Diagnostic enzymology

Laboratory diagnostics department

Chen zhen
The enzymes measured for diagnostic
purposes in serum, plasma, secretions
and tissue fluids are biological
catalysts.
Enzyme is a biocatalyst:

the reaction
Biological catalysts
 Typically are very large proteins.
 Permit reactions to ’go’ at conditions
that the body can tolerate.
 Can process millions of molecules every
second.
 Are very specific – react with one or only
a Few type of molecules (substrates).
 Only small quantities of enzymes are
required to allow a chemical reaction to
take place.
 Classification of Enzymes in Blood
Classification Examples
Plasma-Specific Serine protease ， procoagulants: thrombin, factor XII
enzymes (Hageman factor), factor X (Stuart-Prower factor), and
others
Fibrinolytic enzymes or precursors: plasminogen,
plasminogen proactivator

Secreted Lipase (from pancreas and gastric oxyntic glands), α-

enzymes amylase (from salivary glands and pancreas),
trypsinogen, cholinesterase, prostatic acid phosphatase

Cellular enzymes Lactate dehydrogenase, aminotransferases, alkaline

phosphatase, and others
 Causes of Cell Damage or death
Category Example
Hypoxia (an extremely Loss of blood supply due to narrowing (athermanous
common plaques) or blocking (thrombosis) of artery or vein;
accompaniment of ischemicperfusion injury; inadequate oxygenation due
clinical disease) to cardiorespiratory failure; loss of oxygen-carrying
capacity; CO poisoning; and anemia

Chemicals and drugs Environmental pollutants (lead, mercury); drugs, use

(an important cause of and abuse; alcohol; tobacco
cellular damage

Physical agents Trauma; extremes of heat and cold; radiation; electrical

energy; toxic chemicals
Microbiological agents Bacteria, viruses, fungi, protozoa, and helminths
 Causes of Cell Damage or death
Category Example
Immune mechanisms Immune disorders can cause tissue damage through a
number of the following mechanisms:
Anaphylaxis (causing release of vasoactive amines)
Cytotoxicity (causing the target cell to be lysed)
Immune complex disease (leading to release of
lysosomal enzymes)
Cell-mediated hypersensitivity (leading to cytotoxicity)

Genetic defects Disorders with polygenic inheritance – diabetes

mellitus, gout
Mendelian disorders – X-linked disorders, autosomal
dominant and recessive disorders, disorders with
variable modes of transmission
Nutritional disorders Protein-calorie malnutrition, vitamin deficiencies,
mineral deficiencies, obesity and its consequences
Determination of enzyme activity
The usual methods for determining enzyme
activity are kinetic tests in which the
photometrically measured change in the
absorbance of an indicator per unit of time
is used as a measure of the rate of reaction.
This is proportional to the enzyme activity.
 As the enzymes are determined quantitatively on
the basis of their catalytic activity, the activity is
expressed in kinetic units. The following units have
been defined:

- international unit (U); one international unit is the

quantity of enzyme which catalyzes one micromole
(μmol) of substrate per minute. The catalytic
enzyme activity of a sample is expressed in units
per liter (U/ L), milliunits per liter (mU/L) or
kilounits per liter (kU/ L).
- katal; 1 katal is the quantity of enzyme that
catalyzes the turnover of 1 mole of substrate per
second. The catalytic enzyme activity of a sample
is usually expressed in μkatal/L.
 Enzyme kinetics
 Activity units
 1 katal = 1 mole of a substrate transformed per 1 sec
 1 IU = 1 μmole of a substrate transformed per 1 minute

1 katal = 1 mole / 1 sec

= 106 μmole / 1 sec
= 60 x 106 μmole/1 min (= 60 sec)

Conversion: 1.0 μkatal/L = 60 U/L

1 katal = 6 x 107 IU
Enzyme Assay

Enzyme measurements have traditionally

used kinetic assays to determine activity.
Newer assays have been developed,
however, that measure protein (mass)
concentrations.
Naming enzymes

