Harr MLS Review Chemistry 5.7 Enzymes and Cardiac Markers

The LD pleural fluid:serum ratio for a transudative fluid is usually:

1:2 or less The lactate dehydrogenase activity of body fluids is normally less than serum, and a fluid to serum LD ratio greater than 1:2 is highly suggestive of an exudative process. Elevated lactate dehydrogenase in chest fluid is often caused by lung malignancy, metastatic carcinoma, Hodgkin's disease, and leukemia.

How soon following acute abdominal pain due to pancreatitis is the serum amylase expected to rise?

2-12 hours Serum amylase usually peaks 2-12 hours following acute abdominal pain resulting from pancreatitis. Levels reach 2-6 times the URL and return to normal within 3-4 days. Urinary amylase peaks concurrently with serum but rises higher and remains elevated for up to 1 week.

Which of the following buffers is used in the IFCC recommended method for ALP?

2-Amino-2-methyl-1-propanol The Szasz modification of the Bowers-McComb method measures the hydrolysis of p-nitrophenyl phosphate, and continuously monitors the formation of p-nitrophenol at 405 nm. AMP buffer chelates phosphorus, preventing product inhibition; Zn+2 and Mg+2 are added to the substrate to activate ALP. HEDTA is used to chelate the excess Zn+2, which is inhibitory at high concentrations.

A patient's CK-MB is reported as 18 μg/L and the total CK as 560 IU/L. What is the CK relative index (CKI)?

3.2% The CKI is an expression of the percentage of the total CK that is attributed to CK-MB. CKI = [(CK-MB in μg/L)/(Total CK in IU/L)] × 100 The reference range is 0%-2.5%. Values above 2.5% point to an increase in CK-MB from cardiac muscle.

Which statement about enzymes is true?

A competitive inhibitor will alter the apparent Km of the reaction Enzymes alter the energy of activation by forming a metastable intermediate, the enzyme substrate complex. Enzymes do not alter the free energy or direction of a reaction. Competitive inhibitors bind to the active site where the enzyme binds substrate and are overcome by increasing the substrate concentration.

Which statement best describes the clinical utility of B-type natriuretic peptide (BNP)?

A normal test result (<100 pg/mL) helps rule out congestive heart failure in persons with symptoms associated with coronary insufficiency B-type natriuretic peptide is a hormone produced by the ventricles in response to increased intracardiac blood volume and hydrostatic pressure. It is formed in the heart from a precursor peptide (preproBNP) by enzymatic hydrolysis, first forming proBNP followed by BNP and NT (N-terminal) proBNP which is not physiologically active. Both BNP and NT-proBNP are increased in persons with congestive heart failure (CHF). Levels are not increased in pulmonary obstruction, hypertension, edema associated with renal insufficiency, and other conditions that cause physical limitation and symptoms that overlap CHF. At a cutoff of <100 pg/mL the BNP test is effective in ruling out CHF. Diagnostic accuracy in distinguishing CHF from nonCHF ranges from 83%-95%. In addition, persons with ischemia who have an increased BNP are at greater risk for MI. The NTpro-BNP assay is similar in clinical value, and can be used for persons being treated with nesiritide, a recombinant form of BNP used to treat CHF.

In which liver disease is the DeRitis ratio (ALT:AST) usually greater than 1.0?

Acute hepatitis ALT prevails over AST in hepatitis; however, AST is greater than ALT in carcinoma, alcoholic liver disease, and cirrhosis of the liver.

Which of the following statements regarding transaminases is true?

ALT is far more specific for liver diseases than is AST ALT is far more specific for liver disease than AST. High ALT may result from nonhepatic causes such as AMI, muscle injury or disease, and severe hemolysis, but nonhepatic sources can be ruled out by a high direct bilirubin. Elevated ALT (e.g., >65 IU/L) is used along with immunologic tests for hepatitis to disqualify blood donors. AST is increased in muscle disease, MI, pancreatitis, and lymphoma. Both transaminases are moderately increased in infectious mononucleosis.

Which statement accurately describes serum transaminase levels in AMI?

AST peaks 24-48 hours after an AMI and returns to normal within 4-6 days ALT may be slightly elevated after an AMI. AST levels can be up to 5-10 times the URL after AMI, but elevations of this range are also seen in patients with muscular dystrophy, crush injury, pulmonary embolism, infectious mononucleosis, and cancer of the liver.

What is the typical time course for plasma myoglobin following an AMI?

Abnormal within 2 hours; peaks within 12 hours; returns to normal in 36 hours After AMI, myoglobin usually rises above the cutoff within 1-2 hours, peaks within 8-12 hours and returns to normal within 36 hours. Typically, levels reach a peak concentration that is 10-fold the upper reference limit. Since myoglobin is the first marker to become abnormal after an AMI, it should be measured on admission and if negative, again 2 hours later. If both samples are below the cutoff, the probability of an AMI having occurred is low. If the myoglobin is above the cutoff, a cardiac specific marker such as TnI, or TnT must be performed at some point to confirm the diagnosis.

What is the typical time course for plasma TnI or TnT following an AMI?

Abnormal within 4 hours; peaks within 24 hours; returns to normal in 1 week Troponin is a complex of three polypeptides that function as a regulator of actin and tropomyosin. The three subunits are designated TnC, TnI, and TnT. All are present in both cardiac and some skeletal muscles, but cardiac and skeletal isoforms of TnI and TnT can be differentiated by specific antiseras. TnI and TnT cardiac isoforms in plasma will at least double within 4-6 hours after MI, peak within 24 hours and usually remain elevated for 7-10 days. TnT and TnI have the same sensitivity, but TnT is more commonly elevated in renal failure patients. Both are increased slightly in unstable angina (chest pain while at rest) and cardiac ischemia.

