Clinical Enzymology

Enzymes of Clinical Significance:

OXIDOREDUCTASES
 Lactate Dehydrogenase
• Functions in the EMP that converts glucose to energy
• Highest activities are present in tissues that have high capacity for
utilizing glucose.

Reaction Catalyzed: interconversion of lactic and pyruvic acids

utilizing the coenzyme NAD+
L-Lactate + NAD+ <--LD--> Pyruvate + NADH + H+
*Optimal pH:
Forward/direct reaction (lactate to pyruvate) – pH 8.3 - 8.9
Backward/reverse/indirect reaction (pyruvate to lactate) – pH 7.1 - 7.4
*Component ion: Zinc
 Lactate Dehydrogenase
PERCENT
ISOENZYME TISSUE ASSOCIATED CONDITIONS
CONCENTRATION
Heart Myocardial infarction
LD-1 (HHHH) 14-26
Red blood cells Hemolytic anemia
Hemolyzed specimen
Heart
Megaloblastic anemia
LD-2 (HHHM) 29-39 Red blood cells
Acute renal infarct/Kidney
Kidneys
damage
Pulmonary embolism
Lung
Extensive pulmonary pneumonia
Lymphocytes
LD-3 (HHMM) 20-26 Lymphocytosis
Spleen
Acute pancreatitis
Pancreas
Various carcinoma

LD-4 (HMMM) 8-16

Liver Hepatic injury/inflammation
Skeletal muscle Skeletal muscle injury/dystrophy
LD-5 (MMMM) 6-16
 Lactate Dehydrogenase
Specimen Considerations
1. Non-hemolyzed serum is preferred; serum should be separated
immediately from RBCs
*RBCs LD level is 100-150 times the level in serum
2. Heparinized plasma may also be used but not plasma collected using
other anticoagulants
*Serum LD is 30IU/L higher than in plasma
3. Storage
a) Total LD: 25oC for up to 48 hours (LD-4 and LD-5 are destroyed at
refrigerator temperature)
b) LD isoenzyme analysis: 25oC for up to 24 hours
c) Beyond 2 days: 4oC, add NAD+ or glutathione
 Lactate Dehydrogenase
Laboratory Methods
1. Colorimetric – uses coupled reactions: forward reaction product reacts to
form colored compounds and absorbance measured
spectrophotometrically
a) Pyruvate + 2,4-dinitrophenylhydrazine (2,4-DNPH) –alkaline
pH phenylhydrazone (golden brown) *read at 440 or 525 nm
b) NADH + INT (tetrazolium dye) -–diaphorase -->NAD+ + INTH *read at
340nm
2. Wacker – forward/direct: read absorbance at 340nm *8.3-8.9
*Modification: NADH + phenazine methosulfate and nitroblue tetrazolium
--> blue-purple product -common
for detection of LD isoenzyme fractionation by electrophoresis
3. Wrobleski-La Due – reverse/indirect; common in dry-slide method
*7.1-7.4
Reference Ranges: 100-225 U/L (Forward) *37oC, 80-280 U/L (Reverse)
 Lactate Dehydrogenase
Laboratory Methods - Isoenzyme Assays
1. Electrophoresis – preferred method; requires small sample (1uL) -all
five fractions are identified and quantified but time-consuming
 Lactate Dehydrogenase
Laboratory Methods - Isoenzyme Assays

2. Column chromatography – only isolates LD-1 and LD-2; requires large

sample (0.5mL)

3. Immunoinhibition – antibody to the M subunit inactivates LD isoenzymes

with the M subunits
-only LD-1 is quantified
 Lactate Dehydrogenase
Clinical Significance (Total LD)
1. Pernicious anemia and hemolytic disorders: highest levels
2. Acute myocardial infarction: primary utility
a) LD rises within 12-24 hours of onset, peak levels within 48-72 hours,
remains elevated for 10 days
b) Flipped LD pattern, 36 hours, post-chest pain (higher levels of LD-1
than LD-2): suggestive of AMI
*presence of CK-MB
3. See conditions associated to specific isozenzymes
 Glucose-6-Phosphate
• Involved in theDehydrogenase
first step of the hexose-monophosphate pathway of
glucose metabolism

Reaction Catalyzed: oxidation of G6P to 6-phosphogluconate or 6-

phosphogluconalactone
B-D-Glucose-6-phosphate + NADP+ --G6PD--> 6-phosphogluconate
+ NADPH + H+

