturation (TS) is higher than normal, excess iron is taken up by hepatocytes and other cells. A brief summary of iron metabolism is illustrated.
|What is the protein that carries iron in the blood plasma?||View Page|
|HFE and Other Genes|
A hemochromatosis gene, HFE, was identified in 1996. Mutations in the HFE gene are found in the majority of patients diagnosed with hereditary hemochromatosis (HH). The locus for the gene is on the long arm of chromosome 6 where it codes for a membrane protein, HFE. The exact mechanism of the role of HFE protein in iron metabolism is incompletely understood. It is thought that HFE, along with a second protein, beta-2 microglobin, interacts with transferrin receptors (TfR) on cell membranes. This interaction supresses the affinity of transferrin for TfR, thus lowering the uptake of transferrin--and its attached iron--into the cell. Transferrin receptors have been found on the surface of a variety of cells, with the greatest concentration on cell membranes of intestinal cells, hepatocytes, and RBC precursors. In addition to HFE, HH is also associated with mutations in other genes involved in iron homeostasis, including hemojuvelin (HJV), TfR, hepcidin, and ferroportin. Hepcidin production is reduced in HH due to all of these genetic causes, with a resulting increase in iron absorption. Mutations in HFE are the most common cause of HH.
|Specific HFE Mutations|
Several mutations of the HFE gene have been described. The most common mutation in patients with hereditary hemochromatosis is the C282Y mutation. In the C282Y mutation, a base substitution leads to a change in the amino acid in position 282 from cysteine (C) to tyrosine (Y). The loss of the sulfhydryl-containing amino acid disrupts the tertiary structure of HFE so that it no longer binds to beta-2 microglobulin. Beta-2 microglobulin appears to act along with other proteins to chaperone the newly synthesized HFE out of the Golgi apparatus and to the cell surface where it can then bind to TfR. In the C282Y mutation, HFE remains in the Golgi, never making it to the cell surface. The result is that transferrin binding to TfR is enhanced and excessive amounts of iron enter the cells of the small intestine, liver, and other tissues. A second mutation, H63D, causes a histidine (H) residue in position 63 to be replaced by aspartic acid (D). The mechanism by which this mutation leads to increased iron uptake is less well understood when compared to the C282Y mutation. Unlike the C282Y mutation, the H63D mutation does not seem to affect the binding of beta-2 microglobulin and intracellular movement, since detectable concentrations of the mutated protein are found on cell membranes. Some researchers speculate that the H63D mutation affects the binding of proteins involved in iron regulation and uptake at the cell surface.A third mutation, S65C, leads to a serine-to-cysteine substitution in its associated protein. This mutation has been been found in some compound heterozygotes for C282Y or H63D, but is rarely associated with iron overload in HH.Additional mutations of HFE have been identified, but their clinical significance is unclear. Most laboratories performing molecular assays test for only the C282Y, H63D, and S65C mutations.
|How is the function of HFE protein altered in the C282Y mutation?||View Page|
|Transferrin and Total Iron Binding Capacity|
The test for transferrin (Tf) measures the concentration of the primary carrier protein for iron. Measuring total iron binding capacity (TIBC) is an indirect method of assessing transferrin and provides comparable information. The TIBC (or transferrin) are typically performed along with the SI. Taken together, these determinations are useful in the differential diagnosis of many disorders affecting iron metabolism, including hereditary hemochromatosis (HH) and iron deficiency anemia. Tf and TIBC are typically low-normal or decreased in HH and are increased in iron deficiency. Serum transferrin can be measured directly using immunochemical methods such as nephelometry and turbidimetry. TIBC is performed in a 2-step method by adding ferric iron to the specimen in sufficient quantity to completely fill all of the iron binding sites on transferrin. Excess, unbound iron is removed by adsorption with magnesium carbonate, alumina, or ion resin. The iron content of the saturated binding protein is then measured as described for SI. Serum is the specimen of choice for Tf and TIBC. TIBC is less subject than SI to day-to-day variation and other causes of variability.A typical reference interval for TIBC is 300 - 360 micrograms/dL.(2)
|The protein component that surrounds the genome is called a:||View Page|
|What is the function of the majority of HIV's genes?||View Page|
|Function of HIV Genes|
HIV consists of nine genes. Three of the genes provide genetic information for the capsid proteins, envelope proteins, and viral enzymes. The other six genes are regulatory genes, controlling functions such as uncoating of the HIV genome and the penetration of host cells.
