CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER

LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
PART 1: CREATINE KINASE ISOENZYMES
INTRODUCTION • Dimer containing 2 subunits:
1. M (Muscle)
CREATINE KINASE (CK) 2. B (Brain)
• AKA creatine phosphokinase (CPK) • Isoenzymes (has 3 isoenzymes)
• E.C. 2.7.3.2: ATP:Creatine N- 1. CK1 or CK-BB: brain
Phosphotransferase 2. CK2 or CK-MB: mostly in cardiac
• Molecular weight: 82 kDa (kilo Dalton) muscles (hybrid type)
• Associated with adenosine triphosphate 3. CK3 or CK-MM: In both skeletal and
(ATP) regeneration in contractile or cardiac muscles
transport systems
o Muscle cells: storage and use of
high-energy creatine phosphate to
produce relatively constant levels
of muscle ATP.
- Its predominant physiologic function
occurs in muscle cells where it is involved
in the storage of high-energy creatine
phosphate every contraction cycle of
muscle results in creatine phosphate use
to produce relatively constant level of ATP
in the muscle.

REFERENCE RANGE
• Total CK
➢ Male: 46-171 U/L
➢ Female: 34-145 U/L
- Take note that male has higher
normal values which can be
- This picture shows the reversible
attributed to increase muscle mass
reaction catalyze by creatine kinase.
of males.
Creatine kinase catalyzes the reversible
• CK-MB: ≤ 5% of total CK
transfer of phosphate between creatine
- The normal value of CK-MB is 5% or
and ATP or ADP. This reversible reaction
less compared to that of Total CK.
results to regeneration of
phosphocreatine when ATP is abundant
and regeneration of ATP when ATP is low. CLINICAL CORRELATION

TISSUE SOURCE HIGH CONCENTRATIONS OF CK IN

• Widely distributed in tissue MUSCLE
• Highest activities found in: • Elevated CK level in:
➢ Skeletal muscle ➢ Disorders of cardiac muscle like
➢ Heart muscle myocardial infarction
➢ Brain tissue
• Smaller quantities in bladder, placenta,
gastrointestinal tract, thyroid, uterus,
kidney, lung, prostate, spleen, liver,
pancreas
• Level varies with muscle mass
➢ Gender, race, degree of physical
conditioning, age

KATUSOKS ASSEMBLE 1
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
THREE DISTINCT MOLECULAR FORMS
OF CK
- As mentioned, total CK elevation is found
in numerous disorders hence the
separation of total creatine kinase to its
various isoenzyme fractions is considered
a more specific indicator of various
disorders than total levels typically the
clinical relevance of creatine kinase
activity depends more on isoenzyme
fractionation than on total levels.

➢ Disorder of skeletal muscle like

rhabdomyolysis, muscular
dystrophy particularly Duchenne type
- Extreme elevations of creatine kinase
occur in Duchenne type muscular
• ON ELECTROPHORESIS
dystrophy with values reaching to 50-100
➢ CK-BB (CK1): fastest to migrate
times the upper limit of normal.
toward anode (cathode to anode)
➢ CK-MB (CK2)
➢ CK-MM (CK3): slowest mobility

1. CK-BB
• Present in small quantities in the sera of
healthy people.
• Short half-life of 1-5 hours
- Which result to generally low and
transient CK-BB elevation when
tissue damage occurs and not usually
measurable. However, some
methods of detection can detect CK-
BB enzymes
• Most techniques cannot detect CK-BB
• Highest concentration
➢ Central nervous system (CNS)
➢ Gastrointestinal tract (GIT)
• Sensitive but NOT entirely specific
➢ Uterus during pregnancy
➢ CK elevation is found in various
abnormal cardiac and skeletal
Isoenzyme Tissue Condition
muscle conditions
CK-BB Brain Central nervous
- Although, creatine kinase is a sensitive
system shock
indicator of these disorders like myocardial
infarction or rhabdomyolysis or muscle Bladder Anoxic
dystrophy, it is however not entirely encephalopathy
specific. Lung Cerebrovascular
accident
Prostate Seizure
Uterus Placental or
uterine trauma
Colon Carcinoma
Stomach Reye’s syndrome