Tissue Distribution
• Highest levels in RBCs, adrenal cortex, spleen, thymus, lymph nodes,
lactating mammary glands
 Glucose-6-Phosphate
Dehydrogenase
Specimen Considerations
1.Enzyme elevations: non-hemolyzed serum
2.Enzyme deficiency: red cell hemolysate is used

Reference Values: 10-15 U/g Hgb

Clinical Significance
1.Increased levels: myocardial infarction and megaloblastic anemia
2.G6PD deficiency – X-linked recessive trait which can result to some
various clinical manifestations including hemolytic anemia
 Ceruloplasmin/Ferroxidase I (EC
• 1.16.3.1)
glycoprotein that binds ~95% copper in plasma, acting as the
copper transport protein
• catalyzes the oxidation of ferrous iron to ferric iron
• aids in the synthesis of transferrin
• acute phase protein; thus, increased during physiological stress:
acute infections, MI, surgery, SLE or RA
• decreased in Wilson’s disease, a hepatocellular degeneration which
also causes serum copper decline
 Clinical Enzymology

Enzymes of Clinical Significance:

TRANSFERASES
 Creatine Kinase
• Functions in the EMP that converts glucose to energy
• Highest activities are present in tissues that have high capacity for
utilizing glucose.
• When energy is needed for contraction, phosphate from creatine
phosphate will be transferred to ADP to produce energy (ATP)

Reaction Catalyzed: reversible phosphorylation of creatine by ATP to

form creatine phosphate (energy reserve in muscles);
Creatine + ATP <--CK -->Creatine phosphate + ADP + H+
*Creatine phosphate serves as stored energy in muscles
*Magnesium is needed as activator, but excess level is inhibitory
 Creatine Kinase
Percent
Isoenzyme Tissue Associated Conditions
Conc.
Myocardial infarction
Skeletal muscle disorder; Muscular dystrophy
CK3/ Skeletal muscle Polymyositis
94-100
CK-MM Heart Hypothyroidism
Malignant hyperthermia
Physical activity; Intramuscular injection
Myocardial infarction; Myocardial injury; Ischemia; Angina;
Inflammatory heart disease; Cardiac surgery
Duchenne-type muscular dystrophy
CK2/ Heart Polymyositis
0-6
CK-MB Skeletal muscle Malignant hyperthermia
Reye’s syndrome
Rocky Mountain spotted fever
Carbon monoxide poisoning
Brain
Central nervous system shock; Anoxic encephalopathy
Bladder
Cerebrovascular accident; Seizure
Lung
Placental or uterine trauma
CK1/ Prostate
0 Carcinoma, Reye’s syndrome
CK-BB Uterus
Carbon monoxide poisoning
Colon
Malignant hyperthermia
Stomach
Acute and chronic renal failure
Thyroid
 Creatine Kinase
Other Isoenzymes
1. Mitochondrial CK (CK-Mi or CK-Mt)
-appears in the circulation only when extensive damage occurs
-can form large-molecular-weight aggregates, detected in serum as macro-
CK2
-migrates to a point more cathodal than CK-3
-presence does not correlate with any specific disease state but is an indicator
of severe illness
2. Macro-CK: CK that form large complexes eferred to as macroenzymes
a. Macro-CK1: formed by complexing with an antibody
-CK1 and IgG are most common
-CK2 and IgA
b. Macro-CK2: found predominantly in adults with severe malignancies of the
liver or children with notable tissue distress
-presence in serum is commonly associated with poor prognosis
 Creatine Kinase
Specimen Considerations
1. Non-hemolyzed serum is preferred; slight hemolysis may be tolerated
*Adenylate kinase from RBCs participates in the reactions of CK assays

2. Heparinized plasma may also be used but not plasma collected using other
anticoagulants

3. Storage
a. Serum CK activity is very unstable due to oxidation of sulfhydryl groups
during storage
b. CK assays must be tested within 24 hours or frozen
c. CK is heat labile and light sensitive, storage must be at refrigerator
temperature and unexposed to light
d. Inactivation caused by light may be reversed by storage in the dark at 4oC
for 7 days or -20oC for 1 month when assay is conducted using sulfhydryl activator
 Creatine Kinase
Specimen Considerations