Specific suffix of enzyme names:

-ase

a) substrate name + -ase (e.g. amylase)

b) reaction type + -ase (e.g. dehydrogenase)

= group name, not individual enzyme
 Add class to which each enzyme belongs

AST aspartate aminotransferase

ALT alanine aminotransferase
GGT gamma-glutamyl transpeptidase
ALP alkaline phosphatase
ACP acid phosphatase
AMS -amylase
LPS lipase
CK creatine kinase
CHE cholinesterase
LD lactate dehydrogenase
The tasks of diagnostic enzymology
 Site of the disease (organ localization)

The damaged tissue or organ can be localized by:

- determination of tissue specific enzymes:
- isoenzyme analysis
- evaluation of symptom-oriented enzyme patterns
 hepatitis High level of ALT, AST, ALP, GGT

pancreatitis High level of Amylase, Lipase

 dermatomyositis

Inflammation of the muscles and the skin :

High level of CK, AST, LDH
The tasks of diagnostic enzymology

 Severity of the cell damage

 Diagnosis of the disease

 Differential diagnosis of the disease within

an organ
Diagnostic information is obtained from:

- the level of enzyme activity in the sample

the level of ALT activity
 Case 1

 胡先生 72 yo
   present with gastrointestinal symptoms
including nausea, vomiting, fatigue, abdominal
pain.
 3 days later characterized by jaundice,
hypoglycemia, coma.
 Case 1
胡 ×× ，男， 72 岁 out-patient
ALT 9868 IU/L ＜４０
AST 10211 IU/L ＜４０
ALB 38.5 g/L 35 ～ 55
GLOB 29 g/L
TP 67.8 g/L 60 ～ 90
A/G 1.3 1.2 ～ 2.4
ALP 130 IU/L 45 ～ 132
GGT 36 IU/L 6 ～ 78
TB 87.3 μmol/L 4.3~27.2
DB 20.7 μmol/L 0~6.8
TBA 16.7 μmol/L <10
CHE 124 U/L 200~400
 Interest in eating mushrooms has risen
　

dramatically in recent years as part of the

back-to-nature and organic food
movement.  Mushroom poisoning is
usually the result of ingestion of wild
mushrooms after misidentification of a
toxic mushroom as an edible species.
 Case 2
王 ×× ， 52 yo ， loss of
appetite, nausea, vomiting, mild
fever, and dark urine.

COLOR dark yellow

UBIL +1
URO +2
 Case 2
王 ×× ，男， 52 yo
ALT 1685 U/L ＜４０
AST 1362 U/L ＜４０
ALB 40.7 g/L 35 ～ 55
GLOB 36 g/L
TP 76.5 g/L 60 ～ 90
A/G 1.1 1.2 ～ 2.4
ALP 156 U/L 45 ～ 132
GGT 365 U/L 6 ～ 78
TB 56.0 μmol/L 4.3~27.2
DB 29.0 μmol/L 0~6.8
TBA 39.2 μmol/L <10
CHE 312 U/L 200~400
Case 2
What test would you order next ？

A. serologic marker
B. Liver Function Tests
C. Tumor marker
D. Consider ultrasound
 Case 2
王 ×× ，男， 52yo
HAV-IgM + 阳性
HBsAg -
HBsAb -
HBeAg -
HBeAb -
HBc-IgM -
HBc-IgG -
HCV-Ab -
HEV-IgM -

