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Chapter 8Diagnostic Enzymology And Other Biochemical Markers
Of Organ Damage
Lixia Zhang
Contents Contents
• Basic Concepts For Diagonostic Enzymology
• Enzyme Assay Procedures
• Liver Diseases
* Markers Of Liver Diseases
* Patterns Of Liver Enzymes And Markers For Interpretation Of Disease
* Myocardial And Skeletal Muscle Disease
* Pancreatic Diseases
• Miscellaneous Enzymes
• Suggested Readings
* Important
● Diagnostic enzymology involves the measurement of enzymes in body fluids for the diagnosis of disease. In most cases, serum or blood levels are the most useful, although urine, cerebrospinal, and extracellular fluid levels are sometimes important.
● This chapter focuses on the analytical aspects and clinical significance of important enzymes that are measured for diagnostic purposes.
● The major emphasis is on the use of these
enzymes for the diagnosis of diseases involving the liver, myocardium, skeletal muscle, pancreas, and bone. In the case of myocardial infarction and skeletal muscle injury, nonenzyme markers such as myoglobin and troponin are also discussed.
BASIC CONCEPTS FOR
DIAGONOSTIC ENZYMOLOGY
Pathologic Role
of Enzymes in Blood
Pathologic Role of Enzymes in Blood -1Pathologic Role of Enzymes in Blood -1
● Most enzymes that are used for diagnostic
purposes have no direct physiologic role in the
blood: Their presence under normal
circumstances is the result of natural aging and
turnover of cells.
Pathologic Role of Enzymes in Blood -2Pathologic Role of Enzymes in Blood -2
● The normal level of activity of these enzymes in the serum is a function of the rate of release and clearance.
● The half-lives of these enzymes vary greatly from an estimated 2 hours for the BB isoenzyme of creatine kinase (CK) to 170 hours for placental alkaline phosphatase (ALP).
Pathologic Role of Enzymes in Blood -3Pathologic Role of Enzymes in Blood -3
● High levels of enzymes in the blood can indicate increased cellular turnover and tissue necrosis caused by disease.
● An equally important and largely overlooked cause of high enzyme levels in blood is tissue synthesis of new enzymes that occurs in response to disease, induction by various drugs, and carcinogenesis.
ENZYME
ASSAY PROCEDURES
ENZYME ASSAY PROCEDURESENZYME ASSAY PROCEDURES
● Single-Reagent Kinetic Assays
● Start Reagent Activity Assays
● Enzyme Activity Calculations
● Mass Measurements
Mass MeasurementsMass Measurements
● These assays are particularly useful for isoenzyme analysis, as antisera can be directed toward specific isoenzymes, isoforms, or subunits.
● Mass measurements are also used to measure the concentration of protein markers (e.g., myoglobin) that do not possess enzymatic activities.
LIVER DISEASES
LIVERLIVER
LIVER ANATOMYLIVER ANATOMY
LIVER DISEASES LIVER DISEASES
● The enzymes ALT and AST, ALP, LDH, GGT, and (to a lesser extent) 5'-nucleotidase (5'NT) are commonly measured for the assessment of liver function.
● None of these markers is specific for any single liver disorder.
LIVER DISEASES LIVER DISEASES
● This section covers liver dysfunction,
describes individual liver enzymes and
their laboratory measurement , and
explains how patterns of liver enzyme
data can be used to aid in the diagnosis
of liver diseases.