4. People with higher muscle mass will have higher baseline CK activity

5. Neonates have total CK activity twice of adults which declines rapidly

6. Pregnancy causes gradual increase in CK activity, peaking at the last several

weeks; after 5 days from delivery, the CK activity returns to normal
 Creatine Kinase
Laboratory Methods
1. Colorimetric Methods – forward reaction product reacts to form colored compounds
and absorbance measure spectrophotometrically
a.Hughes Method
Creatine + diacetyl and A-naphthol --CK--> pink end product
b.Sax and Moore Method
Creatine + ninhydrin -–CK--> fluorophore

2. Tanzer-Gilvarg – forward/direct; optimum pH: 9.0

-requires sulfhydryl activator
Creatine + ATP <--CK--> Creatine phosphate + ADP + H+
ADP + phosphoenolpyruvate <--PK--> ATP + pyruvate
Pyruvate + NADPH + H+ <--LD--> lactate + NAD+ (decrease in
absorbance)
 Creatine Kinase
Laboratory Methods
3.Oliver-Rosalki – reverse/indirect; optimum pH: 6.8; most commonl method
-Requires sulfhydryl activator
Creatine phosphate + ADP + H+ <--CK--> Creatine + ATP
ATP + glucose <--hexokinase--> ADP + Glucose-6-phosphate
G6P + NADP+ <--G6PD--> 6-phosphogluconate + NADPH + H+
(increase in absorbance)

4.Szasz, et al. – uses the same reaction as Oliver-Rosalki

-optimized by adding N-acetylcysteine, EDTA, and adenosine pentaphosphate
-basis of a reference method developed by IFCC

Reference Ranges: 15-160 U/L (Males), 15-130 U/L (Females)

 Creatine Kinase
Laboratory Methods - Isoenzyme Assays
1.Electrophoresis – preferred method; reference method for CK isoenzymes
 Creatine Kinase
Laboratory Methods - Isoenzyme Assays
2. Chromatography: Column or ion-exchange chromatography

3. Immunoassays: detects CK-MB with minimal cross-reactivity

-detects concentration rather than enzyme activity, thus able to detect inactivated CK-
MB
*IRMA
-Solid phase with attached anti-B subunit + sample = Only CK with B
subunit will attach
-Anti-M subunit labelled with radioisotope is added, only attaching to
CK-MB
 Creatine Kinase
Laboratory Methods - Isoenzyme Assays
*Immunoenzymatic Sandwich Technique
-same principle as IRMA but label is enzyme instead of radioisotope
*Immunoinhibition
-Antibodies to both M and B subunits used to determine the CK-MB
activity
-Anti-M subunit inhibits M activity but not B activity
-Total activity-->inhibition-->Remaining CK activity-->multiplied by two
-CK-BB is still detected which may cause falsely increased results
-CK-Mi and macro-CK are not inhibited which may also cause errors
*RIA
*Radioisotope method
*Double antibody immunoinhibition assay
 Creatine Kinase
Clinical Significance (Total CK)
1.Duchenne-type muscular dystrophy: highest levels, 50-100x (CK and CK3)
2.Acute myocardial infarction: primary utility
*Total CK rises in 2-4 hours of onset, peak levels within 48-72 hours, remains
elevated for 10 days
3. Others: CNS problems, hypothyroidis, malignant hyperpyrexia, Reye's syndrome,
ectopic pregnancy, V. vulnificus infections
4. See conditions associated to specific isozenzymes

Clinical Significance (Total CK)

1. CK2 rises in 4-8 hours of onset, peak levels within 12-24 hours, remains elevated
for 2-3 days
*In patients with chest pain but has no increase in CK2 within 48 hour, AMI can
be ruled out with 100% accuracy
 The Aminotransferases: AST
• and ALT
Catalyzes the interconversion of amino acids and alpha-oxoacids by
the transfer of amino group
• Result of transamination: donating amino acid becomes a keto-acid
while the acceptor keto-acid becomes a new amino acid
• Transamination functions as the major link between amino acid and
carbohydrate metabolism

*All transamination require Vitamin B6 derivative (pyridoxal-5-

phosphate) as coenzyme
*In some individuals, particularly chronic ethanol abusers, Vitamin B6
deficiency may occur, causing falsely decreased ALT/AST levels
 Aspartate Aminotransferase
Reaction Catalyzed: Transfer of an amino group from aspartate to
alpha-ketoacids with the formation of oxaloacetate and glutamate
Aspartate + A-ketoglutarate <--AST--> Oxaloacetate + Glutamate
*Pyridoxal-5-phosphate or pyridoxine (Vitamin B6) serves as the coenzyme
for AST