KATUSOKS ASSEMBLE 2
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
Thyroid Carbon monoxide Malignant
poisoning hyperthermia
Malignant Reye’s syndrome
hyperthermia Rocky Mountain
Acute and chronic spotted fever
renal failure Carbon monoxide
poisoning
• Serum rarely contains CK-BB of brain
origin • MYOCARDIAL INFRACTION
- Although the brain tissue has high ➢ CK-MB level begin to rise within 4-8
concentrations of CK-BB. What’s the hours
reason for this? ➢ Peak at 12-24 hours
• Big molecular size approx. 80kDa – CK- ➢ Return to normal after 48-72 hours
BB CANNOT cross the blood-brain-barrier - This time frame is important to consider
and does NOT generally appear in the when interpreting CK-MB level
serum. TAKE NOTE:
- It’s passaged across the blood-brain- ➢ Troponins are more sensitive and more
barrier is hindered so even if there is specific than CK-MB, and may elevate
damage to the brain, serum CK-BB in the absence of CK-MB elevation
would not elevate. - Non-enzyme proteins like Troponin I
- Nevertheless, if there is extensive and T are more sensitive and more
damage to the brain especially the specific than CK-MB in detecting acute
integrity of the blood-brain-barrier is myocardial infraction
breached; CK-BB can sometimes be
detected in the serum 3. CK-MM
• Major isoenzyme in the sera of healthy
CREATINE KINASE people (94-100% of total CK)
• Major isoenzyme in striated muscle
2. CK-MB o Skeletal muscle and cardiac
• Undetectable to trace in healthy people o Skeletal muscle: almost entirely
• 20% of all CK-MB comes from cardiac CK-MM isoenzyme, a little CK-MB
tissue isoenzyme in it
• Normally, CK-MB is <6% of total CK o Cardiac muscle: majority CK-MM
➢ ≥6% of CK total is CK-MB: good (80%), 20% CK-MB
indicator of myocardial damage - Hence, injury to the heart and skeletal
muscle accounts to the majority of CK-
ISOENZYME TISSUE CONDITION MM elevation as shown in the table
CK-MB Heart Myocardial infraction
skeletal Myocardial injury ISOENZYME TISSUE CONDITION
muscle Ischemia CK-MM Heart Myocardial
Angina Skeletal Infraction
Inflammatory heart Muscle Skeletal muscle
disease disorder
Cardiac surgery Muscular dystrophy
Duchenne-type Polymyositis
muscular dystrophy Hypothyroidism
Polymyositis Malignant
- Inflammation hyperthermia
of skeletal Physical activity
muscle Intramuscular
injection

KATUSOKS ASSEMBLE 3
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
- These conditions leading to elevation Atypical CK Isoenzyme Bands
of CK-MM is due to the intracellularly
CK-MM isoenzyme is released to the
circulation due to destruction of muscle
fibers

CAUSED OF CK-MM ELEVATION

1. HYPOTHYROIDISM
- Associated with minimal elevation of
CK especially CK-MM isoenzyme. So, • Macro-CK
a lesser degree the CK-MB isoenzyme. ➢ Migrate to a position midway between
The exact pathophysiology is still CK-MB and CK-MM
uncertain. Nevertheless, the possible ➢ 0.8-1.6% of total CK in the serum
explanations include: ➢ An elevated Macro-CK has No
disease correlation
1. Increased muscle membrane permeability
➢ Age- and sex-related: Macro-CK is
resulting to muscle fiber
more frequent among women older
degeneration/destruction
than 50 years old
2. Effect of thyroid hormone on enzyme
1. Largely CK-BB complexed with
activity
immunoglobulin gamma and
3. Slower clearance of CK from the plasma
sometimes immunoglobulin alpha
as a result of slower metabolism caused by
(lgG, and lgA)
Hypothyroidism
2. CK-MM complexed with
2. PHYSICAL ACTIVITY lipoproteins
• Elevation is variable; may be elevated as
long as 48 hours
• Degree of exercise in relation to
exercise capacity of the person: major
determinant of the degree of CK-MM
elevation
➢ Well-conditioned person has lesser
CK-MM elevation compared to less- • Mitochondrial-CK (CK-Mi)
conditioned person o Migrate to a point cathodal or
➢ Levels may be elevated for as long as before the position of CK-MM
48 hours or 2 days o Bound to the exterior surface of the
inner mitochondrial membranes of
3. INTRAMUSCULAR INJECTION muscle, brain, and liver
• CK elevation is <5x the ULN (upper limit o Dimeric form, or the bigger
of normal) following intramuscular OLIGOMERIC FORM aggregates
injections with high MW of 350kDa
• Usually disappears after 48 hours from o Not present in normal serum
injection, but may persist for one week o Present only if there is extensive
tissue damage causing breakdown
of the mitochondria and cell wall
➢ Its presence in the serum has
NO SPECIFIC DISEASE
CORRELATION however it
may indicate severe illness
when detected in the serum

