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Chapter 8
Diagnostic Enzymology And
Other Biochemical Markers
Of Organ Damage
Lixia Zhang
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 -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 -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 -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 PROCEDURES
●
Single-Reagent Kinetic Assays
●
Start Reagent Activity Assays
●
Enzyme Activity Calculations
●
Mass Measurements
Mass 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
LIVER ANATOMY
LIVER
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
●
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 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
Mushrooms
Metals
Drugs
Metabolic
Carbon tetrachloride
Trichloroethylene
Amanita phalloides
Yellow phosphorus
Acetaminophen
Halothane
Isoniazid
Rifampicin
Reye’s syndrome
Wilson’s disease
Galactosemia
α-Antitrypsin deficiency
Liver Diseases -2
Cholestatic Liver Diseases
Name of Diseases
Intrahepatic Obstruction
Extrahepatic Obstruction
Markers
Bilirubin
ALP,GGT,5’-NT
Bilirubin
ALP,GGT,5’-NT
Liver Diseases – 3
Chronic Liver Diseases
Name of Diseases
Chronic hepatitis
Liver Cancer
Markers
Liver enzymes (in active form)
Liver enzymes , AFP
Liver Diseases – 4
Alcoholic liver disease
Name of Diseases
Fatty Liver
Alcoholic
Cirrhosis
Markers
Liver enzymes within normal
AST, ALT are increased
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)
ALT
20%AST
GGT
80% AST
ALP
Specific enzymes of liver -1
■
ALP And ALP Isoenzymes
Enzyme
ALP
distribution
important indication
liver, bone,
hepatobiliary disease,
Intestine, kidney
skeletal disease
Adrenals, placenta
ALP Isoenzymes
liver, bone,
Intestine, kidney
placenta
differentiating between
bone and liver sources
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
ALT
liver
AST
liver
heart
10-40 U/L
10-40 U/L
indication
parenchymal diseases
of liver
AMI
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
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 -1
disease
acute hepatitis
enzyme marker
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
and
HCV
preclinical incubation phase is longer and ALT
and AST may remain normal for 2 to 6months
ALT and AST return to normal within 2 to 3months
ALT and AST in acute liver injury -2
disease
chronic active
hepatitis
end-stage
liver disease
enzyme marker
ALT and AST elevated 5 to10 fold
ALT and AST return to normal or
subnormal
Markers For Liver Function And Disease -1
Disease or function
Markers
Function evaluation
Normal synthesis
capacity
Excretory function
Albumin, retinol binding protein,
prealbumin,prothrombin time,
cholinesterase
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 -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 Hepatitis
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
• 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.
Multiplies of normal
Relationship of AST and ALT to
ALP and GGT in Cholestasis
De Ritis Ratio (AST/ALT)
De 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
De Ritis（AST/ALT)
Disease
Acute disorders of the liver
acute viral hepatitis
infectious mononucleosis
chronic disorders of the liver
alcoholic liver disease
postnecrotic cirrhosis
chronic active hepatitis
chronic persistent hepatitis
-2
AST/ALT
<1.0
>1.0
normal
De Ritis（AST/ALT) -3
Intrahepatic and Extrahepatic obstruction
Disease
AST/ALT
extrahepatic cholestasis
＜1.5
intrahepatic cholestasis
≥1.5
Couses
acute passage
of a stone
biliary cirrhosis
and malignancy
De Ritis（AST/ALT) - 4
Intrahepatic and Extrahepatic obstruction
Other laboratory tests:
ALP
extrahepatic >intrahepatic
Conjugated bilirubin extrahepatic >intrahepatic
Amylase
extrahepatic >intrahepatic
Multiplies of normal
Relationship of AST and ALT to
ALP and GGT in Malignancy
De Ritis Ratio (AST/ALT) -5
Alcoholic Liver Disease
●
●
Disproportionate increases of AST
relative to ALT are observed
AST/ALT>6.0
Multiplies of normal
Relationship of AST and ALT to ALP and
GGT in Alcoholic Live Disease
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 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
A: Stable coronary artery plaque not vulnerable for rupture.
B: Unstable plaque that is vulnerable for rupturing by shear stresses.
Unstable 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 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 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
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 Distribution
CK (creatine kinase) -3
CK Isoforms and CK Variants
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 (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
• 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 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
2-8h 8-24h 24-72h >72h
Myoglobin
CK-MB isoforms
CK-MB
LDH1
Troponin
95
90
60
40
75
75
60
95
85
95
0
0
98
95
98
0
0
50
90
98
specificity
70
90
95
85
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
Extensive skeletal
muscle injury
myoglobin
total CK
in serum
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
Lipase
90%
90 %
40%
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
Chronic pancreatitis
Other disorders
79.5
75.0
92.0
Acute pancreatitis pos 29.0 neg 99.4
Chronic pancreatitis pos 13.6 neg 99.5
87.2
81.3
92.5
87.2
81.3
92.1
Predictive values (%)
pos 32.4 neg 99.4
pos 31.2 neg 99.4
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.