LIVER DISEASESLIVER DISEASES
Inclusion:
● Acute Hepatocellular Injury
● Cholestatic Liver Diseases
● Chronic Liver Diseases
● Alcoholic Liver Disease
Liver Diseases -1
Acute Hepatocellular Injury
Name of Diseases Markers
viral hepatitis Bilirubin
AST,ALT
Acute liver failure AST,ALT
Various substances that can induce acute liver failure
Solvents Carbon tetrachloride TrichloroethyleneMushrooms Amanita phalloidesMetals Yellow phosphorusDrugs Acetaminophen Halothane Isoniazid RifampicinMetabolic Reye’s syndrome Wilson’s disease Galactosemia α-Antitrypsin deficiency
Liver Diseases -2
Cholestatic Liver Diseases
Name of Diseases Markers
Intrahepatic Obstruction Bilirubin
ALP,GGT,5’-NT
Extrahepatic Obstruction Bilirubin
ALP,GGT,5’-NT
Liver Diseases – 3
Chronic Liver Diseases
Name of Diseases Markers
Chronic hepatitis Liver enzymes (in active form)
Liver Cancer Liver enzymes , AFP
Liver Diseases – 4
Alcoholic liver disease
Name of Diseases Markers
Fatty Liver Liver enzymes within normal
Alcoholic AST, ALT are increased
Cirrhosis ALP, AST, ALT and GGT are
elevated
MARKERS
OF LIVER DISEASES
Specific enzymes of liver
● ALP ( Alkaline Phosphatase) and ALP Isoenzymes
● GGT ( g-glutamyl-peptide : amino acid γ- glutamyltransferase)
● 5'-NT ( 5'-ribonucleotide phosphohydrolase)
● AST ( L-aspartate : 2- oxoglutarate amino- transferase )
● ALT (L-alanine: 2-oxoglutarate amino-transferase)
80% AST
ALT 20%AST
GGT
ALP
Specific enzymes of liver -1
■ ALP And ALP Isoenzymes
Enzyme distribution important indication
ALP liver, bone, hepatobiliary disease, Intestine, kidney skeletal disease Adrenals, placenta
ALP Isoenzymes liver, bone, differentiating between Intestine, kidney bone and liver sources placenta
Plasma alkaline phosphatase activity as a function of age and sex(—men ;…… ,
women). Horizontal lines refer to multiples of the adult upper reference limit.
Specific enzymes of liver -2
■ GGT
distribution reference indication
liver (most) men 9-50 U/L hepatobiliary
Prostate (little) women 8-40 U/L diseases
Prostate (little) ( in 37°C ) alcoholism
* Located in the cell membrane
Specific enzymes of liver -3
■ 5'-NT
distribution indication
cytosolic membrane-bound increased activity
enzyme that 5'-phosphate in the serum reflects
esters of nucleotides. hepatobiliary diseases
Specific enzymes of liver -4
■ ALT and AST
distribution reference indication ALT liver 10-40 U/L parenchymal diseases of liver
AST liver 10-40 U/L AMI heart parenchymal diseases of liver and skeletal muscle diseases
PATTERNS OF
LIVER ENZYMES AND MARKERS
FOR
INTERPRETATION OF DISEASE
● Unlike some disorders such as acute pancreatitis and myocardial infarction, for which there are enzyme markers that are primarily used for one disorder and have high diagnostic efficiencies, there are no enzyme markers that are specific for any single liver disease.
● When evaluating these disorders, therefore, it is appropriate to consider a panel of markers, sometimes called live function tests (LFTs).
● usually includes bilirubin, AST, ALT,ALP,and sometimes GGT and 5'NT
● although these tests can reflect various disease processes in the liver, they do not reflect hepatic reserve for synthesis and metabolic functions
Hepatocellular Versus Obstructive Liver Diseases
Acute Injury Acute Injury
When Acute Hepatocellular Injury :
■ AST and ALT and are therefore rarely
used for diagnostic purposes.
■ ALP, GGT, and 5'NT are not as markedly
elevated.
ALT and AST in acute liver injury -1ALT and AST in acute liver injury -1
disease enzyme marker
acute hepatitis ALT and AST elevated>1000U/L (viral or toxic) HAV ALT elevated in 3 to 4 weeks after infection ALT return to normal within 8 to 12 weeks HBV preclinical incubation phase is longer and ALT and and AST may remain normal for 2 to 6months HCV ALT and AST return to normal within 2 to 3months
ALT and AST in acute liver injury -2
disease enzyme marker
chronic active hepatitis ALT and AST elevated 5 to10 fold end-stage liver disease ALT and AST return to normal or subnormal
Markers For Liver Function And Disease -1 Markers For Liver Function And Disease -1
Disease or function Markers
Function evaluation Normal synthesis Albumin, retinol binding protein, capacity prealbumin,prothrombin time, cholinesteraseExcretory function Bilirubin, bile acids, rose bengal and sulfobromophthalein excretion Metabolic function Ammonia, amino acids, serum protein, lipids, electrophoresis,globulins Drug metabolism Antipyrine breath and clearance test, 14CO2 breath test
Markers For Liver Function And Disease -2Markers For Liver Function And Disease -2
Disease or function Markers
Pathological evaluation
Hepatocellular injury ALT, AST,LDH,
Obstruction Bilirubin, ALP, GGT,5’-NT bile acids
Infections Viral and autoimmune serologies
Malignancies Carcinoembryonic antigen,
α-fetoprotein
Relationship of AST and ALT to ALP and GGT in HepatitisRelationship of AST and ALT to ALP and GGT in Hepatitis
Cholestasis -1 Cholestasis -1
● The best markers for intrahepatic and
extrahepatic cholestasis are ALP, GGT,
and 5'NT
● The largest elevations (four- to 10-fold)
of ALP are typically seen in obstruction
owing to gallstones or malignancy and
in biliary cirrhosis.
