CLINICAL CHEMISTRY 2

✔ Enzyme
∙ Biologic proteins that catalyze biochemical reactions
● Are intracellular proteins
● Produced by living cells
● Affect velocity of a chemical reaction/hasten or speed up chemical reaction in organic substance
● Presence can increase reaction between 2 reactants (substrates) to speed up formation of product
● Are not consumed nor changed with reactions
∙ measured in lab in terms of activity, not absolute values
● Activity of enzyme to a substrate is directly proportional to its concentration (↑faster reaction
↑concentration)
∙ serum = enzyme concentration is low
If increased, indicates:
1. Cellular injury/damage
2. Increased membrane permeability
● Enzymes are useful for determining tissue/organ damage in patient
✔ Functions of Enzymes
∙ hydration of carbon dioxide (during respiration)
● Enzymes also hasten carbonic acid formation (important in buffering pH of plasma)
∙ nerve induction (makes it faster)
∙ muscle contraction (accelerate conversion of substrates that contract muscle)
∙ nutrient degradation (digestion)
● Ex. macromolecules like lipids, proteins, carbohydrates require enzymes to convert into simpler
form for absorption process
∙ growth and reproduction
● Works w/ hormones in development of cells
∙ energy storage and use (in the form of creatine phosphate in muscle)
✔ General Properties and Definitions
∙ Components of an Enzyme
▪ Active Site:
● A waterless cavity of an enzyme where substrates bind and undergo chemical reactions
● Where substrates bind
● There is specificity between substrates and enzymes
▪ Allosteric Site :
● Waterless cavity that binds regulatory or effector molecules

✔ Terms associated with enzymes

● Substrates : are substances acted upon by enzymes
 ○ Are specific for each enzyme
● Cofactors : non protein substances added in the enzyme-substrate complex to manifest activity
○ Presence would enhance enzymatic activity
a. Coenzyme : considered as a 2nd substrate
i. An organic cofactor that hastens enzymatic reaction
ii. Prosthetic group - a tightly bound coenzyme on another enzyme
iii. Ex. Nicotinamide adenine dinucleotide (NAD) and NADP
b. Activator : are usually metal and non metal ions that facilitates enzyme-substrate binding
i. Alters spatial configuration of enzyme active site to facilitate substrate binding
ii. Ex. Mg, Ca, Cl, Br
● Isoenzyme: enzymes w/ similar enzymatic activity but differ in physical, biochemical, and
immunologic characteristics
○ Measurement of isoenzyme is preferred than total enzyme activity bc it makes the test highly
specific to target tissue/organ (ex. CK which has 3 isoenzymes)
● Apoenzyme: protein portion of enzyme that is subject to denaturation process
○ Denaturation of apoenzyme = function is lost
● Holoenzyme: active substance formed by the combination of a coenzyme and apoenzyme

✔ Enzyme Nomenclature
∙ Enzyme Commission of IUB
▪ Systematic name (long name): defines substrate acted on, reaction catalyzed, and possibly
the coenzyme involved
● Ex. Orthophosphoric monoester phosphohydrolase
▪ Recommended name: usable or trivial name
● Ex. Acid phosphatase
▪ EC numerical code :
o composed of 4 digits
● Ex. E.C.3.1.3.2

● 4th digit = serial number specific to the enzyme sub subclass

✔ Enzyme Classification

1. Oxidoreductases - catalyze redox reaction between 2 substrates

-hydrogen ion is commonly transferred atom in oxidoreductase
-coenzymes are also part of the oxidoreductase activity
▪ A-+ B → A + B-
 ▪ E.g: dehydrogenases (LD and G6PD)
2. Transferases - catalyze the transfer of chemical group other than hydrogen atom between 2 substrates
▪ A-X + B → A + B-X
▪ E.g: ALT, AST, GGT, kinase group (specific for phosphate group)
3. Hydrolases - catalyze the hydrolysis of various bonds
- can only act on this bond in the presence of water (w/o water, hydrolase wont function)
▪ A–B + H2O → A–OH + B–H
▪ E.g. : amylase, lipase, phosphatases
4. Lyases - enzymes that catalyze the removal of groups from substrates without hydrolysis
- product of enzymatic activity remain to contain double bonds
▪ ATP → cAMP + PPi
▪ E.g. : Fructose bisphosphate aldolase (ALS)
5. Isomerases - catalyze the interconversion of geometric, optical, or positional isomerase
▪A→B
▪ E.g.: Triphosphate isomerase (TPI)
6. Ligases - catalyze the joining of 2 substrate molecules coupled with breaking of the pyrophosphate
bond in ATP
▪ Ab + C → A–C + b
▪ E.g. : Glutathione Synthetase (GSH-S)