Which of the following statements regarding amylase methods is true?

Activity will vary depending on the method used Chloride and Ca2+ ions are required for amylase activity. Samples with high activity should be diluted with NaCl to prevent inactivation. Lipase and CK require sulfhydryl activators. Saccharogenic methods measure the production of glucose, while amyloclastic methods measure the degradation of starch. Starch is a polymer of α-D glucose subunits linked together by both α 1-4 and α 1-6 glycosidic bonds. Different lots may have more or less branching owing to the number of α 1-6 bonds. Since amylase hydrolyzes at the α 1-4 sites only, the amount of product measured is influenced by the extent of branching.

In which condition is the LD most likely to be within normal limits?

Acute appendicitis LD is increased slightly to moderately in most causes of liver disease. Smallest elevations are seen in obstructive jaundice and highest in hepatic carcinoma and toxic hepatitis, where levels can reach 10-fold the upper reference limit. LD is also increased in crush injury and muscular dystrophies due to skeletal muscle damage, and in pulmonary infarction owing to embolism formation. Amylase is increased in a majority of persons with acute appendicitis, but LD is not.

Which condition gives rise to the highest serum level of transaminases?

Acute hepatitis The transaminases usually reach 20-50 times the URL in acute viral and toxic hepatitis. Both transaminases are moderately increased (5-10 × URL) in infectious mononucleosis, toxic hepatitis, diffuse intrahepatic obstruction, lymphoma, and cancer of the liver, and slightly increased (2-5 × URL) in cirrhosis and extrahepatic obstruction

Which of the following is the most effective serial sampling time for ruling out AMI using both myoglobin and a cardiac specific marker in an emergency department environment?

Admission, 3 hours, 6 hours, and a final sample within 12 hours Since the time between the onset of symptoms and arrival in the emergency department is often speculative, serial measurement of cardiac markers is required in order to rule out AMI. Since myoglobin is the first marker to rise after AMI, if used it should be measured on admission. Since TnI, TnT, and CK-MB are more cardiac specific, at least one should be measured starting at 3 hours postadmission, and again at 6 hours postadmission. If all results are negative to this point, a final assay should be performed 6-12 hours postadmission to conclusively rule out the possibility of AMI and evaluate the short-term risk of AMI.

Which of the following laboratory tests is a marker for ischemic heart disease?

Albumin cobalt binding When heart muscle suffers reversible damage as a result of oxygen deprivation, free radicals are released from the cells and bind to circulating albumin. The albumin is modified at the N-terminus, causing a reduced ability to bind certain metals. This ischemia-modified albumin can be measured by its inability to bind cobalt. An excess of cobalt is incubated with plasma followed by addition of dithiothreitol. The sulfhydryl compound complexes with the free cobalt, forming a colored complex. The absorbance of the reaction mixture is directly proportional to the ischemia-modified albumin concentration. In addition to ischemia-modified albumin, glycogen phosphorylase-BB is a marker for ischemia because it is released from heart muscle during an ischemic episode. Myosin light chains and fatty acid-binding protein are released from necrotic heart tissue in the early stages of AMI.

Which of the following enzymes is considered most tissue specific?

Alcohol dehydrogenase (ADH) No enzyme is truly tissue specific and diagnostic accuracy depends upon recognizing changes in plasma levels that characterize different diseases. This includes the mass or activity of enzyme released, its rise, peak, and return to normal, the isoenzyme(s) released, and the concomitant changes of other enzymes. Alanine aminotransferase and alcohol dehydrogenase are primarily increased in necrotic liver disease.

Which enzyme is most likely to be elevated in the plasma of a person suffering from a muscle wasting disorder?

Aldolase 5'-Nucleotidase is increased primarily in obstructive liver disease and liver cancer. When elevated along with ALP, it identifies the liver as the source of ALP. Glutamate dehydrogenase is increased in necrotic liver diseases along with transaminases, but because of its distribution it is elevated to a greater extent in toxic hepatitis and therefore is useful as a marker for halothane (anesthesia) toxicity. Aldolase is found in all tissues and is increased in many conditions including myocardial infarction, viral hepatitis, and myelocytic leukemia. However, like CK, the greatest increase is seen in skeletal muscle-wasting disease such as muscular dystrophies.

Which of the following enzymes is activated by calcium ions?

Amylase Most enzymes require metals as activators or cofactors. CK and ALP require Mg+2 for full activity, and amylase requires Ca+2. Metals required for activity should be components of the substrate used for enzyme analysis. The substrate must also contain anions required (e.g., Cl- for amylase) and should not contain inhibiting cations or anions (e.g., Zn+2 and Mn+2 for CK).

SITUATION: A specimen for CK performed on an automated analyzer using an optimized Oliver-Rosalki method gives an error flag indicating substrate depletion. The sample is diluted 1:2 and 1:4 by the serial dilution technique and reassured. After correcting for the dilution, the results are as follows: 1:2 dilution = 3,000 IU/L 1:4 dilution = 3.600 IU/L Dilutions are made a second time and assayed again but give identical results. What is the most likely explanation?

An endogenous competitive inhibitor is present in the serum When a competitive inhibitor is present in the serum, a dilution of the sample will cause an increase in the reaction rate by reducing the concentration of the inhibitor. Dilution of serum frequently increases the activity of CK and amylase. The same effect will occur when a smaller volume of serum is used in the assay because less inhibitor will be present in the reaction mixture.