治疗：清淡饮食、保肝药物、注意隔离、监测肝功
 Case 3
段 ×× ，男， 42yo check-up
ALT 51 U/L ＜４０
AST 59 U/L ＜４０
ALB 39.5 g/L 35 ～ 55
GLOB 25 g/L
TP 64.2 g/L 60 ～ 90
A/G 1.6 1.2 ～ 2.4
ALP 118 U/L 45 ～ 132
GGT 96 U/L 6 ～ 78
TB 21.3 μmol/L 4.3~27.2
DB 10.2 μmol/L 0~6.8
TBA 1.2 μmol/L <10
CHE 284 U/L 200~400
Diagnostic information is obtained from:
- the determination of enzyme patterns (of all
enzyme activities present in the serum at the
same time)
- evaluation of enzyme activities in relation
to each other, e.g. calculation of enzyme
ratios
monitoring enzyme activities

- determination of isoenzymes.
 Isoenzymes:
 Isoenzymes are different forms of an enzyme
which catalyze the same reaction, but which
exhibit different physical or kinetic properties,
such as isoelectric point, pH optimum.
 Diagram of the origin of isoenzymes, assuming the
existence of two distinct gene loci.
Structural a b
genes
When the active enzymes are
mRNA polymers containing more
than one subunits, hybrid
A B isoenzymes consisting of
Polypeptides mixtures of different subunits
may be formed. One such
A B isoenzyme is formed in the
subunits
case of a dimeric enzyme,
such as CK, and three if the
Possible enzyme is a tetramer, such as
dimers LD.

Possible
tetramers
CKMM CKMB CKBB

Possible dimers
LDH1 LDH2 LDH3 LDH4 LDH5

Possible tetramers
 Isoenzymes:
 the isoenzyme complement of each tissue is
genetically determined. Isoenzyme analysis
makes it possible to identify the tissue of
origin of increased enzyme activity, e.g.
pancreatic amylase, CK-MB, LD-1
 Lactate dehydrogenase (LD)

LD is present in varying amounts in the cytoplasm of all

cells in the body. Elevations of total LD activity are
therefore found in many pathological conditions but are
as such of limited diagnostic and differential diagnostic
value because of the lack of organ specificity. However,
if the total LD is elevated, quantitative differentiation of
the isoenzymes can provide diagnostically useful
organ-related information.
 LD isoenzyme
Isoenzyme Chief sources Possible cause
LD-1;HHHH; Cardiac muscle, Cardiac muscle damage (infarction,surgery)
LD-2;HHHM; erythrocytes
and kidneys Hemolysis (in vivo or in vitro)

Ineffective erythropoiesis

Muscular dystrophy

Renal infarction

Germ cell tumor

LD-3;HHMM Spleen, lung, lymph nodes, platelet destruction

Platelets, endocrine glands Lymphoreficular disease

Splenic infarction

Malignant tumor

LD-4;HMMM Liver, skeletal muscle Hepatobiliary disease

LD-5;MMMM Right heart failure

Skeletal muscle damage

Prostatic carcinoma

Malignant disease
 Principle: electrophoretic separation of the
specimen on agarose gel at an alkaline pH. The
rate of migration to the anode is dependent on the
subunit composition of the isoenzyme.
Isoenzymes containing the H subunit migrate
quickly, those containing the M subunit slowly,
LD-1 therefore has the highest migration rate,
LD-5 the lowest.
Electrophoresis of LD isoenzymes (%)
LD-1 HHHH 15-23
LD-2 HHHM 30-39
LD-3 HHMM 20-25
LD-4 HMMM 8-15
LD-5 MMMM 9-14
LD isoenzyme

 LD1/LD2↑
 Heart attack
 Hemolytic disease
LD isoenzyme

 LD5↑
 Liver damage (hepatitis)
 Skeletal muscle damage
Biological and interfering factors
 Elevated or decreased enzyme activities can
also result from biological effects or
interfering factors which influence the test
in question. Biological effects lead to
changes in the serum enzyme activities in
vivo, i.e. before collection of the specimen,
while interfering factors alter the results in
vitro.
Biological factors
Important biological factors which lead to
changes in enzyme activity are diagnostic
and therapeutic procedures, diet, alcohol,
physical activity, pregnancy, posture and
tourniquet application during sample
collection, and postsynthetic changes in the
enzyme.
 Food intake
 Conditions of specimen collection
 Intraindividual variation
 Age
 Exercise
 Diet
Interfering factors
 Important interfering factors which can lead
to in vitro changes in enzyme activities are
hemolysis, hyperbilirubinemia and
metabolites in the sample.
 Hyperbilirubinemia
 Hemolysis
 Hypertriglyceridemia
 Enzyme stability during storage
Pancreas: Amylase & Lipase
 Function
 Exocrine
 precursor digestive enzymes
 lipases