KATUSOKS ASSEMBLE 4
CLINICAL CHEMISTRY 2 LECTURE
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
SIGNIFICANT OF ATYPICAL CK
ISOENZYME BANDS
• Atypical CK isoenzymes may be
measured as CK-MB resulting to
falsely elevated CK-MB levels
- Overall, there is still indefinite correlation
between these atypical CK forms, the
mitochondrial CK and macro CK and
their specific disease state.
- It appears that their significance related
primarily to the methods used for
detecting CK-MB.
- In certain analytical procedures, this
atypical form may be erroneously
measured as CK-MB resulting to
erroneously high CK-MB levels.
- And that would translate to a wrong
diagnostic result and wrong clinical
diagnosis.
METHODS OF MEASUREMENT
METHODS FOR MEASUREMENT OF CK
ISOENZYMES
1. ELECTROPHORESIS
o Reference method
o Reaction is measured using overlay
technique; bands are visualized
under UV light
o Atypical CK isoenzyme can be
separated
- Like macro CK and mitochondrial CK can
be separated and detected apart from the
three major bands.
o Strong fluorescent band close to
CK-BB which is attributed to binding
of fluorescent drugs or bilirubin by
albumin
o Visualization of adenylate cyclase
KATUSOKS ASSEMBLE
1ST SEMESTER
A.Y. 2022 - 2023
(AK) in electrophoresis cathodal to
CK-MM
▪ Unsatisfactory, hemolyzed
sample
- In addition to visualizing atypical CK bands,
another advantage of electrophoresis
method, includes detecting an
unsatisfactory separation and allowing
visualization of adenylate kinase (AK)
- Adenylate kinase (AK) is an enzyme
released from erythrocytes in hemolyzed
samples and may appear as a band
cathodal or prior to the CK-MM
▪ AK interferes with chemical
and immunoinhibition methods
→ falsely elevated CK or CK-
MB
2. ION-EXCHANGE
CHROMATOGRAPHY
o May be sensitive and precise if
performed with good technique
- More potential to be more sensitive and
precise than electrophoretic procedures if
performed with good techniques.
3. IMMUNOINHIBITON METHOD
o To determine CK-MB
o Anti-M to inhibits M activity
- This method uses Anti-M which inhibits
all M activity but not B activity
- CK activity is then measured before and
after inhibition
o Activity remaining after M subunit
inhibition is due to both CK-MB and
CK-BB
- The activity remaining after inhibition of
M is the result of the B subunit activity both
CK-MB and CK-BB
- CK-BB is seldomly present in the serum.
- Basically, we are just measuring the CK-
MB activity only.
▪ Residual activity is multiplied by
2 to account for MB activity
(50% inhibited)
o Major disadvantage: falsely elevated
CK-MB if CK-BB and/or atypical CK
isoenzymes are present.
- If CK-BB or Atypical isoenzyme like Macro-
CK and Mitochondrial-CK are present it
may result to false elevation of CK-MB
5
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
4. RADIOIMMUNOASSAY
o Detects CK-MB with minimal cross-
reactivity
o Measure enzyme concentration
rather than activity → may detect
SOURCE OF ERROR
inactive CK-MB → detection of
myocardial infraction earlier than • Hemolysis: no CK but rich in AK
other methods activity that reacts with ADP to
- This may detect inactive CK-MB produce ATP which is then available
to participate in the assay reaction
resulting to falsely elevated CK level
ASSAY FOR ENZYME ACTIVITY
- Hemolysis of serum samples may be a
• CK activity in serum is UNSTABLE –
source of elevated creatine kinase activity
rapidly inactivated due to oxidation of
- Erythrocytes are devoid of CK, however,
sulfhydryl groups
they are rich in adenylate kinase activity.
- to remedy this, inactivation can be partially
o Only possible with hemolysis >320
reversed by the addition of…
mg/dL hemoglobin releasing
o Remedy: Addition of SULFHYDRYL
sufficient AK that exhaust AK
COMPOUNDS like N-
inhibitors in the reagent
acetylcysteine, mercaptoethanol,
- Trace hemolysis causes little if any CK
thioglycerol, and dithiothreitol
elevation.
• SERUM SHOULD BE STORED IN
- CK catalyzes both forward and reverse DARK PLACE: CK is inactivated by
reactions involving phosphorylation of light
creatine or ATP as shown in the reaction o If inactivated by light, activity is
equation above. restored after storage in the dark at
• Analysis of CK activity reaction is 4C (centigrade) for 7 days, or -
coupled with other enzyme systems, 20C for 1 month
and determination of change in - When the assay is conducted using
absorbance at 340nm sulfhydryl activator
o FORWARD REACTION:
• Effect of muscular activity and
▪ Coupled with pyruvate kinase- muscle mass on CK level
LDH-NADH system o Physically well-trained people
▪ Optimal pH 9.0 tend to have elevated baseline
▪ By Tanzer and Gilvarg CK
o Bedridden people prolonged
periods have decreased CK
activity.
o REVERSE REACTION:
▪ Coupled with hexokinase-
glucose-6-phosphate
dehydrogenase-NADP
system
▪ Optimal pH 6.8
▪ Reaction proposed by Oliver,
modified by Rosalki: most
commonly performed method
in the laboratory
➢ 2x to 6x faster than
forward reaction
➢ Has Less interference

KATUSOKS ASSEMBLE 6
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
PART 2: LACTATE
DEHYDROGENASE
➢ Lactate Dehydrogenase or simply LDH
or LD has systematic name of
E.C.1.1.1.27: L-Lactate: NAD+
Oxidoreductase.
➢ It is a Zinc-containing enzyme
➢ Catalyzes the interconversion of lactic
acid and pyruvic acid as shown on the
LACTATE DEHYDROGENASE
equation below.
FRACTION
- This table shows the proportion of each
of the lactate dehydrogenase
isoenzyme.
- In this table we can deduce that in the
sera of healthy individual, the major
➢ It is a hydrogen transfer enzyme that isoenzyme fraction is LDH-2.
uses the coenzyme NAD+ Followed by LDH-1, LDH-3, LDH-4,
LDH-5
TISSUE SOURCE OF LDH
➢ Widely distributed in the body, found in
the cytoplasm of all cell and tissue in
the body.
➢ High activities: heart, liver, skeletal
muscle, kidney, erythrocyte
➢ Lesser amount: lung, smooth muscle,
brain