Cholestasis -2 Cholestasis -2
• AST and ALT are generally only slightly elevated in cholestasis, rarely more than
500 U/L.
• Measurement of total and direct bilirubin
are also important in making the diagnosis
of obstructive jaundice.
Relationship of AST and ALT to ALP and GGT in CholestasisRelationship of AST and ALT to ALP and GGT in Cholestasis
Mu
ltip
lies
of
no
rmal
De Ritis Ratio (AST/ALT)
De Ritis Ratio (AST/ALT) -1De Ritis Ratio (AST/ALT) -1
• Further differentiation of specific liver diseases is aided by calculating the ratio of AST to ALT levels. — acute or chronic ? — intra- or extrahepatic ?
• recommended by the International Federation of Clinical Chemistry (IFCC)
• de Ritis ratio is normally approximately 1.15
ALT versus AST levels in various liver diseases ALT versus AST levels in various liver diseases
De Ritis ( AST/ALT) -2De Ritis ( AST/ALT) -2
Disease AST/ALT
Acute disorders of the liver <1.0 acute viral hepatitis infectious mononucleosis chronic disorders of the liver >1.0 alcoholic liver disease postnecrotic cirrhosis chronic active hepatitis chronic persistent hepatitis normal
Disease AST/ALT Couses
extrahepatic cholestasis < 1.5 acute passage of a stone intrahepatic cholestasis ≥1.5 biliary cirrhosis and malignancy
De Ritis ( AST/ALT) -3 Intrahepatic and Extrahepatic obstruction
De Ritis ( AST/ALT) - 4 Intrahepatic and Extrahepatic obstruction
Other laboratory tests: ALP extrahepatic >intrahepatic Conjugated bilirubin extrahepatic >intrahepatic Amylase extrahepatic >intrahepatic
Relationship of AST and ALT to ALP and GGT in Malignancy Relationship of AST and ALT to ALP and GGT in Malignancy
Mu
ltip
lies
of
no
rmal
De Ritis Ratio (AST/ALT) -5
Alcoholic Liver Disease
● Disproportionate increases of AST
relative to ALT are observed
● AST/ALT>6.0
Relationship of AST and ALT to ALP and GGT in Alcoholic Live Disease
Relationship of AST and ALT to ALP and GGT in Alcoholic Live Disease
Mu
ltip
lies
of
no
rmal
De Ritis Ratio (AST/ALT) -6
Muscle Disease
● The largest increases of AST relative
to ALT are seen in myocardial and
skeletal muscle diseases
● AST/ALT>10.0
Markers of Alcohol Use
Markers of Alcohol Markers of Alcohol
• Markers of alcohol use that most widely used marker for alcoholism is GGT
• Significant elevations of GGT are also observed after prolonged drinking episodes of several months or more.
• it is not a specific test • Other markers currently being studied include
carbohydrate-deficient transferrin and fatty acid ethyl esters.
MYOCARDIAL AND SKELETAL
MUSCLE DISEASE
CHD: coronary
heart
disease
UA: unstable
angina
AMI: acute
myocardial
infarction
Unstable Angina and AMI -1 Unstable Angina and AMI -1
A: Stable coronary artery plaque not vulnerable for rupture.
B: Unstable plaque that is vulnerable for rupturing by shear stresses.
Unstable Angina and AMI -2Unstable Angina and AMI -2
● Rupture of this plaque leads to the syndrome
of unstable angina, the disease that immediately
precedes an AMI.
● Both conditions, collectively known as acute
coronary syndromes, can produce chest pain
at rest, lead to specific changes in tile
electrocardiogram, and a mild or severe
release of enzymes and proteins into blood.
Congestive Heart FailureCongestive Heart Failure
● Heart failure is a very common disease, ● Chronic congestive heart failure (CHF) is seen in cardiomyopathies and valve disease. ● New markers are being developed, such as brain natriuretic peptide (BNP) that might have a role in the management and monitoring of these patients.