✔ Enzyme Kinetics
▪ Catalytic Mechanism of Enzymes
▪ Enzymes catalyze physiologic reactions by lowering the activation energy level that the
reactants must reach
- Activation energy level is important for the product formation
- Greater activation energy level required = longer product formation

✔ Catalytic Mechanism of Enzymes

● Relationship between enzyme, substrate, and product
 ▪ Absolute specificity : enzymes combine with only one substrate and catalyze only one reaction
- Ex. lactase would combine w/ lactose to allow release of glucose + galactose
▪ Group specificity: enzymes combine with all substrates containing a particular chemical group
- Ex. kinase → phosphate groups
▪ Bond specificity: enzymes combined with substrates with specific chemical bond
- Ex. lipase → ester bonds
▪ Stereoisomeric specificity: enzymes combine with only one specific optical isomer
✔ Factors that Influence Enzymatic Reactions
▪ Substrate Concentration:
● with amount of enzyme exceeding amount of substrate, the reaction rate steadily increases
● ↑ substrate present = ↑rate of enzyme activity
● Saturation kinetics happen when substrate concentration reaches max value thus addition of
substrate will no longer increase the rate of enzyme activity

1. First order kinetics

a. Reaction rate is proportional to the substrate concentration
2. Zero order kinetics
a. Only a fixed number of substrate (in excess) is converted to product per second
● Used in the laboratory bc the substrate used by the manufacturer of the test reagent will
only have certain concentration of substrate that is used in the reagent
● In lab, ↑ activity + concentration of enzyme = measurement of machine is out of range
○ Dilute the serum to ↓enzyme concentration = machine can read
○ Do not report the result of the diluted serum, multiply first the dilution factor to
the diluted serum result
▪ Enzyme Concentration: ↑ enzyme concentration = ↑ reaction
● ↑ enzyme present, more enzymes can bind to the available substrate
▪ pH :
● Most physiologic reaction occur in range of 7.0 to 8.0 (near plasma pH)
● Extreme pH level may denature protein portion of enzyme
○ Some enzymes can resist ↑acid or ↑base such as phosphatases
■ Ex. acid phosphatase and alkaline phosphatase
▪ Temperature: assay temp should be constant within ±0.1degC at which the enzyme is active at
25degC, 30degC, 37degC
● Optimal is body temp = 37degC
● There are 2 types of temp for enzyme measurement
○ Room temperature = 25degC
■ Incubation time is longer
○ Warm temperature = 37degC
■ Incubation time is shorter
● ↑temp = ↑rate of chemical reaction
○ due to increased movement of molecules
○ Q10 or Temperature coefficient happens when temp is ↑ by 10 degC resulting in a two-fold
increase in enzyme activity
 ● If temp goes beyond body temp (ex. 40 degC), denaturation process starts
○ Inactivation/significant denaturation is seen @ 56 degC
○ Complete inactivation is seen @ 60-65 degC
● Most enzymes are thermolabile (means inactive at ↑temp)
○ Placental (Regan) Alkaline phosphatase is thermostable
● ↓temp = enzymes are reversibly inactive
○ Refrigerator and freezer temp makes enzymes inactive but can be reversed when returned
to RT/body temp
○ Thawing for enzyme serum samples is only done once; multiple freeze-thawing procedure
can cause protein denaturation causing inactivation of enzymes
▪ Cofactors:
● Enhances enzyme activity
1. Activators
a. Enhance binding of substrate to active site
b. Ex. metallic (Ca, Fe, Mg, Mn, Zn) and non metallic (Br and Cl)
2. Coenzymes (prosthetic groups)
a. Serve as second substrates for enzymatic reaction (NAD)
▪ Inhibitors: interferes with the enzymatic reaction with the substrate; prevent catalytic action
● 3 types:
○ Competitive inhibitor
■ Physically bind/compete to active site
■ Can prevent substrate-enzyme binding, affect conversion of substrate to
product
○ Noncompetitive inhibitor
■ Binds to an enzyme other than the active site such as allosteric site of
enzyme
○ Uncompetitive inhibitor
■ Binds with enzyme-substrate complex
▪ Storage of the sample (to preserve enzyme activity)
● Serum @ -20degC or colder (freezer temp)
● Substrate and coenzyme storage @ ref temp (2-8degC) or 1-6degC
● Sample containing LDH @ RT (20-25 degC)
▪ Hemolysis and Milky specimen
● Hemolyzed sample cause erroneous enzyme measurement due to enzymes present in RBC
○ Causes false ↑ result; repeat blood collection
● Milky/lipemic/lactescence spx has ↑ fat content indicating improper fasting
○ ↑fat after proper fasting = lipid metabolism disorder
○ Cause false ↓ result; interferes with colorimetric method
Note: presence of isoenzyme can ↑enzyme measurement specifically the test specificity