In a nonmyocardial as opposed to a myocardial cause of an increased serum or plasma CK-MB, which would be expected?

An increase in CK-MB that is persistent Plasma CK-MB becomes abnormal 4 hours postinfarction, peaks in 16-20 hours, and usually returns to normal within 48 hours. In some noncardiac causes of elevated plasma CK-MB such as muscular dystrophy, there is a persistent elevation of both total CK and CK-MB. TnI and TnT are cardiac-specific markers. They become elevated slightly before CK-MB when a CK-MB URL of 4 μg/L is used, remain elevated for 7-10 days following an AMI, and are not increased in muscular dystrophy, malignant hyperthermia, or crush injuries that are associated with an increase in the concentration of CK-MB. Absolute CK-MB increases are evaluated cautiously, when CK-MB is less than 2.5% of total enzyme because noncardiac sources may be responsible.

Which of the following statements is true?

Apoenzyme + prosthetic group = holoenzyme A coenzyme is an organic molecule required for full enzyme activity. A prosthetic group is a coenzyme that is tightly bound to the apoenzyme and is required for activity. Cofactors are inorganic atoms or molecules needed for full catalytic activity. Pyridoxyl- 5 ́-phosphate is a prosthetic group for ALT and AST. Consequently, patients with low levels of pyridoxal- 5 ́-phosphate (P-5 ́-P) (vitamin B6 deficiency) may have reduced transaminase activity in vitro. Enzymes can have diverse pH (and temperature) optimas.

Which of the following amylase substrates is recommended by the IFCC?

Blocked maltohepatoside Amylase is commonly measured using synthetic substrates. In the IFCC-recommended method, p-nitrophenyl maltohepatiside is used. One end of the polymer is covalently linked to p-nitrophenol and the other is linked to 4,6 ethylidine to prevent its hydrolysis by α-glucosidase. Amylase hydrolyzes the substrate from both ends producing fragments of 2, 3, and 4 glucose subunits. α-Glucosidase hydrolyzes the subunits containing p-nitrophenyl groups, forming glucose and p-nitrophenol. The increase absorbance at 405 nm is proportional to amylase activity.

A serum ALP level greater than twice the elevation of GGT suggests:

Bone disease or malignancy In obstructive jaundice, GGT is elevated more than ALP. A disproportionate increase in ALP points to a nonhepatic source of ALP, often bone disease. GGT is the most sensitive marker of acute alcoholic hepatitis, rising about fivefold higher than ALP or transaminases.

Which of the following statements about the aminotransferases (AST and ALT) is true?

Both transfer an amino group to α-ketoglutarate ALT catalyzes the transfer of an amino group from alanine, a three-carbon amino acid, to α-ketoglutarate (2-oxoglutarate), forming pyruvate. AST catalyzes the transfer of an amino group from aspartate (four carbons) to α-ketoglutarate, forming oxaloacetate. The reactions are highly reversible and regulate the flow of aspartate into the urea cycle. Both transaminases require P-5'-P as an intermediate amino acceptor (coenzyme). Cytoplasmic and mitochondrial isoenzymes are produced but are not differentiated in clinical practice.

Which of the following enzymes is a transferase?

CK Enzymes are identified by a numeric system called the EC (Enzyme Commission) number. The first number refers to the class of the enzyme. There are six classes; in order, these are oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Dehydrogenases are oxidoreductases, whereas kinases and transaminases are transferases. CK is EC number 2.7.3.2, which distinguishes it from other kinases.

Which of the following statements regarding the clinical use of CK-MB (CK-2) is true?

CK-MB is more specific than myoglobin Serum myoglobin becomes abnormal within 1-2 hours after an acute myocardial infarction (AMI) before troponin and CK-MB. CK-MB becomes abnormal shortly after troponin I (TnI) or troponin T (TnT) when a URL of 4 μg/L is used, and peaks at around the same time following AMI. However, cardiac troponins remain elevated significantly longer than CK-MB after AMI, are not increased in crush injuries. There is less than 5 μg/L CK-MB in the serum of healthy adults, while the total CK ranges from 10-110 U/L. Consequently, an abnormal CK-MB can occur in the absence of an elevated total CK.

An international unit (IU) of enzyme activity is the quantity of enzyme that:

converts 1 μmol of substrate to product per minute The IU is a rate expressed in micromoles per minute. Activity is reported as IUs per liter (IU/L) or mIU/mL. The SI unit for enzyme activity is the katal (1 katal converts 1 mol of substrate to product in 1 second).

Which of the following statements regarding the diagnosis of pancreatitis is correct?

Diagnostic sensitivity is increased by assaying both amylase and lipase Amylase is not increased in all persons with pancreatitis and can be increased in several nonpancreatic conditions. Lipase adds both sensitivity and specificity to the diagnosis of acute pancreatitis. Plasma or serum lipase becomes abnormal within 6 hours, peaks at approximately 24 hours, and remains abnormal for about 1 week following an episode of acute pancreatitis. In acute pancreatitis, the rate of urinary amylase excretion increases, and the amylase:creatinine clearance ratio is helpful in diagnosing some cases of pancreatitis. The normal A:C clearance ratio is 1%-4%. In acute pancreatitis, the ratio is usually above 4% and can be as high as 15%. In chronic pancreatitis, acinar cell degeneration often occurs, resulting in loss of amylase and lipase production. This lowers the sensitivity of amylase and lipase in detecting chronic disease to below 50%. Patients with chronic disease have pancreatic insufficiency giving rise to increased fecal fat, and decreased pancreatic digestive enzymes such as trypsin, chymotrypsin, and elastin.