Skeletal Muscle DisordersSkeletal Muscle Disorders
● Evidence of high CK activity in the serum is useful for the diagnosis and evaluation of both acute and chronic skeletal muscle diseases. ● The measurement of enzymes and isoenzymes is useful in the diagnosis of muscle disorders.
Enzymes and Proteins Useful in
Myocardial Disease
Enzymes
Useful in Myocardial Disease
CK (creatine kinase) -1 CK (creatine kinase) -1
The reference range of total CK
at 37°C : 38-174U/L for adult men
96-140 U/L for adult women.
CK (creatine kinase) -2
CK Isoenzyme Tissue DistributionCK Isoenzyme Tissue Distribution
CK (creatine kinase) -3
CK Isoforms and CK Variants
CK (creatine kinase) - 4
CK (creatine kinase) - 4
● CK and CK Isoenzyme Analytical Measurements
● Most normal samples will exhibit
a single band owing to CK-MM.
● Patients with AMI will have high
concentrations of CK-MM and MB.
CK (creatine kinase) - 5
Ck isoforms as measured by agarose electrophoresis
CK (creatine kinase) - 6
CK Isoenzyme Reference values
• CK-MB : Cutoff values for electrophoresis and immunoinhibition are usually set around 5% of total CK or 10 U/L.For immunoassays, decision limits are expressed in mass quantities and are approximately 5 to 10ng/mL.
• CK-BB is normally absent in adult serum.
• CK-MB and CK-BB levels are higher in children
LDH and LDH Isoenzymes LDH and LDH Isoenzymes
LDH (Lactose Dehydrogenase ) Reference
The interval for the forward reaction is approximately 80 to 200 U/L at 37°C. approximately 200 to 400 U/L for the reverse reaction.
LDH tissue distribution
The decision limit most widely used is that LDH1 activity in excess of 40% of total LDH activity supports the diagnosis of
AMI.
Proteins
Useful in Myocardial Disease
Myoglobin Myoglobin
• found in all skeletal muscle and myocardial tissues
• myoglobin was an early marker for AMI
• Reference limits for serum myoglobin vary but are generally less than 100 μg/L
• Myoglobin is also increased in patients with skeletal muscle disease or injury, and in patients with acute or chronic renal failure.
Troponin Troponin
• Troponin is consists of three isotypes
Troponin-T (cTnT)
Troponin-I (cTnI )
Troponin C (cTnC)
• cTnT and cTnI are better diagnostic markers for AMI than existing enzymes such as CK- MB
Use of Cardiac Markers in the Diagnosis of AMI
Cardiac Markers in the Diagnosis of AMI -1
● The diagnosis of AMI is defined by the WHO:
● clinical signs and symptoms
● specific changes in electrocardiographic recordings
● elevated serum enzymes and proteins total CK, CK-MB, LDH, and LDH isoenzymes myoglobin and the cardiac troponins
Cardiac Markers in the Diagnosis of AMI -2
● Cardiac Markers in the Diagnosis of AMI ● Early Diagnosis: Myoglobin CK Isoforms ● Definitive Diagnosis : CK-MB Troponin ● Late Diagnosis : LDH cTnT and cTnI
Cardiac Markers in the Diagnosis of AMI -3
Estimates Of Clinical Sensitivity And Specificity Of Diagnostic Tests For AMI
Markers Sensitivity specificity 2-8h 8-24h 24-72h >72h
Myoglobin 95 75 0 0 70 CK-MB isoforms 90 60 0 0 90 CK-MB 60 95 98 50 95 LDH1 40 85 95 90 85
Troponin 75 95 98 98 90
Cardiac Markers in the Diagnosis of AMI - 4
Activity versus time curves in serum for biochemical markers of acute myocardial infarction.
Cardiac Markers in the Diagnosis of AMI -5
• The cardiac troponins (T or I) are the most efficient markers available today for diagnosis of AMI
• The specificity of troponin is also increased over CK-MB or myoglobin
• cardiac troponin has been recommended by the NACB as the preferred marker for the definitive diagnosis of AMI
• With the development of structural markers such as cTnT and cTnI, the NACB has recommended discontinuance of LDH isoenzymes
Release of cardiac troponin from damaged myocytes
Enzymes and Proteins
in Skeletal Muscle Disease
Enzymes and Proteins in Skeletal Muscle Disease
• The important enzyme markers of skeletal muscle necrosis include total CK, CK-MB, and, to a lesser extent, AST and aldolase.