✔ Measurement of catalytic activity

● Increase in product concentration
○ Enzyme activity is increased to convert substrate to product, as more product is formed more
substrate is converted
○ Conversion activity is made possible through the presence of enzymes
● Decrease in substrate concentration
● Decrease in coenzyme concentration (NADH)
○ Can also be measured since it is also known as the 2nd substrate
○ Enzymes determined with coenzymes are oxidoreductases
● Increase in altered coenzyme concentration
○ In the presence of oxidoreductases, there will be changes in reduced and oxidized NAD
 ○ Formation of reduced NADH, the effect in the absorbance reading is increased
○ Formation of oxidized NAD, the effect in the absorbance reading is decreased

✔ Measurement of catalytic activity

● Dependent on enzyme concentration
● Always performed in zero-order kinetics
● Performed during the linear phase of reaction
✔ General methods of measuring enzymatic reaction
● Fixed-time (two point) assay
○ Combine certain volume of sample and reagent, incubate the tube, then measure the absorbance
○ Reagents are combined, the reaction proceeds, the reaction is stopped and the amount of reaction
is measured.
● Continuous monitoring or kinetic assays
○ Multiple enzyme activity is included in the procedure
○ In actual practice, the interval is 30 to 60 seconds
○ This can be either manual or automated
■ Manual = combine sample and reagent, incubate, read absorbance for 1st absorbance
reading; reincubate again, read absorbance for 2nd absorbance; reincubate again, read
absorbance for 3rd absorbance
■ Automated = has multiple absorbance readings due to multiple measurements at a given
fixed interval (usually 30 to 60 seconds)
○ Deviation from zero kinetics can be observed
○ 2 types of enzymes involved
1. Primary enzyme
a. Target enzyme in the patient’s serum
b. Enzyme of interest
2. Secondary enzyme (aka coupling/indicator enzyme)
a. Rely on the primary enzyme’s activity for the enzymatic activity to proceed
Note:
● Michaelis-Menten Equation
○ Is a mathematical representation of the relationship between the velocity of enzymatic reaction and the
substrate concentration

✔ Calculation of Enzyme Activity

▪ International Unit (EC)
▪ the amount of enzyme that will catalyze the reaction of 1 μmol of substrate per
minute. (μmol/min)
▪ Katal (SI)
▪ the amount of enzyme that will catalyze the reaction of 1 mol of substrate per
Second (mol/s)
✔Measurement of Enzyme Mass
● Quantified by electrophoretic techniques that provides resolution of isoenzymes and isoforms
 ✔ Enzyme Theories - in these 2 theories, enzyme-substrate binding would cause enzyme catalytic action to speed
up conversion of the substrate into a product
▪ Emil Fisher's/ Lock and Key theory :
● The shape of the key, which is the substrate, must fit in the lock, the enzyme
▪ Kochland's Induced Fit Theory :
● Based on the substrate binding to the active site of the enzyme
● Substrate would attach to the binding site, configuration of the substrate would be
manipulated so that it would fit to the active site of the enzyme where the substrates should
attach

Enzymes of Clinical Significance

✔ LIVER ENZYMES
Note: suffixes ending in -ate finishes with -ate (ex. glutamate-oxaloacetate); suffixes ending in -ic finishes with -ic
1. Aspartate Aminotransferase (AST)
● Serum glutamic-oxaloacetic transaminase (SGOT) / Serum
glutamate-oxaloacetate transaminase
● Transfer of an amino group between aspartate and α-keto
acid
○ Product would be glutamate and oxaloacetate
● Involved in the synthesis (anabolism) and degradation
(catabolism) of AA in the liver.
● Widely distributed, highest activities in cardiac, liver and
skeletal muscle.
○ AST is not liver specific
● 2 Isoenzymes:
1. Cytoplasmic AST
a. Originates in the cytoplasm of the cell
b. Predominant isoenzyme in the serum
c. Is released when there is cell membrane
injury
2. Mitochondrial AST
a. Originates in the mitochondria of the cell
b. Elevated when there is severe injury or
damage to the cell (indicating destruction of
mitochondria)

● Diagnostic Significance
○ Associated with acute myocardial infarction
■ After MI, AST levels begin to rise in 6-8 hours, peak at 24 hours, and return to
normal in 5 days.
■ Peak level = highest serum level
○ Also ↑ in hepatocellular and skeletal muscle disease
■ AST is released to a greater degree in chronic disorders of the liver with
progressive damage to hepatocytes
● Assay for Enzyme Activity
○ Karmen Method
■ Uses malate dehydrogenase and monitors ↓ in absorbance at 340 nm
● Malate dehydrogenase is used as the secondary/coupling/indicator
 enzyme
● Form of coenzyme is oxidized
■ Falsely ↑ in hemolyzed sample
■ Reference Range: 5 – 30 U/L
● Reagent manufacturer’s RR must be used, RR varies