Isoenzyme %
LACTATE DEHYDROGENASE
LDH-1 14-26
- Molecular weight: 128 kilodalton (kDa)
LDH-2 29-39
- It is a tetramer which means that it has 4
subunits. LDH-3 20-26
o Each subunit is 32 Kilodalton (kDa) LDH-4 8-16
- Each subunit can either be designated LDH-5 6-16
as H for heart and M for muscle.
o The combination of H and M in 5 - LDH-2: major fraction of LDH
arrangements yielding 5 major isoenzyme
isoenzymes of LDH - Followed by the following in
▪ LDH-1: migrates fastest toward descending order:
the anode o LDH-1, LDH-3, LDH-4, LDH-5
▪ LDH-5: migrates slowest toward - LDH-1 and LDH-2: lower KM
the anode (Michaelis constant) so higher affinity
for lactate than pyruvate.
- LDH-4 and LDH-5: lower KM (Michaelis
constant) so higher affinity for
pyruvate than lactate.

REFERENCE RANGE
• Normal range: 125-220 U/L (units per
liter)

KATUSOKS ASSEMBLE 7
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
RELATIVE PERCENTAGE OF LDH • Elevated total LDH = non-specific
ISOENZYMES finding
• Highest level of total LDH
o Pernicious anemia
o Hemolytic disorders

LDH ISOENZYME AND TISSUE

SOURCE
- For now, let us discuss LDH
isoenzyme, their tissue sources and
the disorder associated with elevated
level of a particular LDH isoenzyme.

- This table shows to you the relative ISOENZYME TISSUE DISORDER

percentage of LDH Isoenzymes in various SOURCE
tissues. LDH-1 ▪ Heart ▪ Myocardial
- Serum: it is predominantly LDH-2, (HHHH) ▪ red blood infarction
followed by LDH-1, -3, -4, and -5. cells ▪ hemolytic anemia
- Heart: primary contains LDH-1 and LDH-
2, but nevertheless, the heart muscle LDH-2 ▪ Heart ▪ Megaloblastic
contains a small amount of LDH-3 and (HHHM) ▪ red blood anemia
LDH-4. cells ▪ Acute renal
- Red Blood Cells: primary contains LDH-1 infarct
and LDH-2, but also contains a small ▪ Hemolyzed
amount of LDH-3 and LDH-4. specimen
- Renal cortex: contains LDH-1, -2, -3 and
-4 LDH-3 ▪ Lung ▪ Pulmonary
- Lung: the lungs primarily contain LDH-3, (HHMM) ▪ Lymphocytes embolism
followed by LDH-4, -2, -1 and lastly LDH-5 ▪ Spleen ▪ Extensive
- Skeletal muscle: primarily with LDH-5 and ▪ Pancreas ▪ Pulmonary
small amount of LDH-4 and LDH-3 pneumonia
- Liver: primarily with LDH-5 and followed ▪ Lymphocytosis
by LDH-4 and LDH-3 ▪ Acute
- Therefore, we can say that the tissue pancreatitis
distribution of lactate dehydrogenase ▪ Carcinoma
varies primarily in its isoenzyme
composition rather than its content of LDH-4 ▪ Liver ▪ Hepatic Injury or
LDH. (HMMM) Inflammation

CLINICAL CORRELATION OF LDH LDH-5 ▪ Skeletal ▪ Skeletal muscle

Widespread activity in Numerous Body (MMMM) muscle injury
Tissue
- Because of its widespread activity in
numerous body tissue, Lactate
dehydrogenase is elevated in a variety
of disorder.
- Increase level are found in cardiac,
hepatic, skeletal muscle and renal
diseases. As well as in several
hematologic and neoplastic disorder.

KATUSOKS ASSEMBLE 8
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
LDH-1 and LDH-2 - So, if there is heart attack or
hemolysis, LDH-1 becomes more
predominant than LDH-2. So, there
will be LDH Flip pattern.
o *LDH-1/LDH-2 ratio more than 1 may be
observed in hemolyzed blood sample

ACUTE MYOCARDIAL INFARCTION

• LDH rises within 12-24 hours
• Peaks within 48-72 hours
• Remains elevated for 10 days

LDH-4
• Elevated in liver damage
▪ LDH-4 levels have greatest clinical
significance in the detection of hepatic
disorders or damage particularly intra-
hepatic disorders
- Heart and red blood cells ▪ This can be appreciated as a significant
o Contain both LDH-1 and LDH-2 peaking of LDH-4 fraction in the
▪ LDH-1 contain is higher
electrophoretogram.
than LDH-2 content
o Acute myocardial infarction and
intravascular hemolysis result to
higher elevation of LDH-1 than
LDH-2 → LDH Flip pattern

LDH-5
• Elevated in skeletal muscle damage or
disorders as depicted in muscle
- What do we mean by LDH Flip dystrophies
pattern? Normally, LDH-2 is more - This can be appreciated as a significant
predominant than LDH-1 in the serum. peaking of LDH-5 fraction in the
But when there is destruction of heart electrophoretogram
and red blood cells which contains
predominantly LDH-1 compared to
LDH-2.