Isoenzymes
Isoenzymes Tissue Notes
Predominant form in serum
Cell cytoplasm Liver
Half-life: 16 hours
Skeletal muscle
Heart Significantly increased in disorders causing
Mitochondria Kidney, Pancreas, RBC cellular necrosis
Spleen, Lung, Serum Half-life: 87 hours
 Aspartate Aminotransferase
Specimen Considerations
1. Non-hemolyzed serum is preferred; serum should be separated immediately from
RBCs
*Turbid and icteric specimen interfere with the assay

2. Heparinized, EDTA, oxalated or citrated plasma may also be used

3. Storage: Stable for up to 4 days at ref temperature (longer if frozen)

*Stability can be enhanced by adding pyridoxal-5-phosphate
 Aspartate Aminotransferase
Laboratory Methods: All methods are based on measuring the change in the
amount of product (oxaloacetate) formed

1.Karmen– kinetic UV (enzymatic) method; preferred

Oxaloacetate + NADH + H+ <--MDH--> malate + NAD+
Optimal pH: 7.3-7.8
Measured at 340nm (decrease in absorbance)
2.Reitman-Frankel – colorimetric; not specific; lipemia and icterus interferes
Oxaloacetate + 2,4-dinitrophenylhydrazone --alkaline--> reddish brown color
(hydrazone)
Measured at 505nm
3.Babson, et al – colorimetric
Oxaloacetate + diazonium salt --> violet color (diazo)
Reference Ranges: 5-30 U/L
 Aspartate Aminotransferase
Clinical Significance
1.Liver disorders: primary utility
a.Acute hepatocellular disorders (e.g. viral hepatitis): highest levels 100x
b.Cirrhosis 4x
c.Cholestatic diseases (gall bladder)
2.Acute myocardial infarction: not very useful due to wide tissue distribution 2-3x
-AST rises in 6-8 hours of onset, peak levels within 24 hours, remains elevated
for 5 days
3.Congestive heart failure, probably due to inadequate blood supply to the liver
4.Pulmonary embolism
5.Skeletal muscle disorders 4-8x
 Alanine Aminotransferase
Reaction Catalyzed: Transfer of an amino group from alanine to
alpha-ketoacids with the formation of pyruvate and glutamate
Alanine + A-ketoglutarate <--ALT--> Pyruvate + Glutamate
*Pyridoxal-5-phosphate or pyridoxine (Vitamin B6) serves as the coenzyme
for ALT

Isoenzymes
No mitochondrial isoenzyme *Half-life: 47 hours

Specimen Considerations
*Same with AST except that ALT is relatively unaffected by
hemolysis
 Alanine Aminotransferase
Laboratory Methods: All methods are based on measuring the change in the
amount of product (pyruvate) formed

1.Walker et al – kinetic UV (enzymatic) method; preferred

Pyruvate + NADH + H+ LDH lactate + NAD+
Optimal pH: 7.3-7.8
Measured at 340nm
2.Henry-Pollard – same principle with Walker et al method
3.Reitman-Frankel – same principle with AST assay
4.Babson, et al – same principle with AST assay

Reference Ranges: 6-37 U/L

 Alanine Aminotransferase
Clinical Significance
1. ALT is considered a more specific liver marker than AST
a. Hepatocellular disorders
b. Inflammatory conditions of the liver: higher elevations than AST
-tend to remain elevated longer due to its longer half-life (24 hours)
c. Obstructive liver disease and Cirrhosis
*De Ritis Ratio: ratio of ALT to AST
-may be useful in determining the cause of liver disease using established facts:
a. ALT is only present in the cytoplasm
b. Only 30% of AST is present in the cytoplasm, the rest are found in the
mitochondria
c. Relative activity of AST in the liver tissue is several times higher than ALT
 Alanine Aminotransferase
Clinical Significance
AST>ALT *mitochondrial damage
a.Cirrhosis
b.Alcoholic hepatitis*
c.Liver cancer
d.Acute fulminant hepatic failure
ALT>AST
a.Infectious hepatitis
b.Inflammatory conditions of the liver
 Gamma-Glutamyltransferase
• Significant in some aspects of control of peptide and protein synthesis,
regulation of tissue glutathionevels and the transport of amino acids across cell
membranes of the kidneys and intestines