Gene NumberAbbreviationGene Function1gagcapsid proteins2polviral enzymes3envenvelope proteins4vifregulatory gene5tatregulatory gene6vpuregulatory gene7nefregulatory gene8vprregulatory gene9revregulatory gene
|Basic Structural Components|
Human immunodeficiency virus (HIV) belongs to the Family Retroviridae and consists of two basic components: a core of ribonucleic acid (RNA), called the genome, and a protein component that surrounds the genome, called a capsid. The genome carries the genetic information of the virus, while the capsid gives the virus its shape and protects the genome. The HIV genome consists of three major genes: group specific antigens (Ags) or capsid proteins (gag); polymerase gene proteins: reverse transcriptase, protease, and integrase enzymes (pol); and envelope glycoproteins (env).The capsid is made up of subunits called capsomeres. Viral proteins are identified as either "gp" for glycoprotein or "p" for protein followed by the molecular weight in kilodaltons. For example, HIV-1 includes the envelope proteins gp160, gp120, and gp41; the gag core gene proteins, p55, p24, and p17; the polymerase gene proteins, p66, p51, and p31. HIV-2 proteins are similar to HIV-1 proteins. However, some of the proteins differ in molecular weight from those found in HIV-1.
|Spread of Infection (2)|
At this time an enzyme called protease, using enzymes and proteins from preliminary protein molecules, forms capsomere segments which unite to form an icosahedral capsid.The capsid then changes into a bullet-shaped capsid and surrounds the viral RNA.Next some of the host cell's membrane joins with the viral glycoproteins gp120 and gp41 to form the spikes.Last, part of the host cell's surface membrane encloses the virus and becomes the envelope.
|Spread of Infection (1)|
The proviral DNA provides genetic coding that instructs cellular enzymes to construct new HIV genomes, capsid proteins, and reverse transcriptase molecules.All of these are assembled near the edge of the host cell.
|Proteins Involved in Adsorption|
The joining of the HIV and the host cell involves a spike on the HIV envelope and a CD4 molecule on the T-lymphocyte, macrophage, or brain cell.The molecule on the HIV spike is called glycoprotein 120 or gp120. The "120" refers to the molecular weight of the glycoprotein.While the CD4 site is important in viral binding, there is evidence that there are other molecules called co-receptors also involved.These molecules are embedded in the membranes of T-lymphocytes, macrophages, and brain cells. In the T-lymphocyte the abbreviated name of the protein molecule is CXCR4.
|Penetration and Infection|
After penetration of the cell membrane by the gp41, the HIV capsid enters the cell's cytoplasm. Next, cellular enzymes strip away the capsid so that the HIV genome is released. Also stripped away are proteins p24 and p17. Protein 24 coats the HIV genome and protein 17 lines the inside of the capsid.