Which of the following statements regarding ALP is true?

Elevated serum ALP seen with elevated GGT suggests a hepatic source ALP isoenzymes can result from different genes or from modification of a common gene product in the tissues. Some differ mainly in carbohydrate content and cannot be identified by immunologic methods. Highest levels of ALP are seen in Paget's disease of bone, where ALP can be as high as 25 times the URL. GGT in serum is derived from the hepatobiliary system and is increased in alcoholic hepatitis and hepatobiliary obstruction. It is not increased in diseases of bone or in pregnancy. When the increase in GGT is twofold higher than the increase in ALP, the liver is assumed to be the source of the elevated ALP. Serum ALP is a sensitive marker for extrahepatic obstruction, which causes an increase of approximately 10 times the URL. A lesser increase is seen in intrahepatic obstruction. ALP is only mildly elevated in acute hepatitis as a result of accompanying obstruction.

Which enzyme is measured in whole blood?

Glucose-6-phosphate dehydrogenase Glucose-6-phosphate dehydrogenase deficiency is the most common inherited RBC enzyme deficiency and is X linked. The enzyme is measured on a whole- blood hemolysate using glucose-6-phosphate as the substrate, and forms 6-phosphogluconate as NADP+ is converted to NADPH. Persons with the deficiency are prone to a hemolytic episode upon exposure to certain oxidative drugs and fava beans and as a result of infections. Heinz bodies form in the RBCs, bite cells are seen in the circulation, and plasma haptoglobin is reduced in severe cases.

Which substrate concentration is needed to achieve zero-order conditions?

Greater than 99 × Km A zero-order reaction rate is independent of substrate concentration because there is sufficient substrate to saturate the enzyme. V = Vmax × [S]/Km + [S] where V = velocity, Vmax = maximum velocity, [S] = substrate concentration, and Km = substrate concentration required to give 1/2 Vmax. If [S] >>> Km, then the Km can be ignored. V = Vmax × [S]/[S] = Vmax × [S]° or velocity approaches maximum and is independent of substrate concentration.

Which of the statements below regarding the methods of Henry for AST and ALT is correct?

Hemolysis will cause positive interference in both AST and ALT assays RBCs are rich in AST and to a lesser extent in ALT. Hemolysis causes positive interference in both assays, although the effect on AST is greater. Samples are stable for up to 24 hours at room temperature and up to 3 days at 4°C, and should be frozen if kept longer. The starting absorbance should be at least 1.5 A for both assays. Substrates with lower concentrations of NADH are subject to NADH depletion during the lag phase due to side reactions or high transaminase activity. When P-5'-P is added, a significant increase in activity sometimes occurs because some of the enzyme in the serum is in the inactive apoenzyme form.

Which of the following liver diseases produces the highest levels of transaminases?

Hepatic cancer Elevation of transaminases is greatest in acute hepatitis (20-50 × URL). Levels are moderately elevated (5-10 × URL) in hepatic cancer. They are slightly elevated (2-5 × URL) in chronic hepatitis, hepatic cirrhosis, alcoholic hepatitis, and obstructive jaundice.

In the Oliver-Rosalki method, the reverse reaction is used to measure CK activity. The enzyme(s) used in the coupling reactions is (are):

Hexokinase and G-6-PD The Oliver-Rosalki method for CK is based upon the formation of ATP from creatine phosphate. Hexokinase catalyzes the phosphorylation of glucose by ATP. This produces glucose-6-PO4 and adenosine diphosphate (ADP). The glucose-6-PO4 is oxidized to 6-phosphogluconate as NADP+ is reduced to NADPH. ATP + glucose in the presence of Hexokinase goes to ADP + glucose-6-PO4 glucose-6-PO4 + NADP+ in the presence of G-6-PD goes to 6-phosphogluconate + NADPH + H+

Which statement best describes the clinical utility of plasma homocysteine?

High plasma levels are associated with atherosclerosis and increased risk of thrombosis Homocysteine includes the monomeric amino acid as well as the dimers such as homocystine that contain homocysteine. Plasma levels are measured as an independent risk factor for coronary artery disease. High levels of homocysteine are toxic to vascular endothelium and promote inflammation and plaque formation. Plasma levels are independent of LDL and other cholesterol fractions and help explain why approximately 35% of people with first-time AMI have LDL cholesterol levels < 130 mg/dL.

Which test becomes abnormal in the earliest stage of the acute coronary syndrome?

High-sensitivity C-reactive protein The acute coronary syndrome (ACS) refers to the evolution of coronary artery events that lead up to AMI. Coronary artery disease (CAD) begins with formation of a plaque comprised of lipid from dead endothelium that proliferates into the artery lumen. The plaque becomes disrupted and the vessel wall inflamed in the asymptomatic stage of CAD. If platelet activation occurs and results in thrombosis, blood flow becomes significantly reduced, resulting in angina. This signals the transition to more advanced disease in which ischemia to heart muscle occurs and eventually to AMI. Myoglobin and CK-MB isoforms are not increased until the end stage of ACS. High-sensitivity C-reactive protein (hs-CRP) is an ultrasensitive CRP assay that accurately measures CRP below 1 mg/L. CRP is an acute-phase protein increased in inflammation. Levels of CRP between 3.2-10 mg/L signal low-grade inflammation, which occurs in the asymptomatic phase of ACS. Such inflammation occurs when coronary artery plaques become disrupted, and therefore, persons with CAD who have a mildly increased CRP are at high risk of disease progression. Glycogen phosphorylase-BB and albumin cobalt binding are increased by ischemia. Troponins are increased when there is unstable angina and ischemic injury, and indicate an increased risk for AMI.