 Endocrine
 metabolic hormones
 Insulin from β cells
 Glucagon from α cells
 Amylase

 Amylase refers to a group of enzymes

which break down sugars and starches
 Amylase is produced by the pancreas and
salivary glands and released into the mouth,
stomach, and intestines.
Urinary amylase

• Due to its small size, amylase is easily filtered by

the glomerulus of the kidney and excreted in the
urine.

• Therefore, urinary amylase should be increased in

pancreatitis. levels of amylase in the urine to

remain high for several days longer than blood
amylase levels.
Reference interval ：
 Serum : 30-110 U/L
 Urine : 32-640U/L
 Clinical significance
 higher than normal amylase levels may
indicate inflammation of the pancreas
(pancreatitis). Pancreatitis can be caused by
excessive use of alcohol, infection, injury,
some medications, or blockage of the
pancreatic duct.
 Clinical significance
 higher than normal amylase levels may
also indicate inflammation of the salivary
glands because of conditions such as
mumps or blockage of the glands.
Parotitis :
 Appearance of a
swollen face
 Pain, headache ,fever
 Loss of appetite
 Clinical significance
 macroamylasemia, an uncommon and
harmless condition in which amylase is
bound to a protein in the blood, can cause
high blood amylase levels and low or
normal urine amylase levels.
 high amylase levels may also result from
cancer of the pancreas.
 Clinical significance
 hyperamylasemia is also observed in
several other pancreatic as well as
nonpanceatic disorders, and the diagnostic
value of a single elevated amylase level
without regard to other clinical findings is
limited.
 Lipase
 Lipase is a enzyme produced in the
pancreas to help digest fats. If the pancreas
is damaged, or if the pancreatic duct leading
from the pancreas to the duodenum is
blocked, large amounts of lipase may be
released into the bloodstream.
Reference interval ：
 Serum : 23-300 U/L (depending on the lab)
 Clinical significance
 the clinical specificity for the diagnosis of
pancreatitis is only 40% for amylase and 60%
for lipase. The specificity for amylase is low
because of the existence of nonpancreatic
sources of the enzyme such as the salivary
gland. The specificity of lipase is influenced by
the design of the assay used.
 Clinical significance

 Amylase and lipase should be tested

simultaneously. Amylase by itself is relatively
insensitive for making the diagnosis of
pancreatitis. It tends to fall to normal more
rapidly than lipase, so that in late pancreatitis
only the lipase is elevated.
 Clinical significance
 Acute pancreatitis is highly likely when the
plasma amylase is increased to more than
twofold normal and lipase is increased more
than fivefold normal.
 Clinical significance
 When plasma amylase is increased, but the
lipase is normal, a nonpancreatic condition
is almost always the cause. When amylase
is increased more than twofold and lipase is
increased but less than fivefold, renal
failure, intestinal obstruction, and acute
pancreatitis are possible causes.
 Alkaline phosphatase (ALP)
ALP is an enzyme found in several tissues throughout
the body, including liver, bone and kidney.
In the liver , it is found on the edges of cells that join to
form bile ducts-tiny tubes that drain bile from the liver
to the bowels where it is needed to help digest fat in the
diet.
ALP in bone is produced by special cells called
osteoblasts that are involved in the formation of bone.
 Clinical significance
 Elevations of ALP are most commonly caused by
liver disease or bone disorders.
Any conditon causing excessive bone formation,
including bone disorders such as Paget`s disease
and others such as rheumatoid arthritis and healing
fractures, can cause increase ALP levels.
 Acid phosphatase (ACP)
ACP is an enzyme found throughout the body ,but
primarily in the prostate gland.
It is a phosphatase, a type of enzyme, used to free
attached phosphate groups from other molecules
during digestion.
 Clinical significance
 Elevations of total ACP can be an indicator of
carcinoma of the prostate, bone disease.
 In suspected prostatic carcinoma and for the
evaluation of treatment immunochemical
determination of the concentration of prostate
specific antigen (PSA) is now more usual than
determination of the activity of total ACP .
 In patients with malignant tumors and elevated
levels of ALP, elevation of ACP is a further sign
of skeletal involvement.
 Cholinesterase (CHE)
 An enzyme that splits acetylcholine into
acetic acid and choline, that help the
nervous system work properly.
Cholinesterase (CHE)