Reaction Catalyzed: Transfer of gamma-glutamyl residue from gamma-

glutamyl peptides (*Glutathione serves as the gamma-glutamyl donor in
most biologic system) to amino acids, water and other small peptides
Glutathione (gamma-glutamyl-cysteinyl-glycine) + peptide <--GGT-->
Glutamyl peptide + L-cysteinylglycine

Isoenzymes
No isoenzymes
 Gamma-Glutamyltransferase
Specimen Considerations
1. Non-hemolyzed serum is preferred; relatively unaffected by hemolysis

2. Heparin causes turbidity while citrate, oxalate and fluoride inhibits GGT

3. Storage: Stable for up to 7 days at ref temperature (2-3 months if frozen)

4. Neonate have higher levels than adults but declines within 6 months

5. Values are lower in females due to estrogen/progesterone

 Gamma-Glutamyltransferase
Laboratory Methods
1. Most widely accepted substrate: G-glutamyl-p-nitroanilide
G-glutamyl-p-nitroanilide + glycylclycine --GGT G-glutamyl-glycylclycine + p-
nitroaniline
*The end-product (p-nitroaniline) is a chromogenic substance with strong absorbance
at 405-420nm
*May be used for either end-point or kinetic assay

2. Szasz assay, the IFCC reference method for GGT and modified from the above
reaction, uses G-glutamyl-3-carboxy-4-nitroanilide with end-product as 5-amino-2-
nitrobenzoate read at 410nm

Reference Ranges: 6-45 U/L (Males); 5-30 U/L (Females) *37oC

 Gamma-Glutamyltransferase
Clinical Significance
1. Considered one of the most sensitive hepatobiliary marker, but least specific:
elevates in virtually all forms of hepatobiliary disorders
a.Biliary tract obstruction: highest elevations (more sensitive than ALP, AST,
ALT, Bilirubin)
b.Liver disease especially during puberty and pregnancy (as ALP is
physiologically increased)
c. Liver inflammation
2.Detection and monitoring of alcoholism: GGT returns to normal 2-3 weeks after
cessation from alcohol consumption but rises again upon resumption of alcohol
consumption
3.Medications: warfarin, phenobarbital, phenytoin
4.Other conditions: pancreatitis, DM, MI
 Clinical Enzymology

Enzymes of Clinical Significance:

HYDROLASES
 The Phosphohydrolases: ALP
and ACP
• Enzymes of low specificity characterized by their ability to hydrolyze
a large variety of organic phosphate ester with the formation of an
alcohol and a phosphate ion

• Two main types:

1. Alkaline phosphatase
2. Acid phosphatase
 Alkaline Phosphatase
Reaction Catalyzed: liberation of inorganic phosphate from organic
phosphoester with the production of alcohol

Phosphomonoester + water <--ALP at pH 9.0-10.0 -->alcohol +

phosphate ion

*Magnesium as activator, zinc as constituent metal ion

 Alkaline Phosphatase
Isoenzyme Electrophore Heat Chemical Inhibitors Notes
sis Stability
20% ethanol;
Liver 1 (fastest) 3 Levamisol;
L-homoarginine
4 (most 2M urea; Levamisol;
Bone 2 Enhances bone formation
heat labile) L-homoarginine
1 (most
Observed in pregnant women
Placenta 3 heat L-phenylalanine
during the third trimester
stable)
Associated with the renal
Kidney 4 - -
reabsorption of phosphate
Involved with fatty acid
Intestine 5 (slowest) 2 L-phenylalanine transport; calcium and
phosphate absorption
 Alkaline Phosphatase
Other Isoenzymes (Nearly identical to placental ALP)
1. Regan isoenzyme: 3-15% incidence in lung, breast, ovarian and colon cancer
-highest incidence in ovarian and gynecologic cancers
-useful in monitoring therapy as it disappears on successful treatment
-heat stability and inhibition characteristics same as with placental isoenzyme
but migrates to the same position as the bone fraction

2. Nagao isoenzyme: may be considered a variant of the Regan isoenzyme

-detected in patients with metastatic cancer of the pleural surfaces, as well as
in patients with cancer if the pancreas and the bile duct
-electrophoretic, heat stability and inhibition characteristics same as with
Regan isoenzyme; may also be inhibited by L-leucine