Which of the following statements regarding total CK is true?

Highest levels are seen in Duchenne's muscular dystrophy Total CK is neither sensitive nor specific for AMI. An infarct can occur without causing an elevated total CK. Exercise and intramuscular injections cause a significant increase in total CK. Crush injuries and muscular dystrophy can increase the total CK up to 50 times the URL.

Which definition best describes the catalytic activity of amylase?

Hydrolyzes second α 1-4 glycosidic linkages of starch, glycogen, and other polyglucans Amylase in humans is a hydrolase that splits the second α 1-4 glycosidic bonds of polyglucans forming maltose. There are two major types of amylase: P-type derived from the pancreas and S-type derived from the salivary glands. These can be differentiated by both electrophoresis and immunoassay. In healthy persons, the principal form in plasma is the salivary isoenzyme. There are several genetic variants of the salivary isoenzyme, which in part accounts for the broad reference range.

The increase in the level of serum enzymes used to detect cholestatic liver disease is caused mainly by:

Increased production and secretion by cells The amount of enzyme in the serum can be increased by necrosis, altered permeability, secretion, or synthesis. It is also dependent upon tissue perfusion, enzyme half-life, molecular size, and location of the enzyme within the cell. Most enzymes are liberated by necrosis, but a few, such as ALP and γ-glutamyltransferase, are produced and secreted at a greater rate in obstructive liver disease.

In the Oliver-Rosalki method for CK, adenosine monophosphate (AMP) is added to the substrate in order to:

Inhibit adenylate kinase Positive interference in the Oliver-Rosalki method can occur when adenylate kinase is present in the serum from hemolysis or damaged tissue. Adenylate kinase hydrolyzes ADP, forming AMP and ATP (2 ADP in the presence of AK goes to AMP + ATP). This reaction is inhibited by adding AMP and diadenosine pentaphosphate (Ap5A) to the substrate.

Which of the following statements describes a non kinetic enzyme assay?

Initial absorbance is measured followed by a second reading after 5 minutes A kinetic assay uses several evenly spaced absorbance measurements to calculate the change in absorbance per unit time. A constant change in absorbance per unit of time occurs only when the rate of the reaction is zero order (independent of substrate concentration). Enzyme activity is proportional to rate only under zero-order conditions

Which enzyme is least useful in differentiating necrotic from obstructive jaundice?

LD GGT and 5 ́ nucleotidase are markedly elevated in both intra- and posthepatic obstruction. ALT is slightly elevated in obstructive jaundice but is markedly elevated in necrotic jaundice. Although LD is usually greater in necrotic jaundice than in obstructive jaundice, elevations in these conditions overlap frequently and result from many other causes.

Which statement best describes the clinical utility of plasma or serum myoglobin?

Levels below 100 μg/L on admission and 2-4 hours post admission help to exclude a diagnosis of AMI Myoglobin is a heme-containing pigment in both skeletal and cardiac muscle cells. The upper limit of normal is approximately 90 μg/L for males and 75 μg/L for females. The plasma myoglobin is a sensitive marker for AMI. Over 95% of affected persons have a value higher than the cutoff (typically >110 μg/L). However, specificity is approximately 75%-85% owing to skeletal muscle injury or renal insufficiency. For this reason, a plasma myoglobin below the cutoff on admission, and within the first 3 hours following chest pain helps to rule out AMI. A value above the cutoff must be confirmed using a cardiac specific assay such as TnI or TnT.

Which statement about the clinical utility of plasma or serum lipase is true?

Lipase is not increased in mumps, malignancy, or ectopic pregnancy Lipase elevation is of greater magnitude (2-50 × N) and duration than amylase in acute pancreatitis. When the lipase method is optimized by inclusion of colipase and bile salts, the test is more sensitive and specific than serum amylase for detection of acute pancreatitis. However, lipase is also increased in peptic ulcers, renal insufficiency, and intestinal obstruction. Lipase levels are often low in chronic pancreatitis, and are low in cystic fibrosis.

Which isoenzyme of ALP migrates farthest toward the anode when electrophoresed at pH 8.6?

Liver Liver ALP isoenzymes migrate farthest toward the anode, but fast and slow variants occur. The slow liver ALP band is difficult to distinguish from placental and bone ALP. The order from cathode to anode is: -Renal→Intestinal→Bone→Placental→Liver + Improved separation of bone and liver isoenzymes can be achieved by incubating the serum with neuraminidase prior to electrophoresis. The enzyme reduces the sialic content of the bone isoenzyme, causing it to migrate at a slower rate.

Select the coupling enzyme used in the kinetic AST reaction of Henry.

Malate dehydrogenase The method of Henry for AST uses malate dehydrogenase (MD) to reduce oxaloacetate to malate. The electrons come from NADH forming NAD+. Aspartate + α-ketoglutarate in the presence of AST goes to Oxaloacetate + Glutamate Oxaloacetate + NADH + H+ in the presence of MD goes to Malate + NAD+

In which condition is the measurement of acid phosphatase clinically useful?