Armstrong C. Practice guidelines AHA and NHLBI review diagnosis and management of the metabolic syndrome. Am Fam Physician. 2006;74:891-1062.D'Amore PJ. Evolution of c-reactive protein as a cardiac risk factor. Lab Med. 2005;36:234-238.Devaraj, S, Swarbrick MM, Singh U et al. CRP and adiponectin and its oligomers in the metabolic syndrome evaluation of new laboratory-based biomarkers. Am J Clin Pathol. 2008;129:815-822.Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet. 2005;365:1415-1428.Expert Panel in Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (authors). Executive summary of the third report of the national cholesterol education program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III). JAMA.2001;285:2486-2497.Gade W, Gade J, Collins M et al. Failures of feedback: rush hour along the highway to obesity. Clin Lab Sci. 2010;23:39-50.Gade W, Gade J, Collins M et al. Beyond obesity: the diagnosis and pathophysiology of metabolic syndrome. Clin Lab Sci. 2010;23:51-61.Grundy SM. Does a diagnosis of metabolic syndrome have value in clinical practice? Am J Clin Nutr. 2006;83:1248-1251.Grundy SM, Brewer HB, Cleeman JI, et al. Definition of metabolic syndrome: report of the national heart, lung, and blood institute/american heart association conference on scientific issues related to definition. Circulation. 2004;109:433-438.Grundy SM, Cleeman JI, Daniels SR, et al. Diagnosis and management of the metabolic syndrome: An American Heart Association/National Heart, Lung, and Blood Institute scientific statement. Circulation. 2005;112:2735-2752.Grundy SM. Obesity, metabolic syndrome, and cardiovascular disease. J Clin Endocrinol Metab. 2004;89:2595-2600.Mathew B, Francis L, Kayalar A, et al. Obesity: effects on cardiovascular disease and its diagnosis. J Am Board Fam Med. 2008;21:562-568.Metabolic Syndrome. National Heart Lung and Blood Institute. Diseases and Conditions Index. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/ms/ms_whatis.html. Accessed December 5, 2011.Mittal S. The Metabolic Syndrome in Clinical Practice. London, England. Springer-Verlag Springer Science; 2008.Molinaro RJ. Metabolic syndrome: an update on prevalence, criteria, and laboratory testing. MLO. 2007;39:24-27.Ronti T, Lupattelli G, Mannarino E. The endocrine function of adipose tissue: an update. Clin Endocrinol. 2006;64:355-365.
IL-6 responds to tissue injury. IL-6 is synthesized and secreted by many different cells in addition to adipocytes including immune cells, fibroblasts, endothelial cells and skeletal muscle. IL-6 is increased in obesity and insulin resistance and those with elevated levels are at higher risk for type 2 diabetes and myocardial infarction. Similar to TNF-a, IL-6 increases NEFA release and reduces adiponectin secretion. IL-6 increases insulin resistance by inhibiting insulin receptor signal transduction in liver cells. It also increases other inflammatory cytokines, interleukin-1 (IL-1) and TNF-a, and stimulates the liver to produce C-reactive protein (CRP), an important protein marker of inflammation.
|Which adipokine is synthesized and released by adipocytes and many other body cell types, is an inflammatory cytokine that stimulates the liver to produce C-reactive protein (CRP), and is increased in obesity and insulin resistance?||View Page|
Insulin is a pancreatic hormone that plays a vital role in carbohydrate and lipid metabolism. Insulin regulates glucose concentrations by: Promoting glycolysis - the uptake of glucose by cells for energy Stimulating glycogenesis - the conversion of excess blood glucose to glycogen storage in the liver Inhibiting glycogenolysis - the conversion of glycogen back to glucose Inhibiting gluconeogenesis - the formation of glucose from noncarbohydrates Insulin increases lipid synthesis in the liver and fat cells and inhibits lipolysis, the release of non-esterified fatty acids (NEFAs) from triglycerides in fat and muscle cells. Insulin also promotes protein synthesis.If insulin resistance occurs, carbohydrate and lipid metabolism are impaired. Insulin resistance ordinarily results in increased insulin levels as the body senses a need for more insulin action. The impaired insulin action results in elevated plasma glucose levels. The increase in lipolysis increases blood concentrations of NEFAs and causes abnormal blood lipid levels.
According to the American Heart Association, the risk factors for metabolic syndrome include:Abdominal obesity (excessive fat tissue in and around the abdomen) Atherogenic dyslipidemia (blood fat disorders – high triglycerides, low HDL cholesterol and high LDL cholesterol – that foster plaque buildups in artery walls) Elevated blood pressure Insulin resistance or glucose intolerance (the body can't properly use insulin or blood sugar) Prothrombotic state (e.g., high fibrinogen or plasminogen activator inhibitor–1 in the blood) Proinflammatory state (e.g., elevated high sensitivity C-reactive protein in the blood) Reference: Metabolic syndrome.The American Heart Association website. Available at:http://www.heart.org/HEARTORG/Conditions/More/MetabolicSyndrome/Metabolic-Syndrome_UCM_002080_SubHomePage.jsp#. Accessed December 5, 2011.
|C-Reactive Protein (CRP)|
CRP is an important acute phase reactant protein synthesized and released by the liver. In obesity, increased secretion of cytokines results in increased stimulation of the liver and increased CRP. Elevated CRP levels are a marker of inflammation. In metabolic syndrome and obesity, CRP levels can be used to identify a proinflammatory state. Increased CRP levels are correlated with an increased risk for cardiovascular disease, particularly myocardial infarction and stroke.