key physiological role

in the turnover of the
neurotransmitter
acetylcholine
 Cholinesterase (CHE)
 There are 2 cholinesterases: one is synthesized in
the liver and present in the serum, and the other,
now formally known as acetylcholinesterase, is
synthesized in the RBCs; both are used to
determine the extent of organophosphate
exposure, which are used in pesticides; the serum
form is more useful in detecting acute toxicity
while acetylcholinesterase better reflects chronic
exposure.
Reference interval ：
 Serum : 200 - 400 U/L (depending on the lab)
 Clinical significance

Decreased cholinesterase levels may be due to:

 Liver damage
 Poisoning from organophosphates (chemicals
found in some pesticides) ,which can inhibit
cholinesterase activity.
 Acute infection
 Common Questions

 Do elevated lipase level always mean I have a pancreatic

condition?
In some cases ,an elevated lipase may be due to a
condition other than pancreatitis. Moderately increased
lipase values can occur in other conditions, such as kidney
disease, due to decreased clearance from the blood , a
bowel obstruction, or peptic ulcer disease , although the
lipase test is not usually used to monitor these conditions.
Your doctor will determine whether you have a pancreatic
disorder. He will make a diagnosis based on your
symptoms, medical history, and test results.
 Common Questions

 Why are lipase and amylase tests usually ordered together?

Blood amylase levels are sensitive for pancreatic disorders
but are not specific. Lipase levels are usually more specific
than amylase for diseases of the pancreas. Evaluating the
results of the two tests together help to diagnose or rule out
pancreatitis and other conditions.
 Case 1

* 37 yo male with acute intense abdominal

pain associated with nausea and vomiting
* No significant PMH
* Excessive alcohol in dinner
 Case 3
 WBC 8,000
 Total bilirubin 10.2 (normal < 17)
 AST 35, ALT 45
 Lipase 541 (normal < 300)
 Amylase 425 (normal < 110)
 What is the likely cause of this
patient’s acute clinical symptoms?

A. Appendicitis
B. Kidney stone
C. Pancreatitis
D. Cholycystitis
 Case 4
* 26 yo apple picker
complained of skin rashes,
nausea, headache, sweating
 Case 4
The Lab Data :

WBC 10,100
RBC 4.5
HB 150
ALT 42 (normal < 40)
ALP 136 (normal < 150)
CHE 72 (normal 200 ~ 400)
GGT 55 (normal < 30)
Amylase 80 (normal < 110)
What is the likely cause of this
patient’s clinical symptoms?
A. Liver diseases
B. Kidney diseases
C. Pesticide poisoning
D. Blood diseases
The following enzyme patterns,which
are usually used for screening
hepatobiliary diseases?
A. ALT ALP ACP
B. AST ALT Lipase
C. Amylase CHE Lipase
D. ALT GGT CHE
 Myocardial infarction can cause
elevations of LD. in this case it is
mainly the ( ) that is increased.
A. LD1/LD2
B. LD2/LD1
C. LD5
D. LD3
E. LD4