Measuring the enzyme in a vaginal swab extract The PSA test is clinically more sensitive than prostatic acid phosphatase in detecting prostatic cancer. The clinical use of prostatic acid phosphatase is confined to the investigation of sexual assault. Acid phosphatase activity > 50 IU/L establishes the presence of seminal fluid in the vaginal sample. Tartrate-resistant acid phosphatase is used as a cytochemical marker for hairy-cell leukemia, and may be measured in serum to identify diseases with increased osteoclast activity, particularly malignancies involving bone.

Which of the following conditions is associated with a high level of S-type amylase?

Mumps Both salivary and pancreatic amylases designated S-type and P-type, respectively, are present in normal serum. High amylase occurs in mumps, ectopic pregnancy, biliary obstruction, peptic ulcers, alcoholism, malignancies, and other nonpancreatic diseases. Isoenzymes can be separated by electrophoresis (S-type is faster than P-type), but more commonly immunoinhibition of S-type amylase is used to rule out mumps, malignancy, and ectopic pregnancy, which give rise to high S-type amylase.

Which of the following cardiac markers derived from neutrophils predicts an increased risk for myocardial infarction?

Myeloperoxidase (MPO) All of the answer choices are markers for acute coronary syndrome and increased risk of AMI. MPO is released from neutrophils and is thought to destabilize the arterial plaque by oxidizing both LDL and HDL and reducing nitric oxide levels in the coronary arteries. Levels in the upper third quartile predict an increased risk of a coronary event even when troponin is normal. GPBB is released from myocytes early in an ischemic episode and becomes abnormal about 2 hours after an AMI. sCD40l is released from activated platelets and indicates an unstable plaque. PLA2 is produced by the arterial wall. It removes a fatty acid from phospholipids and increases the amount of oxidized LDL, leading to foam cell formation. Like hs-CRP, it is a marker for an inflamed plaque.

Which substance is used in the CK assay to activate the enzyme?

N-acetylcysteine In addition to Mg+2, CK requires a thiol compound to reduce interchain disulfide bridges and bind heavy metals that inactivate the enzyme. N-acetylcysteine is an activator of CK used for this purpose in the IFCC recommended method. Pyridoxyl-5 ́-phosphate is a prosthetic group of AST and ALT. FAD is a prosthetic group of glucose oxidase. Imidazole is used to buffer the CK reagent.

Which of the following statements about enzymatic reactions is true?

NADH has absorbance maximal at 340 and 366 nm Most enzymes are measured by monitoring the rate of absorbance change at 340 nm as NADH is produced or consumed. This rate will be proportional to enzyme activity when substrate is in excess. When the enzyme is present in excess, the initial reaction rate will be proportional to substrate concentration. This condition, called a first-order reaction, is needed when the enzyme is used as a reagent to measure a specific analyte.

Which substrate is used in the Bowers-McComb method for ALP?

p-Nitrophenyl phosphate The method of Bowers-McComb (Szasz modification) is the IFCC-recommended method for ALP. This method uses 2-amino-2-methyl-1-propanol, pH 10.15, and measures the increase in absorbance at 405 nm as p-nitrophenyl phosphate is hydrolyzed to p-nitrophenol.

A patient has a plasma myoglobin of 10 μg/L at admission. Three hours later, the myoglobin is 14 μg/L and the troponin I is 0.02 μg/L (reference range 0-0.03 μg/L). These results are consistent with which condition?

No evidence of myocardial or skeletal muscle injury This person displays very low plasma myoglobin (reference range for females is approximately 17-75 μg/L). The TnI result is also within normal limits. These results are consistent with baseline levels and no evidence of cardiac or skeletal muscle injury. TnI cutpoints for diagnosis of AMI are dependent upon the method, and may be higher than the upper limit of normal. Troponin results above the upper reference limit but below the cutpoint for AMI indicate myocardial injury and increased risk for AMI.

Which condition is least likely to be associated with increased serum ALP?

Osteoporosis ALP is elevated in osteomalacia (rickets), bone cancer, and bone disease secondary to hyperthyroidism and hyperparathyroidism, but total ALP it is high in less than 30% of osteoporosis patients. Pancreatic disease associated with biliary obstruction, such as cancer at the head of the pancreas, is associated with elevated ALP.

The reference method for lipase uses olive oil as the substrate because:

Other esterases can hydrolyze triglyceride and synthetic diglycerides Triglycerides may be hydrolyzed by nonspecific esterases in serum as well as lipase. Lipase acts only at an interface of oil and H2O and requires bile salts and colipase for activity. Colipase is a protein secreted by the pancreas.

Select the products formed from the forward reaction of AST.

Oxaloacetate and glutamate AST forms oxaloacetate and glutamate from aspartate and α-ketoglutarate (2-oxoglutarate). Both transaminases use α-ketoglutarate and glutamate as a common substrate and product pair. Both aspartate and alanine can be used to generate glutamate in the central nervous system, where it acts as a neurotransmitter.

Which condition produces the highest elevation of serum lactate dehydrogenase?

Pernicious anemia Serum LD levels are highest in pernicious anemia, reaching 10-50 times the upper reference limit (URL) as a result of intramedullary hemolysis. Moderate elevations (5-10 × URL) usually are seen in acute myocardial infarction, necrotic liver disease, and muscular dystrophy. Slight increases (2-3 × URL) are sometimes seen in obstructive liver disease.

The most commonly employed method of assay for plasma or serum lipase is based on:

Peroxidase coupling Although all of the methods cited are available, the most commonly used method for lipase assay is based upon the hydrolysis of a synthetic diglyceride substrate yielding 2-monoglyceride. This is hydrolyzed and forms glycerol, which is phosphorylated and forms glycerol-3-phosphate. This is oxidized by glycerophosphate oxidase, yielding hydrogen peroxide.