KATUSOKS ASSEMBLE 9
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
TOMBSTONE PATTERN METHODS OF MEASUREMENT OF LDH
- In cases when the total LDH levels is
increased and when the relative Analysis of LDH Isoenzymes
amount….will produce a TOMBSTONE 1. Electrophoresis
PATTERN 2. Immunoinhibition Method
• Isomorphic pattern is produced when total 3. Chemical Inhibition Meth
LDH is increased, and the relative amount 4. Differences in Substrate Affinity
of each isoenzyme is roughly the same.
• Noted in patients with diffuse tissue DIFFERENCES IN SUBSTRATE
damage often accompanied by shock or AFFINITY
hypoxemia.

- The H subunit and M subunit of LDH

LDH-6 have different affinity to alpha-
• Sixth LDH isoenzyme that migrates hydroxybutyrate.
cathodic to LDH-5. • H subunit – has greater affinity for a-
• Migrates cathodic to LDH 5 – located hydroxybutyrate than M subunit.
before LDH 5. - This has led to the use of this substrate
• LDH-6 is alcohol dehydrogenase which in an attempt to measure the LDH-1
can also metabolize lactate activity.
• Elevated in arteriosclerotic • LDH-1 consists of entirely H subunits
cardiovascular failure, liver injury due to ▪ LDH-1: HHHH
severe circulatory insufficiency. • Measurement of a-hydroxybutyrate
• Appearance signifies grave prognosis activity represents LDH-1 activity of
and impending death. total LDH.

LDH COMPLEXED WITH However, a-

IMMUNOGLOBULINS hydroxybutyrate
• LDH can be complexed with dehydrogenase
Immunoglobulin alpha (IgA) and activity is not
Immunoglobulin gamma (IgG) entirely specific
- This can reveal atypical bands on for LDH-1 fraction
electrophoresis. because LDH-2,
• Usually migrates between LDH-3 and LDH- LDH-3, and LDH-4
4. also contain
• Elevation is NOT ASSOCIATED with any varying amounts
abnormality. of H subunit.

KATUSOKS ASSEMBLE 10
CLINICAL CHEMISTRY 2 LECTURE
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
ASSAY FOR ENZYME ACTIVITY
• LDH catalyzes the interconversion of lactic
and pyruvic acids using the coenzyme
NAD+ as shown in this equation:
• REACTION CAN PROCEED IN EITHER:
1. Forward (lactate-to-pyruvate)
reaction
a. Optimal pH 8.3 to 8.9
b. Used by most of the laboratories
1ST SEMESTER
A.Y. 2022 - 2023
• LDH activity is unstable in serum
regardless of temperature at which it is
stored.
o Loss of LDH activity occurs more
quickly at 4C (4 degree centigrade)
than at 25C
o LDH-5 is the most labile isoenzyme
▪ Serum sample for LDH
determination should not be frozen.
• If blood sample cannot be analyzed
immediately, store the blood sample at
25C and analyzed within 24-48 hours.

2. Reverse (pyruvate-to-lactate) • LDH changes after three days of storage

reaction of serum at room temperature.
a. Optimal pH 7.1 to 7.4 o Total LDH decreases by 20%
b. 3x faster than forward reaction o LDH-1 increases by 20%
c. More susceptible to substrate o LDH-5 decreases by 18%
exhaustion and loss of linearity of
reaction kinetics
- Both forward and reverse reaction are
being use in clinical assay
- Although REVERSE REACTION IS 3X
FASTER than forward reaction

SOURCE OF ERROR
- These are the possible sources of error
when measuring LDH.

• RBC contains LDH concentration approx.

100x too 150x than found in serum
o Any degree of hemolysis is
unacceptable sample → FALSE
elevation of LDH

• Artifactual Elevation of LDH

o Exercise: at most 25% rise in average
values when strenuous activity
o Contact with clot
o Physical agitation of specimens
▪ Like what occurs in most
pneumatic tube systems in the
hospital
o Blood transfusion: transient only

KATUSOKS ASSEMBLE 11
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
PART 3: AMYLASE ● The structure of glycogen has similar
AMYLASE (CLINICAL CHEMISTRY) but more branched structure than
● Systematic Name: amylopectin. Approximately every 10
○ E.C. 3.2.1.1 glucosyl residues are being branched.
○ 1,4-D-Glucan
Glucanohydrolase
● Smallest Enzyme
● Molecular weight: 50 - 55 kDa
● Because of its small size, it is readily
filtered by the renal glomerulus and
appears in the urine. ● Α-amylase attacks only the α-1,4-
glycosidic bonds producing
degradation products consisting of
glucose, maltose, and intermediate
chain dextrin that contains α-1,6-
glycosidic bond.
● CO-FACTORS FOR ACTIVATION:
Ca2+(calcium), Cl- (chloride)

● Amylase is an enzyme that belongs to the

class of hydrolases. Hydrolase enzyme
that catalyzes the breakdown of starch TISSUE SOURCE
and glycogen ● Major Sources:
● Starch consists of both amylose and ○ Acinar cells of the salivary
amylopectin. glands
○ AMYLOSE: long and branched ○ Acinar cells of the pancreas
chain of glucose molecules linked ● Minor Sources:
by an α-1,4-glycosidic bond ○ Skeletal muscle
○ AMYLOPECTIN: a branched chain ○ Small intestine
of polysaccharide with linked by an ○ Fallopian tubes
α-1,6-glycosidic bond at the
branch points.