Note: When reading in spectrophotometer, reading of absorbance between 400-700 is visible light
● < 400 = ultraviolet; >700 = infrared (both colorless)

1. AST is the primary enzyme (enzyme of interest in the serum)

2. MD is the secondary enzyme which is not measured in the serum and is part of the reagent
3. The product of AST activity on aspartate + a-ketoglutarate would become the substrate for the MD
4. ↑AST = ↑oxaloacetate produced = ↑MD reaction
a. AST is directly proportional to MD
b. MD is of the oxidoreductase class

2. Alanine Aminotransferase (ALT)

● Also known as serum glutamic-pyruvic transaminase (SGPT)/ serum
glutamate-pyruvate transaminase
● Transfer of an amino group between alanine and α-ketoglutarate
● Increased in hepatocellular disorders.
○ Is more liver specific than AST
○ ALT is a significant marker in the evaluation of hepatic disorder,
specifically in acute inflammatory conditions
○ ALT is also used to monitor the course of hepatitis treatment to
determine if the patient is responding well
■ ↓ALT = positive treatment response
○ ALT is also used to monitor effect of drug therapy, specially
among patients with chronic diseases
■ ↑ALT = ↓dosage is done
○ ALT is considered a more sensitive/specific screening test for
post transfusion hepatitis and occupational toxic exposure
○ ALT is also used to screen blood donors
Note: to know if ALT or AST; remember “if a guy AST (ask), dapat may SGOT”

● Assay for Enzyme Activity

○ Uses Lactate Dehydrogenase and monitors↓ in absorbance at 340 nm
■ LD as secondary enzyme
■ Coenzyme form is oxidized
■ Product is colorless
○ Product which is measured.Reference Range: 6-37 U/L
 ● De Ritis Ratio
○ The AST/ALT Ratio
○ Differentiates the cause of hepatic disorder
○ Ratio > 1 🡪 non viral origin → AST is ↑ than ALT
○ Ratio < 1 🡪 viral in origin → ALT is ↑ than AST
SGOT/AST SGPT/ALT

MAJOR ORGAN AFFECTED Heart, Muscle, Liver Liver

SUBSTRATE Aspartic Alpha Ketoglutaric Acid Alanine Alpha Ketoglutaric Acid

END PRODUCTS Glutamic acid + oxaloacetic acid Glutamic acid + pyruvic acid

COLOR DEVELOPER 2,4 Dinitrophenylhydrazine (DNPH)

COLOR INTENSIFIER 0.4 NaOH

COLORIMETRIC METHODS Reitman and Frankel

Conditions of increase Transaminases

● Toxic hepatitis - both
○ Chronic liver conditions = ↑AST than ALT
○ Acute liver conditions = ↑ALT than AST
● AMI - AST
● Wolff-Parkinson White syndrome (involuntary contraction of muscle) - AST
● Trichinosis - AST
● Chronic alcoholism - AST
● Dermatomyositis - AST
● Hepatic cancer - both
● Reye’s syndrome - both
● Viral hepatitis - ALT
● Muscular dystrophy - AST
● Acute pancreatitis - AST

3. Gamma-Glutamyltransferase (GGT)
● Catalyze the transfer of the γ-glutamyl residue from γ-glutamyl peptides to amino acids, H20, etc.
○ In the biologic system, the most common donor of Gamma-glutamyl peptide is glutathione
 ● Used for diagnosis hepatobiliary disorders and chronic alcoholism
● GGT is a sensitive marker for determination of ethanol intoxication and occult alcoholism (hidden
type of alcoholism)
● Aminotransferases require coenzymes called pyridoxal phosphate (aka vitamin b6)

● Assay for Enzyme Activity

○ Szaz Assay
■ the absorbance of p-Nitroaniline is measured at 405-420 nm
■ Visible color observed

✔ M.I. Profile
4.Creatine Kinase (CK)
● Aka Creatine phosphokinase
○ When an enzyme is a kinase, it is a transferase that focuses on the transfer of
phosphate group
● Involved in the storage of high-energy creatine phosphate in muscle cells
○ Creatine phosphate is the energy reservoir in the muscle; when it is
consumed during muscle metabolism → creatinine is generated (which is
excreted at a constant rate in the glomerular membrane)
● Widely distributed, highest activities in skeletal muscle, heart, and brain
○ Not specific to the heart
● In terms of structure, CK is a dimeric enzyme (means it is composed of 2 monomer
subunits)
○ Which is either the M (muscle) or the B (brain) subunit

● Methods of Determination
○ Forward Reaction (Tanzer-Gilvarg)
■ ↓ in absorbance at 340 nm is determined
■ Product is oxidized NAD
■ Optimum pH is 9.0 (alkaline)
■ CK is the primary enzyme; PK and LD are secondary enzymes (included in the
reagents)
● CK activity would directly influence the activity of PK and LD
 ○ Reverse Reaction (Oliver-Rosalki)
■ Direction of reaction is from right to left
■ ↑ in absorbance at 340 nm is determined
■ Product is reduced NADH
■ Optimum pH: 6.8 (acidic)
■ CK is the primary enzyme; Hexokinase and G-6-PD is the secondary enzyme