Which isoenzyme of ALP is most heat stable?

Placental Placental ALP and tumor-associated isoenzymes such as the Regan isoenzyme associated with lung cancer are the only isoenzymes that retain activity when serum is heated to 65° C for 10 minutes. Heat inactivation is used primarily to distinguish liver ALP from bone ALP. If less than 20% activity remains after heating serum to 56°C for 10 minutes, then bone ALP is most likely present.

Which of the following statements regarding ALP is true?

Pregnant women have a higher level of serum ALP than other adults ALP is higher in children than in adults due to bone growth. Children and geriatric patients have higher serum ALP due to increased bone isoenzyme. Serum ALP levels are often two- or threefold higher than the URL in the third term of pregnancy. In nonpregnant normal adults, serum ALP is derived from liver and bone. Liver, bone, placental, renal, and intestinal isoenzymes of ALP can be separated by electrophoresis, and many other ALP isoenzymes have been identified by isofocusing.

Which of the following enzymes is usually depressed in liver disease?

Pseudocholinesterase Pseudocholinesterase is found mainly in the liver and functions to hydrolyze acetylcholine. It is depressed by organophosphate insecticides and drugs that function as cholinesterase inhibitors and the serum assay is used to presumptively identify cases of insecticide poisoning. Levels of pseudocholinesterase are decreased in patients with liver disease as a result of depressed synthesis. In cirrhosis and hepatoma, there is a 50%-70% reduction in serum level and a 30%-50% reduction in hepatitis. Elastase-1 is a pancreatic digestive enzyme that breaks down connective tissue protein. Its level in feces is reduced in persons with pancreatic insufficiency. GLD is increased in necrotic jaundice, and aldolase in necrotic jaundice and muscle disease.

Select the products formed from the forward reaction of ALT.

Pyruvate and glutamate Because glutamate is a common product for transaminases, pyruvate (a three-carbon ketoacid) and glutamate would be generated from the transamination reaction between alanine and α-ketoglutarate.

What is the purpose of LD in the kinetic method of Henry for AST?

Rapidly exhausts endogenous pyruvate in the lag phase Patients with liver disease often have high levels of pyruvate and LD. The LD can catalyze the reaction of pyruvate with NADH in the substrate, forming NAD+ and lactate. This would give a falsely high rate for AST because NAD+ is the product measured. Adding LD to the substrate causes pyruvate to be depleted in the first 30 seconds, before AST and MD reactions reach steady state.

SITUATION: A physician calls to request a CK on a sample already sent to the laboratory for coagulation studies. The sample is a 2-hour-old citrated blood and has been stored at 4°C. The plasma shows very slight hemolysis. What is the best course of action and the reason for it?

Reject the sample because the citrate will interfere CK activity is lost with excessive storage, the most labile isoenzyme being CK-1. However, CK in serum is stable at room temperature for about 4 hours and up to 1 week at 4°C provided that an optimized method is used. Slight hemolysis does not interfere because CK is absent from RBCs. More significant hemolysis may cause positive interference by contributing ATP, glucose-6-PO4, and adenylate kinase to the serum. Calcium chelators remove magnesium as well as calcium and should not be used.

Which of the following statements regarding the naming of transaminases is true?

SGPT is the older abbreviation for ALT SGOT refers to the products measured in the in vitro reaction, and is more correctly named AST for the four-carbon amino acid substrate aspartate. SGPT is the older name referring to the products of the reaction for ALT. SGPT is more correctly named ALT for the three-carbon amino acid substrate alanine.

Which of the following conditions will interfere with the measurement of LD?

Slight hemolysis during sample collection RBCs are rich in LD-1 and LD-2, and even slight hemolysis will falsely elevate results. Hemolytic, megaloblastic, and pernicious anemias are associated with LD levels of 10-50 times the URL. LD is stable for 2 days at room temperature or 1 week at 4°C; however, freezing causes deterioration of LD-5. The activity of LD is inhibited by EDTA, which binds divalent cations; serum or heparinized plasma should be used.

In which condition would an elevated serum alkaline phosphatase be likely to occur?

Small cell lung carcinoma The primary diagnostic utility of ALP is to help differentiate necrotic jaundice (↑ALT) from obstructive jaundice (↑ ALP). ALP is also increased in several bone diseases. Large increases are seen in Paget's disease, moderate increases in bone cancer, and slight increases in rickets. Total ALP may be slightly increased in osteoporosis but often it is not. In addition to obstructive jaundice and bone diseases, alkaline phosphatase is a tumor marker. In most cases, the alkaline phosphatase is the product of fetal gene activation, and resembles placental ALP (e.g., hepatoma, small cell carcinoma of the lung, ovarian cancer). Leukemia and Hodgkin's disease may cause an elevated leukocyte or bone-derived ALP.

Which of the following statements regarding amylase methods is true?

Synthetic substrates can be conjugated to p-nitrophenol (PNP) for a kinetic assay Many endogenous inhibitors of amylase, such as wheat germ, are found in serum. Diluted samples often show higher than expected activity caused by dilution of the inhibitor. Units of amylase activity vary widely depending upon the method of assay and calibration. Synthetic substrates such as maltotetrose or 4-nitrophenyl maltohepatoside can be used for kinetic assays. Maltotetrose is hydrolyzed to maltose by amylase, and the maltose hydrolyzed by α-glucosidase or maltose phosphorylase, forming glucose or glucose-1-phosphate, respectively. These can be measured by coupling to NADH-generating reactions. Antibodies to the salivary isoenzyme can be added to synthetic substrate assays to inhibit S-type amylase.