Elevated CRP levels were a marker for non-specific inflammation and were used to monitor acute inflammatory diseases. Recently, highly sensitive measurements of CRP have been developed that detect this protein in lower levels. This measurement known as high sensitivity-CRP (hs-CRP) detects levels seen in chronic and non-acute inflammation. Hs-CRP levels are reported as low, moderate, or high risk for future cardiovascular disease. Hs-CRP concentrations > 3 mg/L indicate a proinflammatory state.
|Which of the following most likely causes the hypertension that often occurs in metabolic syndrome?||View Page|
Most drugs are bound to proteins when they circulate in the body. Albumin is a major drug-binding protein in serum. Albumin is an alkaline protein, so acidic and neutral drugs primarily bind to it. If albumin binding sites become saturated, acidic and neutral drugs can bind to lipoproteins. Alkaline drugs tend to bind to globulins, particularly to the globulin, alpha-1 acid glycoprotein. Only free, unbound drugs are able to bind drug receptors and have therapeutic effects. An equilibrium exists in the systemic circulation between a free and protein-bound drug and between a free and receptor-bound drug. This is illustrated in the image to the right.
|Other Factors Affecting Drug Absorption and Distribution|
In addition to protein availability, other factors may affect drug absorption and distribution in the body as a whole or at specific sites within the body. The following table highlights some of these other factors. Factor Discussion Regional blood flow Reduced area blood flow can be seen in diabetics and enhanced blood flow can be seen in tumors. Lipid solubility of the drug The more lipophilic a drug is, the more likely it will enter the central nervous system. The integrity of the GI tract In a diseased gut, an orally-administered drug may not be absorbed as expected. Age Drug kinetics and dispositions change throughout life. In general, metabolism of drugs is reduced in the elderly. Genetics Mutations or deletions in drug metabolizing enzymes can greatly affect a drug's disposition.
|Given what you have learned thus far, which of the following statements below do you think is true?||View Page|
Pharmacologists determine a drug's pharmacokinetic characteristics empirically during clinical drug trials. From these studies, they are able to determine the solubility and distribution, the average half-life, the levels of protein binding, and the effective concentrations needed for treatment.
TDM provides a quantitative measure of the circulating concentration of a drug. The physician determines if the dosage of the drug needs to be adjusted based on this information.If a drug concentration is determined to be outside the therapeutic range, it may be for one of the reasons listed in the table below. Reason Discussion Noncompliance Patients may (intentionally or unintentionally) not take the drug. TDM can thus help monitor compliance. Dosing errors The dose may have been erroneous or inappropriate given the patient's condition. Malabsorption The TDM result will reveal if the drug cannot be absorbed well through the gut and an alternative route of administration will be needed. Drug interactions Many drugs interfere with the absorption or metabolism of other drugs. These interactions will be revealed by TDM. Kidney or liver disease Any pathology that affects elimination will cause an elevation in a drug level that will be unmasked by TDM. Altered protein binding Changes in serum proteins can lead to big changes in the amount of free drug in serum. Variations in the genetics of drug-metabolizing enzymes can also affect drug concentrations in the body. This is the field of pharmacogenomics that will be discussed later in the course.
|TDM for all drugs?|
Can all drugs benefit from TDM? Not really. For TDM to be effective and useful, one or more of the following should apply: The effective concentration and toxic concentrations must be well-defined. The pharmacokinetics of the drug are known to be variable. The drug is given chronically. There is the potential for drug-to-drug interactions. The drug exhibits high protein binding. The toxicity will mimic the indication for the drug; toxicity may not be visible during an exam but will only be revealed with TDM. The patient is pregnant, very young, or elderly. Compliance or history with the drug is poor.