✔ Source of Error
o Hemolysis cause false ↑ CK activity
● Inside RBC, there is adenylate kinase (AK) enzyme where activity of AK and CK is the same
● Lysed RBC releases AK which causes false elevated CK activity
o CK is inactivated by light = leads to false ↓
● CK is photosensitive
● Prolonged light exposure is not advisable
o Physical activity and IM injections cause ↑ CK
o Reference Range
● CK-MM = 94-98%
● CK-MB = 2-6%
● CK-BB = very low (increase in CK-BB indicates damage in BBB)
● M 🡪 15-160 U/L : F 🡪 15-130 U/L
● CK-MB: <6% of total CK
Ck-3 / CK-MM / Muscle type CK- 2 / CK-MB / Hybrid Type CK-1 / CK-BB / Brain Type

Slowest mobility toward the 2nd fastest to migrate toward the Migrate fastest toward the anode
anode anode
Least anodic (farthest from the Closest to the anode
anode)

Major isoenzyme in striated Isoenzyme that is high in the cardiac Highest concentration is seen in CNS, GI
muscle and normal serum muscle tract, and uterus (among pregnant
High in healthy individuals Significant quantities are found in heart women)
tissues

● Diagnostic Significance of CK Isoenzymes

○ Total CK activity - measure CK-MM, MB, BB are measured
○ CK isoenzyme is the one being used for diagnosis bc it is specific compared to total CK
measurement
■ Isoenzyme measurement would improve the diagnostic capacity of the lab bc of
 specificity of an isoenzyme to an organ/tissue
○ After MI, CK-MB (>6%) levels begin to rise within 4-8 hours, peak at 12-24 hours, and
return to normal levels within 48-72 hours.
■ Normal CK-MB level is <6%
■ CK-MB is the first enzyme to elevate among patients w/ MI
■ Myoglobin Is the first protein to level among patients w/ MI; which is faster than
CK-MB
● Other CK Isoenzymes
○ Macro-CK
■ Called macro-ck bc the ck enzyme is complexed with antibodies/lipoproteins
■ ex. CK-BB that is complexed with antibodies (IgG or IgA); CK-MM that has
lipoprotein attached to it
■ In terms of migration, it is present between CK-MM and CK-MB
○ Mitochondrial CK (CK-Mi)
■ Type of CK that is bound to the mitochondrial membrane
■ ↑ level indicates damage to the mitochondrial membrane; suggesting severe
damage/destruction of cell
■ Migrates cathodal to CK-MM
● Means furthest (opposite side of anode - going to the cathode part)

5.Lactate Dehydrogenase (LD)

● Type of oxidoreductase that catalyzes the interconversion of lactic and pyruvic acids
● Widely distributed, highest activities in heart, hepatic, skeletal muscle and RBC
○ Heart and skeletal muscles are the main source for the monomers
○ There are 2 active subunits for LDH; H and M subunits

● Assay of Enzyme Activity

○ Wacker method
■ Forward Reaction (Lactate 🡪 Pyruvate)
■ Increase in absorbance is monitored at 340 nm
● Measures the reduced NADH
■ Optimal pH is 8.3 – 8.9 (alkaline)
○ Wrobleuski La Due
■ Reverse Reaction (Pyruvate 🡪 Lactate)
■ Decrease in absorbance is monitored at 340 nm
● Measures the oxidized NAD
■ Three times faster but more susceptible to substrate exhaustion
● Means in prolonged testing, the probability of the substrate being unable
to optimally react with enzyme is high
● Therefore, testing must be done immediately
■ Optimal pH is 7.1 to 7.4
 ○ α-hydroxybutyrate dehydrogenase (α-HBD)
■ Has greater affinity of H subunits
● ↑H subunits = ↑affinity of a-HBD
■ Represent LDH-1
● LDH-1 is composed only of H subunit

✔ After MI, LD begin to rise within 10-24 hours, peak at 48-72 hours, and remains elevated for 10 days.
● Among the 3 MI markers, LD is the slowest to elevate
● Since it is elevated for 10 days, measurement can be useful in determining patient response to therapy
✔ Reference Range: 100-225 U/L

✓ LDH isoenzymes
● Is a tetramer (4 monomers) containing two active sub-units (H and M)
● In electrophoresis, LDH-1 is the fastest to migrate; nearest to the anode; LDH 5 is the slowest and least
anodic

● Relative concentration in normal serum:

○ LDH-2 > LDH-1 > LDH-3 > LDH-4 > LDH-5
● In AMI and Intravascular hemolysis, LDH-1 and LDH-2 demonstrate a Flipped pattern (LDH-1 > LDH-2)