KATUSOKS ASSEMBLE 12
CLINICAL CHEMISTRY 2 LECTURE
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
ISOENZYMES
● S-isoamylase: derived from salivary
gland, fallopian tube, and lungs
○ It is “S” because it is derived
from the salivary gland.
● P-isoamylase: derived from pancreas
○ The letter “P” stands for the
pancreas
- The digestion of starches usually begin
in the mouth with the hydrolytic
reaction of the salivary amylase.
- Salivary amylase activity however
has a short duration because upon
swallowing it is inactivated by the
acidity of the gastric contents
- Now, the pancreatic amylase performs
the major digestion action of starches
once the polysaccharides reach the
intestine.
REFERENCE RANGE
SERUM AMYLASE
• 28-100 U/L
URINE AMYLASE
• 1-15 U/h
- There is no uniform expression of amylase
activity
SOMOGYI UNIT
• Frequently used expression of amylase
activity
• Expression of number of milligrams of
glucose released in 30 minutes at 37C
• 1 Somogyi unit = 1.85 IU
CLINICAL CORRELATION
ACUTE PANCREATITIS
• After the onset of abdominal pain,
SERUM AMYLASE
o Begins to rise after 5-8 hours
o Peaks at 24 hours
o Return to normal within 3-5
days
• Values ranging from 250-1,000
Somogyi units per dL (2.55 X ULN
(upper limit of normal)) or higher
• Level of serum amylase does not
correlate with severity of acute
pancreatitis
KATUSOKS ASSEMBLE
1ST SEMESTER
A.Y. 2022 - 2023
- Now, the elevation of amylase level is
non-specific to acute pancreatitis
because amylase can also be found in
other tissues of the body like the
salivary glands, skeletal muscle, small
intestine and the fallopian tubes
• Hence, when diagnosing acute
pancreatitis, serum amylase or lipase is
only one out of the three criteria in
diagnosing acute pancreatitis
OTHER DISORDERS ASSOCIATED WITH
ELEVATED AMYLASE LEVEL
• Salivary gland diseases like mumps,
parotitis
• Intra-abdominal disease like
perforated peptic ulcer, intestinal
obstruction, cholecystitis, mesenteric
infarction, acute appendicitis
o Important to take note: that
intraabdominal causes of
serum amylase elevation other
than acute appendicitis would
yield a serum ….
o Serum amylase is usually less
than 500 Somogyi units/dL
• Ruptured ectopic pregnancy
• Renal insufficiency
o Excreted through the kidneys
• Diabetic ketoacidosis
• Neoplastic diseases
- Result to hyperamylasemia
with levels high as 50x upper
limit of normal
• Asymptomatic hyperamylasemia
- Occur in 1 to 2 population
13
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
MACROMYLASEMIA
• Condition when an amylase molecule
combines with IgG or IgA or a
polysaccharide to form a complex
that is too large to be filtered across the
renal glomerulus - this is the column for condition mainly
• Common among men in the fifth pancreatic hyperamylasemia, salivary,
across the renal glomerulus hyperamylasemia, macroamylasemia
- What will happen to the amylase
when combined with an
immunoglobulin or a
polysaccharide?
- Since amylase become big and cannot
be filtered by the kidneys there would
be reduction of the urinary excretion of
• this is the column for serum amylase
amylase resulting to elevated serum
level
amylase level but decrease urine
amylase level
• Macroamylase cannot be filtered by
the renal glomerulus -> elevation of
serum amylase, low urine amylase

• for serum lipase level

- -for urinary amylase

• Diagnostic significance: differentiate

of the benign macroamylasemia from
other clinically significant causes of
hyperamylasemia

DIFFERENTIAL DIAGNOSIS OF - amylase clearance creatinine

HYPERAMYLASEMIA AND clearance ratio
MACROAMYLASEMIA

- for serum macroamylase

• Pancreatic hyperamylasemia, salivary,

hyperamylasemia, macroamylasemia
have all elevated serum amylase
• among the three conditions mentioned
only the pancreatic hyperamylasemia
have high serum amylase, serum lipase,

KATUSOKS ASSEMBLE 14
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
urinary amylase, amylase clearance
creatinine clearance ratio, serum • In acute pancreatitis P3 is
macroamylase predominant isoenzyme….
• while the other two have low to normal level • P3: predominant isoenzyme in acute
of serum lipase, urinary amylase, amylase pancreatitis but not specific since P3 is
clearance creatinine clearance ratio also found in cases of renal failure
• Salivary hyperamylasemia high serum
amylase, normal serum lipase, low or
normal urinary amylase, low or normal
amylase clearance creatinine clearance
ratio and no serum macroamylase
• Macroamylasemia have high serum
amylase, normal serum lipase, and since Assay for Enzyme Activity
big macroamylasemia is not filtered by the Amyloclastic Measures the
glomerulus urinary amylase and amylase disappearance of starch
clearance creatinine clearance ratio are substrate
both low and serum macroamylase is Saccharogenic Measures the appearance
high of the product
Chromogenic Measures the increasing
METHODS OF MEASUREMENT color from production of
Amylase Isoenzyme Measurements product coupled with a
• Electrophoresis chromogenic dye
• Chromatography Continuous Coupling of several
• Isoelectric focusing monitoring enzyme systems to
• Serum amylase is a mixture of a monitor amylase activity
number of isoenzymes and they can be
separated on the basis of differences in AMYLOCLASTIC METHOD
physical properties. • Iodine is added to attach to starch
substrate forming dark-blue color:
starch-iodine complex
• Amylase is allowed to hydrolyze
starch molecule into smaller units
• Iodine is released resulting to
decrease in dark-blue color intensity
• The decrease in color concentration
is proportional to the amylase
concentration.
ELECTROPHORESIS OF AMYLASE
ISOENZYMES
• S-amylase (S1, S2, S3, S4, S5)
▪ Migrates most quickly
▪ 2/3 of amylase activity of
normal serum SACCHAROGENIC METHOD
• P-amylase (P1, P2, P3, P4) • The classic reference method for
▪ Migrates slower determining amylase activity which is
▪ Predominating amylase in supported in Somogyi units.
normal urine • The use of starch substrate that is
• Most commonly observed fractions of hydrolyzed by amylase to its
amylase in the electrophoresis: P2, constituent carbohydrate molecules
S1, S2 that have reducing properties