Immunoassay is the most common technique used by clinical laboratories for therapeutic drug monitoring. Antibodies that recognize drugs can be developed. Although most drugs are much too small to evoke an immune response, scientists can conjugate drugs to immunogenic proteins to produce antibodies that recognize drug-specific epitopes. There are several methods that utilize the principals of immunoassay for detection and quantification of therapeutic drugs in serum. Some of these methods are: Particle-enhanced turbidimetric inhibition immunoassay (PETINIA) Fluorescence Polarization Immunoassay (FPIA) Chemiluminescent assays
|Protein Availability and Drug Dosing|
Drug-binding proteins in serum can fluctuate in disease states. For example, if albumin levels fall, as can occur in liver failure or nephrotic syndrome, less albumin will be available for drug binding; a subsequent dose may produce a toxic concentration of free drug.The image on the right illustrates the loss of equilibrium between a protein-bound drug and a free drug when drug-binding proteins are diminished.Doses of drugs that are highly protein-bound may need to be adjusted in patients with lower drug-binding protein levels. Examples of some common drugs that are highly protein-bound include thyroxine, warfarin, diazepam, heparin, imipramine and phenytoin.
It has been said that we live in a new era of "individualized medicine." One of the primary drivers for this idea is the emerging field of pharmacogenomics (PGx). PGx is the study of how individual variations in the human genome affect responses to medications. The term "pharmacogenetics" is also used for this discipline (people in the field use both terms); however, the term 'pharmacogenomics' is becoming more popular since we now know the entire human genome. The primary reason that individuals metabolize and respond to drugs differently is the inter-individual differences in receptor proteins and enzymes that metabolize the drugs. Mutations in these receptor proteins and enzymes can give rise to very different responses to drugs. In PGx, these mutations are referred to as variants.
|Warfarin Metabolism, continued|
The genes involved in warfarin metabolism are CYP2C9 and vitamin K epoxide reductase complex subunit 1 (VKOR). Warfarin owes its anticoagulant action to its inhibition of VKOR. This enzyme recycles vitamin K, a critical element for the clotting factors II, VII, IX, and X, as well as for proteins C, S, and Z. There are six CYP2C9 alleles that are known to cause prolonged metabolism of warfarin: CYP2C9 *2, *3, *4, *5, *6, and *11. (Polymorphisms in CYP450 genes are denoted with asterisks.)One-third of the patients that receive warfarin metabolize it differently than expected and experience a higher risk of bleeding.Genetic testing for the two most common polymorphisms (CYP2C9*2 and *3) as well as for VKOR may be able to reduce the variability associated with warfarin dosing response. Labs performing PGx testing can provide general warfarin dosing recommendations based on the patient's genotype analysis. The lab report will indicate whether a patient has a normal, mild, moderate, high, or very high sensitivity to warfarin. For example, a patient who has one CYP2C9 normal wild-type allele (CYP2C9 *1), one polymorphism (CYP2C9*3), and also a VKOR polymorphism is predicted to have a moderate sensitivity to warfarin. This patient should have frequent INR monitoring and possible warfarin dose reduction. It is important to recognize that knowing a genotype does not necessarily guarantee accurate dose prediction; other drugs and/or environmental or disease factors can also alter CYP2C9 activity. Therefore, monitoring the INR is still very important.
CYP2D6 has received the most attention: It is estimated that about 25% of common drugs are metabolized by CYP2D6. CYP2D6 accounts for only about 1% of all CYP450 enzymes, but it is important in the metabolism of about 100 drugs. There are more than 80 genetic variants that have been described in the CYP2D6 gene. The normal, wild-type allele displays normal metabolic activity whereas some of the variant forms have enhanced or diminished activity. The variants can be grouped generally according to the resulting alterations in protein function. The groupings correlate with four major enzyme metabolic capacities (phenotypes): poor, intermediate, extensive (normal), or ultra-rapid metabolizers.
|Hepatic function panel|
Alkaline phosphatase (Alk Phos)
Total protein (TP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
|Comprehensive metabolic panel (CMP)|
Consists of a basic metabolic panel, plus:Albumin (Alb) and Bilirubin (Bili)
Alkaline Phosphatase (Alk Phos)
Total protein (TP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Blood clots when the coagulation factor proteins within the plasma are activated.Blood starts to clot almost immediately after it is drawn unless it is exposed to an anticoagulant.Clots within the blood specimen, even if not visible to the naked eye, will yield inaccurate results.