CK-MB AST LDH

Appearance 4-8 hrs 6-8 hrs 10-24 hrs

 Peak 12-24 hrs 24 hrs 48-72 hrs

Stay Elevated 3 days 5 days 10 days

✔ Other Liver Enzymes

6. Alkaline Phosphatase
● Other nameAlkaline orthophosphoric monoester phosphohydrolase
● A hydrolase that catalyze the hydrolysis of various phosphomonoesters at an alkaline pH
○ pH is 9-10
● Liberate inorganic phosphate from an organic phosphate ester with production of alcohol
● Requires Mg2+ activator
○ In healthy human sera, most of alkaline phosphatase is derived from liver and bones
(osteoblast)
○ Major tissue sources of alkaline phosphatase are
■ Liver
■ Bone
■ Placenta (among pregnant women)
■ Intestine
■ Kidneys
● For evaluation of hepatobiliary and bone disorders.
● Assay for Enzyme Activity
○ Bowers and McComb
■ Based on molar absorptivity of p-Nitrophenol
■ Absorbance is measured at 405 nm
● Seen in visible light spectrum

Other methods:

Methods Substrate End Product

(1-4) Bodansky, Shinowara, Inorganic PO4 + Glycerol

β-glycero-phosphate
Jones, Reinhart

(5) Bessy, Lowry & Brock p-nitrophenyl phosphate p-nitrophenol (yellow)

(6) Bowers & McComb

(7) King and Armstrong Phenyl phosphate phenol

✔ Reference Range
▪ 30 – 90 U/L (Adult)
 ▪ 70 – 220 U/L (0 – 3 months)
▪ 50 – 260 U/L (3 - 10 years)
▪ 60 – 295 U/L (10 - puberty)
● Highest bc it is the peak of growth and development of bones
● During bone development, osteoblast is active resulting in alkaline phosphatase is released

✔ ALP Isoenzymes
● Differentiation by electrophoresis, chemical inhibition, heating method
● When serum is electrophoresis, the ALP isoenzymes are differentiated according to migration
characteristics
○ Order of fastest to slowest: Liver (most anodic), bone, placental, intestinal (least anodic)
1. Liver ALP
a. Fastest and most anodic; ↑ in liver diseases
b. Two fractions: major liver (contributing to the abundance of ALP in the circulation among
healthy individuals) and fast liver (α1) band (responsible for the fast migration)
2. Bone ALP
a. 2nd fastest to migrate; heat labile fraction (when temp is ↑, the activity of bone ALP is
significantly ↓ and is seen in 56degC)
b. ↑bone ALP activity may be correlated w/ pathologic or physiologic condition
i. Bone disease (pathologic) such as Paget’s disease/osteitis deformans
1. Bone ALP is significantly ↑
ii. Healing of bone fractures and physiologic bone growth (physiologic)
1. Physiologic bone growth is regulated by Growth hormone
2. ↑bone growth during puberty
3. Placental ALP
a. ↑ pregnancy specifically @ 16 to 20th week of gestation; most heat stable fraction
i. Given @ higher temp, activity is stable and is measurable
b. Also associated with malignancy (specifically the Carcino Placental ALP)
4. Intestinal ALP
a. Slowest moving fraction; least anodic; farthest from the anode
b. ↑ specifically in blood type B and O secretors during fatty meal consumption (physiologic)
and GIT disorders
● Differentiation using chemical inhibitors
○ Chemical inhibitors inhibits the activity of certain protein/enzymes so that specific isoenzymes are
measured
1. Placental and intestinal ALP are inhibited by phenylalanine (chemical inhibition as a method of
differentiation)
2. 3M urea reagent is the chemical inhibitor for bone ALP
3. Levamisole reagent is the chemical inhibitor for bone and liver ALP
● Differentiation using Heat stability test
○ Total ALP elevations by liver or bone ALP is differentiated by heating of serum at 56degC for 10
mins
○ Heat stability characteristics of ALP isoenzymes in order:
■ Placental (heat stable)
■ Intestinal
■ Liver
 ● ALP residual activity is ↓ to >20%
■ Bone (heat labile)
● ALP residual activity is ↓ to <20%
○

✔ CARCINOPLACENTAL ALP
● Variants of ALP that are associated with cancer
1. REGAN ALP : lung, breast, and gynecological cancers, bone ALP co-migrator, most heat
stable ALP
a. Remains stable even after heating of serum sample at 65degC for 30 mins
2. NAGAO ALP : Adenocarcinoma of the pancreas and bile duct, pleural cancer
3. Inhibitors: Phenylalanine, L-leucine
a. Phenylalanine reagent can inhibit regan ALP
b. Phenylalanine and l-leucine reagent can inhibit nagao ALP