KATUSOKS ASSEMBLE 15
CLINICAL CHEMISTRY 2 LECTURE
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
• Amount of reducing sugar is
measured
• Concentration of reducing sugars is
proportional to amylase activity
• WHEAT GERM LECTIN
CHROMOGENIC METHOD
• Chromogenic dye is added to starch
substrate forming insoluble dye-
substrate complex
• Amylase hydrolyzes the starch
substrate, forming smaller water,
soluble dye-substrate fragments
resulting to increase in color
intensity
• The increase in color intensity of the
soluble dye-substrate solution is
proportional to amylase activity
1ST SEMESTER
A.Y. 2022 - 2023
- In COUPLED ENZYME SYSTEM it
detects both the salivary and
pancreatic amylase together
- Nevertheless, there is also a way of
specifically determining the salivary
amylase level and pancreatic amylase
level by using a salivary amylase
inhibitor which is the ….
➢ preferentially inhibits salivary
amylase
- so, what remains for measurement
is the pancreatic amylase activity
only
➢ Hence, salivary and pancreatic
amylase can be estimated by
measuring total amylase in the
presence and absence of wheat germ
lectin and the total amylase level in
the presence of the inhibitor which is
the wheat germ lectin

SOURCES OF ERROR
Amylase in serum and urine is STABLE
● Little loss of activity at room
temperature for 1 week, or at 4C° (4
degrees centigrade) for 2 months.

Triglyceride can suppress or inhibit serum

CONTINUOUS MONITORING METHOD
amylase activity
- Hypertriglycemia itself can cause acute
pancreatitis hence,
● Amylase may be normal or falsely
low in acute pancreatitis with
hyperlipidemia.

The administration of:

• Morphine and other opioids except
• Coupled enzyme systems the pyridine as analgesic or pain
- Recently, this is being used to reliever before blood sampling will
determine amylase activity by lead to: falsely elevated serum
continuous monitring technique amylase level
➢ Primary enzyme: AMYLASE ● This class of drug causes constriction
➢ Coupling enzyme: a-glucosidase, of sphincter of Oddi and pancreatic
hexokinase, and glucose-6- ducts → elevation of inarticulate
phosphate dehydrogenase pressure causing regurgitation of
• Optimum pH 6.9 amylase into the serum thereby
• The increase in absorbance of causing falsely elevated serum
NADH/reduced NAD at 340nm is amylase level.
measured which is proportional to the
amylase activity

KATUSOKS ASSEMBLE 16
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
CAUTION: Specimen should NOT be PART 4: LIPASE
contaminated with saliva INTRODUCTION
● Saliva amylase content is 700x that • E.C. 3.1.1.1: Triacylglycerol
of serum which may cause falsely high Acylhydrolase (Lipase’s systematic
serum or urine amylase level name)
• RBCs do NOT contain amylase so • Hydrolyzes the ester linkages of fats to
hemolysis has no effect on most produce alcohols and fatty acids
amylase determination methods
EXCEPT those coupled-enzyme
methods in which released peroxide is
determined by a coupled-peroxidase
reaction.

• Plasma specimens should be

anticoagulated with HEPARIN ONLY.
- If plasma is used in determining the • Specific for the fatty acid residues at
amylase level blood should be positions 1 (first arrow from the top) and
coagulated with HEPARIN ONLY. 3 (second arrow) of the triglyceride
molecules (Pancreatic lipase’s
● Citrate or oxalate as anticoagulants enzymatic activity)
should be avoided at all times if we want
to determine the amylase activity because
they are calcium chelators.
- Amylase is a calcium containing
enzyme. And citrate and oxalate are
calcium chelators which potentially
decreased amylase activity.