3. Kasahara isoenzyme: formerly Regan variant-1; hepatoma and in tumors of GIT

 Alkaline Phosphatase
Specimen Considerations
1. Non-hemolyzed serum is preferred; hemolysis causes slight elevations
*RBCs has 6x more ALP than serum

2. Heparinized plasma may be used

3. Assays should be run as soon as possible; ~10% increase in activity upon standing
either RT or 4oC

4. Diet induces ALP activity elevations in patients with either B or O blood group who
are secretors. Up to a 25% increase may occur after ingestion of a high-fat
meal
 Alkaline Phosphatase
Laboratory Methods
*Generally, assays parallel that of ACP with the optimum pH being the difference
*The buffer is critical for accurate measurement; all buffers function as phosphate
acceptors and reaction proceeds much more rapidly in their presence

1.Bowers-McComb: uses phosphate-accepting buffer (2-amino-2-methyl-1-propanol);

Reference; Continuous monitoring method
p-nitrophenylphosphate + water ALP at pH 10.0 p-nitrophenol +
phosphate ion
a. p-Nitrophenylphosphate (colorless) is hydrolyzed to p-nitrophenol (yellow)
b. Increase in absorbance at 405 nm, which is directly proportional to ALP
activity is measured.

2.Bessey-Lowry-Brock: also uses p-nitrophenylphosphate substrate

 Alkaline Phosphatase
Laboratory Methods

3. Assays not using p-nitrophenylphosphate:

a. King-Armstrong: phenylphosphate
b. Huggins-Talalay: phenolphthalein diphosphate
c. Klein-Babson-Read: buffered phenolphthalein phosphate
d. Sinowara-Jones-Reinhart: beta-glycerophosphate

Reference Ranges: 30 to 90 U/L (30°C)

 Alkaline Phosphatase
Laboratory Methods - Isoenzyme Assays
1. Electrophoresis: most useful single technique; must be combined with other
techniques due to overlap
*there is no clear separation between the liver and bone isoenzyme
*liver isoenzyme has two fractions:
a. Liver: the major fraction; frequently the cause of elevated total ALP levels
-increases are caused by various hepatobiliary conditions, usually at
the onset of the disease
b. Fast liver/A-1-liver: smaller fraction migrating ahead of the liver fraction
-reported in liver cancer and other hepatobiliary diseases
-important indicator of obstructive liver disease
-occasionally present in the absence of any disease
 Alkaline Phosphatase
Laboratory Methods - Isoenzyme Assays
2. Heat Stability: imprecise and depend on the following factors: temp control, timing,
method sensitivity *liver and bone isoenzymes overlap
*Procedure:
a. ALP activity is measured pre- and post-heating of serum 56oC for 10
minutes
b. Less than 20% residual activity: elevation is *due to the bone isoenzyme
c. Greater than 20% residual activity: elevation is *due to the liver isoenzyme
d. Placenta isoenzyme will resist heat denaturation at 65oC for 30 minutes

3. Selective Chemical Inhibition: see table

4. A direct immunochemical method for the measurement of bone-related ALP is now

available, making ALP electrophoresis unnecessary in most cases
 Alkaline Phosphatase
Clinical Significance
1. Hepatobiliary disorders: primary utility
a. Biliary tract obstruction: 3-10x
b. Hepatocellular disorders <3x

2. Bone conditions, especially when osteoblasts are involved

a. Physiologic increases should always be considered:
i. First year of life and puberty/period of growth
ii. Healing bone fractures
iii. Adults beyond 50 years old
b. Paget’s disease – highest elevations ~25x
c. Others: Osteomalacia, rickets, hyperparathyroidism, osteogenic sarcoma
 Alkaline Phosphatase
Clinical Significance
3. Placental ALP
a. Pregnancy – increases beyond normal limits at 16-20 weeks of gestation
persisting until the onset of labor, returning to normal
within 3-6 days
b. Complications in pregnancy: Hypertension, Pre-eclampsia, eclampsia,
possible abortion
c.Tumor marker (carcinoplacental ALP) in serum and CSF for germ cell
tumors*

4.Intestinal ALP
a. Diet in B or O blood group secretors
b. Few hours after consumption of a high-fat meal
c. GIT disease: intestinal infarction, inflammation, ulceration, cirrhosis

5. Others: hyperthyroidism, diabetes mellitus

 Alkaline Phosphatase
Clinical Significance (Total ALP)