7. Acid Phosphatase (ACP)

● Same with ALP in terms of activity but differ in pH and tissue source
○ Source of ACP is prostate gland, RBC, platelets, bones (osteoclast)
● Was used as a tumor marker for prostate gland
○ When prostate specific antigen (PSA) was discovered, it replaced ACP
and is now the tumor marker
○ ACP has lower specificity compared to PSA
● Catalyze the hydrolysis of various phosphomonoesters at an acid pH
● Liberate inorganic phosphate from an organic phosphate ester with production
of alcohol
● Also found in seminal fluid
○ For evaluation of metastatic carcinoma of prostate.
○ Forensic investigation of rape
■ Specimen collected is vaginal washing which is tested for
prostatic ACP activity bc there is not prostate gland in female
■ ACP in the seminal fluid only persist for up to 4 days

● To differentiate the prostatic form from the non-specific form like RBC acid Phosphatase
inhibitors are added
● Same as chemical inhibition procedure as ALP
○ L-tartrate ions – inhibits specific prostatic ACP
○ Formaldehyde and Cupric ions – inhibits red cell ACP

▪ Assay for Enzyme Activity

○ Bowers and McComb
■ Based on molar absorptivity of p-Nitrophenol
■ Absorbance is measured at 405 nm
● Seen in visible light spectrum
▪ Reference Range: Prostatic ACP: 0 -3.5 ng/ml
 Methods Substrate

(1) Quantitative end point Thymolphthalein monophosphate

(2) Continuous monitoring ⍺-naphthyl phosphate

METHODS SUBSTRATE END PRODUCTS

GUTMAN AND GUTMAN PHENYL PO4 INORGANIC PHOSPHATE

SHINOWARA PNPP P-NITROPHENOL

(para-nitrophenyl-phospha
te)

BABSON, READ AND PHILLIPS ALPHA NAPHTYL ALPHA NAPHTOL

PO4

ROY AND THYMOLPHTHALEIN FREE THYMOLPHTHALEIN

HILLMAN MonoPO4

✔ Pancreatic Enzymes
1. Amylase (AMS)
● Aka alpha-1,4-glucan-4-glucohydrolase
● Smallest enzyme present in the plasma
○ Normally filtered by the renal glomerulus; normally seen in urine
○ An increase in amylase level in plasma indicates an increase in urine amylase
■ Amylase in urine remain elevated for 7 days among patients with acute
pancreatitis
■ Renal disease causes ↓urine amylase, ↑plasma amylase bc most amylase is
reabsorbed
● Type of hydrolase that catalyzes the breakdown of starch and glycogen via α, 1-6 branching
linkages
○ Important for digestion and absorption of carbohydrates specifically polysaccharides
○ Polysaccharides are broken down into simpler form; amylase is a digestive enzyme
● Major tissue sources of amylase:
○ Pancreas
○ Salivary gland
● Minor tissue sources of amylase
○ Adipose tissues
○ Small intestine
○ Skeletal muscle
 ○ Fallopian tube
● Increased in acute pancreatitis, renal failure and parotitis (inflammation of parotid gland)
○ Pancreatitis usually occurs during deep sleep
■ Manifestations of pancreatitis: bangungot while sleeping which indicates extreme
pain
○ Amylase is not specific to pancreatitis bc of salivary gland
■ Amylase can also be increased during parotitis and mumps
○ Fastest and first to elevate during pancreatitis is amylase
■ 2-12 hrs rise of amylase after onset of acute pancreatitis
■ Peak level of amylase is 24 hrs during acute pancreatitis
■ Level of amylase normalizes within 3-5 days

● Amylase Isoenzymes
○ Salivary Amylase 🡪 ptyalin (fast moving; S type)
○ Pancreatic Amylase 🡪 amylopsin (slow moving; P typeSkype)

Amylase Methodologies

Amyloclastic Measures the disappearance of starch substrate

Concentration of glycogen or starch, as pancreatic amylase acts on
it, decreases (due to degradation of polysaccharide)

Starch-Iodine Complex (Dark-blue) 🡪 decrease in color intensity

Note: only complex sugars react with iodine; simple sugars will not
react with iodine
● Activity of amylase is inversely proportional to the
absorbance of the test

Alternative substrate for starch is glycogen

When iodine is made to react with glycogen, the product is
mahogany brown

Saccharogenic Measures the appearance of the product

Product is directly proportional to the activity of amylase
(Amylase activity is directly proportional to the absorbance)

Starch 🡪 reducing sugars (ex. glucose)

Reducing sugars is directly proportional to the activity of amylase
to the polysaccharide substrate

Chromogenic Measures the increasing color from production of

product - chromogenic dye fragment
 Insoluble starch-dye 🡪 soluble starch-dye fragments
Amylase activity is directly proportional to the absorbance