• It catalyzes the partial hydrolysis of

dietary triglycerides in the intestine to
one mole of 2-monoglyceride
intermediate + two moles of long-chain
fatty acids
o Reaction rate is accelerated by the
presence of colipase, and bile salt
for fat emulsification
▪ In the absence of
emulsification, lipase is
ineffective

LIPASE
• Molecular weight: 45 kDa
• Readily filtered by the glomerulus
(owing to its low molecular weight) but
lipase is normally completely
reabsorbed by the proximal
convoluted tubules – LIPASE IS
ABSENT IN NORMAL URINE
o In patients with failure of renal
tubular reabsorption caused

KATUSOKS ASSEMBLE 17
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
renal disorders, lipase is found LIPASE ISOENYMES
in the urine.by • Three isoenzymes
• Calcium: necessary for maximal lipase o L1
activity o L2: (thought to be the) most
o Inhibition of lipase at higher calcium clinically specific and
concentration due to the sensitive
interference of high calcium level o L3
with the action of bile salts at the • Normal: <38U/L
water/substrate interface. o Normal serum lipase level
▪ Way higher concentration of
calcium has paradoxically
CLINICAL CORRELATION (Of elevated
inhibitory effect to lipase
lipase level)
activity
• In acute pancreatitis, serum lipase
o Increases in 4-8 hour
TISSUE SOURCE
o Peaks at 24 hours
• Pancreas: major and primary source of
o Remains elevated for 8-14
serum lipase
days, rarely last longer than
o Better first-line test for diagnosis of
14 days
acute pancreatitis than serum
▪ In acute pancreatitis
amylase
Serum lipase
- The major and primary source
increases in 4-8 hours
of serum lie basically pancreas
*NOTE: The extent of elevations does not
compared to serum amylase
correlate with severity of disease
which has various fractions of
isoenzymes lipases which
OTHER CAUSES OF ELEVATED SERUM
makes it the better first-line
LIPASE
test.
• Chronic pancreatitis
- Although elevated lipase levels
• Obstruction of pancreatic duct
also may be found in other
• Non pancreatic conditions: renal
intra-abdominal conditions
disease, acute cholecystitis,
aside from acute pancreatitis,
intestinal obstruction or infarction,
but it has less frequency than
duodenal ulcer, liver disease,
the elevations of serum
alcoholism, post-ERCP
amylase.
- Post procedure complication
• Lipase level is normal in conditions of
like in endoscopic retrograde
salivary gland involvement
colangio pancreatic
o Lipase level is normal in conditions of
chromatography or eRCP
salivary gland involvement, unlike
amylase level.
RENAL DISORDER
- Therefore, lipase can
As mentioned, lipase can increase in renal
differentiate serum amylase
disorder. How does this happen?
elevation due to pancreatic
• Lipase is filtered by the glomeruli
disorders versus salivary gland
owing to its low molecular weight
disorders.
• Lipase is small with molecular weight of
• Other source of lipase:
45kDa
• Liver, stomach, intestine, white blood
o Readily filtered by the
cells, fat cells and the milk
glomerulus but normally
completely reabsorbed by
the proximal convoluted
tubules – LIPASE IS
ABSENT IN NORMAL URINE

KATUSOKS ASSEMBLE 18
CLINICAL CHEMISTRY 2 LECTURE 1ST SEMESTER
LECTURE | PROF: CHARLIE CLARION, RMT, MD, FPCP | PRELIMS A.Y. 2022 - 2023
LE S S ON 1: CRE AT INE K I NA SE , L A CT A TE DE HY DR OGE N A SE , AM YL A SE & L IP A SE
• Patients with failure of renal • Modified Cherry -Crandall Method
reabsorption ➢ Substrate: TRIOLEIN – a
o Lipase may now be found in purer form of triglyceride
the urine
• Urine lipase activity in the absence of
pancreatic disease in inversely related
to creatinine clearance
- Which means that, the
higher the urine lipase
activity the lower the
creatinine clearance 2. TURBIDIMETRIC METHOD
- The higher the urine lipase • Simpler and more rapid than titrimetric
activity the more severe the method
renal failure
• Fats in solution create a cloudy
• MUMPS (virus) emulsion
- Does not only affect the • Lipase is added in the emulsion of fats
salivary glands • As fats are being hydrolyzed by lipase
- It also affects other tissues or the particles dispersed and the
organs in the body, such as: emulsion becomes less turbid
the Pancreas among others
• The decrease in turbidity at 400nm
o Elevation of lipase in a patient with
after incubation is proportional to
mumps strongly suggest significant
lipase activity in the specimen
pancreatic involvement by the
mumps virus itself
3. COLORIMETRIC METHOD
• Based on coupled reactions with
METHODS OF MEASUREMENT (of
coupling enzymes like glycerol
lipase level)
kinase, glycerophosphate oxidase,
ASSAY FOR ENZYME ACTIVITY
and peroxidase
• Titrimetric method (estimation of
liberated fatty acids) and turbidimetric
method
o With the reaction principle
shown in the picture below.

SOURCES OF ERROR
1. TITRIMETRIC METHOD
• Lipase is stable in the serum
• Classic Cherry-Crandall Method
➢ Room temperature for 1
Titrimetric assay of lipase measurement
week (negligible loss in the
used in:
activity)
➢ 4 degrees centigrade for 3
weeks
• Hemolysis should be avoided because
it causes falsely low lipase level
➢ Substrate: Olive oil (despite the stability of lipase)
➢ Measurement: Liberated fatty ➢ Hemoglobin inhibits the
acids by titration after 24-hour lipase activity (in serum)
incubation • Other inhibitors of lipase activity
➢ Heavy metals, quinine

KATUSOKS ASSEMBLE 19