When a blood sample is left standing without anticoagulant, it forms a coagulum or blood clot.
The clot contains coagulation proteins, platelets, and entrapped red and white blood cells.
Plasma is the liquid portion of the blood. It contains many substances including:Water
Drugs & Toxins
Numerous types of proteins are dispersed in the plasma. These include:
Coagulation proteins (blood clotting factors), which, if activated, will form a blood clot , and
Serum proteins, which are left dispersed in liquid after the clot is formed. Serum proteins include:
Albumin, a marker of nutrition, and
Globulins, or antibodies.
|Whole blood: components|
Circulating whole blood is a mixture of:
Plasma (which contains fluid, proteins, and lipids), and
Formed elements, consisting of red cells, white cells, and platelets.
Serum is the fluid that is left over the coagulum after the specimen is centrifuged (spun down).
Serum contains all the same substances as plasma, except for the coagulation proteins, which are left behind in the blood clot.
Platelets are small cell fragments present in large numbers in blood.They work together with the blood coagulation proteins to form a blood clot.
|Which of the following macroscopic findings is MOST consistent with the microscopic finding of casts?||View Page|
|The patient was a female and the urinalysis was completed within two hours of collection. Which of the following findings are inconsistent with the rest of the report?||View Page|
|Formation and Significance of Casts|
Casts are cylindrical bodies formed either in the distal convoluted tubules or the collecting ducts of the kidney. Since the walls of the tubule act as a mold for cast formation, the width of the tubule determines the width of the cast. Thus, narrow casts are formed in the distal tubules while broad casts are formed in the collecting ducts.
The matrix of all casts is thought to be Tamm-Horsfall protein, a glycoprotein secreted by the distal loop of Henle and the distal tubule. This protein entraps cells and granular material of tubular origin.
Very few casts are seen in the urine of a person without renal disease, except for hyaline casts, which may be transiently present after strenuous exercise, and during fever, diuretic therapy, and congestive heart failure.
A significant number of urinary casts usually indicates the presence of renal disease.
Granular casts are composed of plasma protein aggregates and cellular remnants. Granular casts appear as irregularly-shaped cylinders of coarse, or fine, highly refractive particles. A granular cast containing coarse brown granules is indicated by the yellow arrow in the image on the right (brightfield, 400X magnification). A hyaline cast can be seen just to the left of the granular cast (blue arrow). The presence of an occasional granular cast is not considered pathologic.
|All of the following factors favor cast formation EXCEPT:||View Page|
|Factors Promoting Cast Formation|
The following factors promote the formation of casts in the kidney:Larger than normal amounts of plasma proteins entering the tubulesDecreased pHDecreased urinary flow rateIncreased urine concentrationAfter formation, casts are loosened from the tubules and discharged into the urine.
|The formed elements which may be present in urine sediment include: (Choose all that apply.)||View Page|
Cholesterol crystals may be seen in renal tubular disease. These crystals look like plates of glass, sometimes with a notch out of one corner. Under polarized light, they exhibit a stained glass effect. These crystals are rarely seen unless the specimen has been refrigerated, because the lipids remain in droplet form. Large amounts of protein, lipid droplets, fatty casts or oval fat bodies should be found along with cholesterol crystals. Cholesterol crystals are found in acid or neutral urine.
|Specimen #4 - Adult Male|
The results of this specimen are abnormal but the abnormalities correlate with each other. The turbidity can be explained by the presence of bacteria and crystals. The presence of RBCs in the microscopic explains the blood found on the dipstick. The casts, bacteria and WBCs can account for the increased protein. The results may be reported.