Continuous monitoring Coupling of several enzyme systems to monitor amylase activity

Oxidoreductase is the last enzyme

● Assay for enzyme activity (continuous monitoring)

○ Increase in absorbance at 340 nm is measured
○ A-glucosidase, hexokinase, G-6-PD is part of the reagent

2. Lipase (LPS)
● Aka Triacylglycerol acylhydrolase
● Type of hydrolase that hydrolyzes the ester linkages of fats to produce alcohols and fatty acids
○ Lipase targets the ester bond that connects different fats
● Hydrolysis of dietary triglycerides in the intestine to 2-monoglyceride and fatty acids
○ When consuming food high in TAG, small intestine converts complex molecules must be
converted into simpler form to be absorbed, in this case, lipase act on TAG to convert it;
making lipase a digestive enzyme
● Lipase is the most specific pancreatic marker
○ Major tissue source of lipase is only the pancreas
○ Level of lipase in plasma is not affected by salivary gland and renal disorder
○ Lipase elevates in acute pancreatitis 6 hrs after onset, peaks at 24 hrs, remains elevated for
7 days
■ Lipase last for 1 week bc molecule is larger (compared to amylase) and cannot be
easily filtered in the plasma
● Presence of hemoglobin (bc of lysis) in sample could affect lipase activity
○ Hgb inhibits enzymatic activity of lipase to the substrate
○ Hgb can false ↓ lipase measurement

● Assay for Enzyme Activity

Cherry Crandall Tietz
(reference method for lipase)

Substrate 50% olive oil (triolein - more pure form of TAG which can serve as a
substrate for TAG measurement)
● Other half contains water
● Water is needed bc lipase is a type of hydrolase
 Titrating agent 0.4N NaOH
- Which is used for
measuring fatty
acid release

Indicator Phenolphthalein Thymolphthalein + Veronal

Endpoint Fatty acid (Oleic acid)

End Color Pink Blue

Note: ↑ lipase activity = ↑ fatty acid generated/liberated from TAG

● Turbidimetric methods
○ Is the measurement of light blocked/absorbed by solution
■ Molecules present in the suspension blocks light
■ ↑ concentration of insoluble molecules (aka more turbid) = ↑ absorbance
○ Estimation of liberated fatty acids
○ TAG is insoluble to water, so when lipase is present to hydrolyze TAG to release product
which are 2-monoglyceride + 2 fatty acids,
■ Result of turbidity of solution will ↓= Absorbance reading will be ↓
■ Activity of lipase to TAG is inversely proportional to the absorbance reading

✔ Miscellaneous Enzymes
1. Nucleotidase (5’N)
● A phosphoric monoester hydrolase
● Predominantly secreted in the liver; can be used as a liver enzyme
● Marker for hepatobiliary disease and infiltrative lesions of the liver
● Reference value: 0-1.6 units
2. Cholinesterase/Pseudocholinesterase
● Index of parenchymal function
● Secreted by the liver
● Used to monitor the effect of muscle relaxants (succinylcholine) after surgery
● Marker for insecticide/pesticide poisoning
○ Specific component that could lead to poisoning = organophosphate
● Reference values: 0.5-1.3 units (in plasma)
3. Angiotensin-Converting enzyme (ACE)
● Aka Peptidyl dipeptidase or Kininase II
● Responsible for converting angiotensin I to angiotensin II within the lungs
○ Importance of conversion is for the activation of inactive angiotensin I to active
angiotensin II which is for regulation of Na by the aldosterone and water retention
● Increased in:
○ Sarcoidosis
○ Acute and chronic bronchitis
○ Leprosy
● Main source
○ Macrophage
 ○ Epithelioid cells
● ACE is a possible indicator of neuronal dysfunction (which causes dementia and alzheimer’s)
4. Ceruloplasmin
● Is a copper-carrying protein which acts as an enzyme
● Marker for Wilson’s disease (which is an ex. of hepatolenticular disease)
5. Ornithine Carbamoyl Transferase (OCT)
● Enzyme for hepatobiliary disease
● Reference value: 8-20 mU/mL
6. Glucose-6-Phosphate Dehydrogenase (G-6-PD)
● Maintains NADPH in the reduced form in the erythrocytes
○ Reduced NADP is important to stabilize cell membrane which would resist cell damage
caused by harmful agents/drugs
● Newborn screening marker
● Found in the adrenal cortex, spleen, RBC, and Lymph nodes
● Deficiency can lead to drug induced hemolytic anemia (ex. primaquine, antimalarial drug)
○ Without G-6-PD, reduced NADP is not maintained therefore cell membrane would be
susceptible to lysis
○ Lysis occurs in the blood vessels
● Increased levels: myocardial infarction, megaloblastic anemia
● Spx: red cell hemolysate, serum
● Reference values: 10-15 U/g Hgb or 1200-2000 mU/mL packed RBC