6. Decreases may be caused by:

a. Hypophosphatasia
b. Hypothyroidism
c. Scurvy
d. Gross anemia
e. Post-transfusion or cardiopulmonary bypass
f. Zinc deficiency
 Acid Phosphatase
Reaction Catalyzed: liberation of inorganic phosphate from organic
phosphoester with the production of alcohol

Phosphomonoester + water <--ACP at pH 9.0-10.0 -->alcohol +

phosphate ion

*Has no clearly defined specific physiological substrate

Isoenzymes
1. Prostatic
2. RBC
3. Platelets
4. Other tissues
 Acid Phosphatase
Specimen Considerations
1. Non-hemolyzed serum; hemolysis causes elevations; serum ACP slightly higher
than plasma ACP
2. Citrated plasma may be used to lessen the possibility of platelet ACP to be
released
3. Separate serum/plasma immediately to decrease chance of release of ACP from
RBCs and platelets
4. Labile enzyme, decreases activity within 1-2 hours at room temperature if
without preservative
5. If not used immediately, sample may be frozen or acidified to a pH less than 6.5
6. Acidification makes ACP stable for up to 2 days at room temperature.
7. When using RIA procedures, serum must not be acidified, and it will be stable for
2 days at 4oC
8. Newborns can have twice to thrice ACP that of adults
 Acid Phosphatase
Laboratory Methods
*Quantitation of ACP is complicated as the physiological role of this enzyme is
unknown. Consequently, a substrate that is specific for the enzyme and reflect its
biochemical function cannot be designated. Therefore, the methods developed only
focused on the precision, sensitivity and the ease of measurement.

1.Roy-Hillman:
-uses the substrate of choice for quantitative endpoint reactions, highly specific
for prostatic ACP
0.2 mL serum + thymolphthalein monophosphate
After 30 minutes, NaHOCl is added-->thymolphthalein (yellow color measured
at 590 nm)
 Acid Phosphatase
Laboratory Methods
2. Most commonly used substrate:
p-nitrophenylphosphate (colorless) + water <--ACP at pH 5.0 -->p-nitrophenol
+ phosphate ion
p-nitrophenol + OH –-Alkaline pH--> p-nitrophenolate ion (yellow) + water
*What is the purpose of alkaline pH?
Methods that uses p-nitrophenylphosphate:
a. Bessey-Lowry-Brock
b. Hudson
3. Assays using phenylphosphate:
a. King-Armstrong
b. Gutman-Gutman
c. Fishman-Betner
 Acid Phosphatase
Laboratory Methods

4. Other substrates:
a. Babson-Read-Phillips: A-napthyl phosphate
-preferred for continuous monitoring, less specific but with increased
specificity
b.Bodansky: B-glycerolphosphate
c.Rietz-Guilbault: 4-methylum-belliferone phosphate

Reference Ranges: 0 to 3.5 ng/mL (Prostatic ACP)

 Acid Phosphatase
Laboratory Methods - Isoenzyme Assays
1. Electrophoresis: RBC isoenzyme stays at point of origin, all others have high
mobility
2. Chemical Inhibition:
a. L-tartrate ions: inhibits prostatic ACP
-not entirely specific, but other ACP isoenzymes are mostly uninhibited
-Total ACP – ACP after inhibition = prostatic ACP
b. Formaldehyde and cupric ions: inhibits RBC ACP
3. Immunochemical method: best approach to detect prostatic isoenzyme; both
sensitive and specific
Ex: RIA, Counter-immune electrophoresis, Immunoprecipitation
 Acid Phosphatase
Clinical Significance
1. Prostatic carcinoma: primary utility
-prostatic ACP increases only in 50% of patients with metastasized tumor *PSA
2. Other conditions of the prostate:
-hyperplasia, prostatitis, surgery, prostatic massage, rectal exam
3. Other malignancies: breasts and others
4. Forensic clinical chemistry: investigation of rape; persists in vaginal washings for
up to 4 days
5. Bone diseases: Paget’s disease, diseases involving osteoclasts
6. Some cases of liver disease, biliary obstruction and kidney disorders
7. Gaucher’s disease
8. Thrombocytopenia due to platelet destruction (ITP)
 5' Nucleotidase
• A phosphoric monoester hydrolase which acts only on nucleotides,
believed to function in the extracellular production of adenosine, nutrient
absorption and cell proliferation

Reaction Catalyzed:
5’ ribonucleotide + water --5’NT ribonucleoside + phosphate
Zinc is an integral part of the enzyme

Tissue Distribution
• Widely distributed; plasma 5’NT predominantly comes from the liver
 5' Nucleotidase
Specimen Considerations
1. Non-hemolyzed serum is preferred
2. Chelating agents inhibit the enzyme

Laboratory Methods
Measurement is generally difficult as ALP can cleave its substrate.
Methods uses the principle of competitive inhibition with ALP.