Which statement regarding bone-specific ALP is true?

The bone isoenzyme can be measured immunochemically Bone ALP assays (Ostase and Alkphase-B) use monoclonal antibodies to measure the bone isoenzyme in mass units. The assays may be used to monitor bone remodeling by osteoblasts in osteoporosis, and thus, are useful for following treatment. Bone specific ALP is not sufficiently sensitive to diagnose osteoporosis, and antibodies may cross-react with other ALP isoenzymes, depending on their source.

Which of the following statements regarding enzymatic reactions is true?

The enzyme increases the rate of the reaction An enzyme will accelerate the rate of a reaction, reducing the time required to reach equilibrium. The concentration of reactants and products at equilibrium will be the same with or without the enzyme.

SITUATION: An EDTA sample for TnI assay gives a result of 0.04 ng/mL (reference range 0-0.03 ng/mL). The test is repeated 3 hours later on a new specimen and the result is 0.06 ng/mL. A third sample collected 6 hours later gives a result of 0.07 ng/mL. The EKG showed no evidence of ST segment elevation (STEMI). What is the most likely explanation?

The patient has suffered cardiac injury EDTA is the additive of choice for troponin assays because it avoids microclots that can lead to false positive results when serum or heparinized plasma is used. Spurious false positives caused by matrix effects usually revert to normal when the test is repeated on a new sample. An AMI will cause the TnI to increase in subsequent tests. Results between 0.04-0.10 ng/mL are the result of cardiac injury, and indicate either AMI or an increased short-term risk of AMI.

Which statement about methods for measuring LD is true?

The pyruvate-to-lactate reaction proceeds at about twice the rate as the forward reaction Although the rate of the reverse reaction (P → L) is faster, the L → P reaction is more popular because it produces a positive rate (generates NADH), is not subject to product inhibition, and is highly linear. The pH optimum for the forward reaction is approximately 8.8.

What is the recommended troponin T and I cutoff (upper limit of normal) for detecting myocardial infarction?

The upper 99th percentile or lowest level that can be measured with 10% CV The American College of Cardiology recommends the cutoff for an abnormal troponin test be set at the 99th percentile of the normal population, 0.013 ng/mL, or if the assay precision at this level is >10% then the cutoff should be the lowest value measurable with a CV of 10% (typically 0.03 ng/mL). An abnormal result (0.04 ng/mL or higher) in a patient with other evidence of ischemic changes indicates cardiac damage. This typically occurs when a pattern of increasing troponin concentration is seen over the first 6 hours after initial testing.

Which of the following statements about the phosphatases is true?

They are divided into two classes based upon pH needed for activity Phosphatases are classified as either alkaline or acid depending upon the pH needed for optimum activity. The phosphatases hydrolyze a wide range of monophosphoric acid esters. ALP is inhibited by phosphorus (product inhibition). The International Federation of Clinical Chemistry (IFCC) recommended method employs 2-amino-2-methyl-1-propanol, a buffer that binds Pi.

A patient has a plasma CK-MB of 14 μg/L at admission and a total CK of 170 IU/L. Serum myoglobin is 130 μg/L and TnI is 1.6 μg/L. Three hours later, the TnI is 3.0 μg/L. Which statement best describes this situation?

This patient has had an AMI and further testing is unnecessary Results on admission indicate strongly that the patient has suffered an MI. The 3-hour TnI confirms this and rules out the possibility of a sample collection or transcription error for the admission sample. Repeat testing of other cardiac markers at 3 hours was not necessary because admission results were significantly increased for all three markers. Skeletal muscle damage or crush injury does not cause an increase in cardiac TnI.

The reference method for serum lipase is based upon:

Titration of fatty acids with dilute NaOH following controlled incubation of serum with olive oil The reference method of Cherry and Crandall is based upon the titration of fatty acids formed by the hydrolysis of an emulsion of olive oil after incubation for 24 hours at 37°C. Because most of the activity occurs within the first 3 hours, the incubation time may be shortened to as little as 1 hour without loss of clinical utility.

In which type of liver disease would you expect the greatest elevation of LD?

Toxic hepatitis Liver disease produces an elevated LD-4 and LD-5. Levels may reach up to 10 times the URL in toxic hepatitis and in hepatoma. However, LD levels are lower in viral hepatitis (2-5 × URL), only slightly elevated in cirrhosis (2-3 × URL) and not significantly elevated in alcoholic liver disease.

Which of the following cardiac markers is consistently increased in persons who exhibit unstable angina?

Troponin T Persons with unstable angina (angina at rest) who have an elevated TnT or TnI are at eight times greater risk of having an MI within the next 6 months. This property is being used to identify short-term risk patients who should be considered for coronary angioplasty. The reference range for troponin is very low (0-0.03 ng/mL); persons with unstable angina usually have values between 0.04 and 0.1 ng/mL without clinical evidence of AMI. CK-MB and myoglobin have not been useful in identifying persons with unstable angina.

Select the most sensitive marker for alcoholic liver disease.

γ-Glutamyltransferase (GGT) Although AST and ALT are elevated in alcoholic hepatitis, GGT is a more sensitive indicator of alcoholic liver disease. Levels of GGT can reach in excess of 25 times the URL in alcoholic hepatitis. It is also markedly elevated in obstructive jaundice; a high GGT supports the inference that liver is the tissue source of an elevated ALP.