• In cases of concomitant skeletal muscle injury and AMI, measurement of troponin allows differentiation between skeletal muscle injury and myocardial damage.
Enzymes and Proteins in Skeletal MuscleDisease Enzymes and Proteins in Skeletal MuscleDisease
Extensive skeletal
muscle injury
total CK in serum myoglobin
in serum and urine (is observed in patients with rhabdomyolysis)
May induce acute renal failure
PANCREATIC DISEASES
Enzymes of Pancreatic Origin
• Amylase versus Marcroamylase• Amylase Isoenzymes (s type and p type) Amylase Isoenzymes Isoforms (S1 and P2 )• Lipase • Trypsin
Clinical Uses of Pancreatic Enzymes
In Acute Pancreatitis
In Chronic Pancreatitis
In Other Disorders
Clinical Uses of Pancreatic Enzymes -1
● In Acute Pancreatitis
● Laboratory findings include an elevated serum
amylase, amylase clearance, and lipase.
● Amylase and lipase levels rise within a few hours
after onset and remain elevated for 36 to48 hours.
● In Acute Pancreatitis
Sensitivity and specificities in Acute Pancreatitis
Sensitivity specificities accuracy
Amylase 90% 40% -
Lipase 90 % 60% 95.8%
Clinical Uses of Pancreatic Enzymes -2
● In Chronic Pancreatitis
● Serum enzyme levels in chronic pancreatitis are less useful than in the acute presentation.
● Amylase and lipase levels are very often within normal limits and may actually be low during the end stages of the disease
Clinical efficiency of tests in acute and chronic pancreatitis
Disorder α-Amylase total pancreatic amylase lipase
Clinical sensitivity (%)
Acute pancreatitis 79.5 87.2 87.2
Chronic pancreatitis 75.0 81.3 81.3
Other disorders 92.0 92.5 92.1 Predictive values (%)
Acute pancreatitis pos 29.0 neg 99.4 pos 32.4 neg 99.4 pos 31.2 neg 99.4
Chronic pancreatitis pos 13.6 neg 99.5 pos 15.5 neg 99.7 pos 14.8 neg 99.1
Clinical Uses of Pancreatic Enzymes -3
● In Other Disorders
● Injury to or obstruction of surrounding tissue, such as a perforated ulcer or peritonitis, may also cause compression of the pancreas, resulting in enzyme release. ● Elevations of amylase, immunoreactive trypsin, and, to a lesser extent, lipase also occur in other nonpancreatic disorders.
● Amylase of high levels are seen in patients with renal failure.
MISCELLANEOUS ENZYMES
MISCELLANEOUS ENZYMES
ACP (Acid Phosphatase)
CHE (Cholinesterase)
ACE (peptidyl-dipeptide hydrolase)
G6PD (Glucose-6-Phosphate Dehydrogenase)
MISCELLANEOUS ENZYMES -1
● ACP ● Measurement of acid phosphatase was primarily used for monitoring patients with prostatic cancer.
● With the development of prostate-specific antigen (PSA), assays for acid phosphastase, if used at all, are only used on rare occasions.
MISCELLANEOUS ENZYMES - 2
● CHE ● In the serum, however, only
pseudocholinesterase (PCHE)
acylcholine acylhydrolase.
● Low levels of PCHE are found
in patients with various forms
of liver disease.
MISCELLANEOUS ENZYMES - 2
● CHE ● Nonhepatic disorders, such as AMI, infections, and pulmonary embolism, have also been shown to decrease PCHE levels.
● The activity of PCHE is also important
in monitoring industrial and agricultural
workers who use and are exposed to
organophosphate insecticides.
MISCELLANEOUS ENZYMES - 3
● ACE ● It is produced by the lungs and catalyzes the conversion of the decapeptide angiotensin I to the decapeptide angiotensin II. ● It is a vital enzyme in the control of aldosterone secretion and regulation of blood pressure.
MISCELLANEOUS ENZYMES - 3
● ACE
● It is elevated in approximately 50% to 80% of cases of sarcoidosis, a multisystem granulomatous disease that most commonly involves the lungs.
● Several other conditions can produce
elevated activities.
MISCELLANEOUS ENZYMES - 4
● G6PD
● It is an important erythrocyte enzyme
● G6PD deficiency affects black men and Whites of Mediterranean descent.
● These individuals develop varying degrees of hemolytic anemias.