Clinical Significance
1. Determine the source of an increased ALP
2. Most commonly increased in bile duct obstructions; also, in acute
hepatitis
3. Others: Rheumatoid arthritis, Ovarian cancer
 Amylase
• Smallest enzyme with a MW of ~50,000 Daltons

Reaction Catalyzed: Digestion of starch and glycogen to form

maltodextrins
Starch/glycogen -–AMS, Ca, Cl--> glucose, maltose, dextrin
Calcium is an integral part of the enzyme
Calcium and chloride is required for activation

Isoenzymes Tissue Notes Associated Conditions

Migrates slowly Acute pancreatitis: P-type
P – P1, P2, Acinar cells of the Dominates the normal urine activity, especially P3
P3 pancreas amylase May also increase in renal
Common fraction: P2 failure
Salivary gland Migrates fast
S – S1, S2, Fallopian tube 2/3 of the normal serum
S3 Lungs amylase
Others Common fraction: S1, S2
 Amylase
Specimen Considerations
1. Both serum and urine amylase are relatively stable; RT: 1 week, 4oC: 2 months
2. Serum or heparinized plasma may be used
3. Hemolysis does not affect most methods, except the enzyme assays that make use
of peroxide and peroxidase reaction
4. Triglycerides suppresses AMS activity; thus, the possibility of normal AMS activity
in acute pancreatitis cases with lipemia
5. Pain relievers such as morphine, codeine and other opiates used prior to sampling
leads to false elevations of serum AMS activity
6. Administration of glucocorticoids, dexamethasone or oral contraceptives may also
cause false elevations of serum AMS activity
 Amylase
Laboratory Methods
1. Amyloclastic: measures the disappearance of starch substrate
Starch-iodine complex (dark blue) -–AMS-->
breakdown of starch, release of iodine (decrease in color)

2. Saccharogenic: measures the appearance of product formed

-classic reference method; reported in Somogyi units
-Somogyi units are an expression of the number of milligrams of glucose
released in 30 minutes at 37°C under specific assay conditions
Starch –-AMS--> glucose and other sugars
*Reducing sugars are detected
 Amylase
Laboratory Methods
3. Chromogenic: measures increasing color from production of product coupled with
a chromogenic dye
Starch-chromogen dye complex (water-insoluble) -–AMS-->
smaller starch-chromogen dye complex (water-soluble,
increases color)

4. Continuous monitoring: coupling of enzyme systems to monitor amylase activity;

Optimal pH = 6.9
Maltopentose --AMS--> maltotriose + maltose
Maltotriose + maltose --A-glucosidase--> 5-glucose
5-glucose + 5 ATP --hexokinase--> 5 ADP + 5-Glucose-6-phosphate
G6P + NAD+ --G6PD--> 6-phosphogluconate + NADH + H+
(increase in absorbance at 340nm)
Reference Ranges: 25–130 U/L (Serum); 1-15 U/h (Urine)
 Amylase
Clinical Significance
1. Pancreatitis:
Acute pancreatitis: AMS activity rises 2-12 hours after onset, peak at 24
hours, remain elevated for 3 days
Chronic pancreatitis: remains elevated
Viral hepatitis: produces secondary pancreatitis
2. Salivary gland diseases: mumps, parotitis, lesions
3. Intraabdominal conditions: perforated/penetrating peptic ulcer, intestinal
obstruction, cholecystitis, ruptured ectopic pregnancy, mesenteric infarction,
acute appendicitis, etc.
4. Renal diseases: insufficiency, ketoacidosis
5. Macroamylasemia: asymptomatic condition of hyperamylasemia seen in 1-2% of
the population
-Amylase molecules combine with immunoglobulins IgG or IgA, forming a
complex too large to be filtered across the glomerulus
 Amylase
Clinical Significance

6. Decreased in:
Extensive destruction of the pancreas
Advanced cystic fibrosis
Marked hepatic insufficiency
 Lipase
• For the metabolism of triglycerides

Reaction Catalyzed: Preferentially hydrolyzes glycerol esters of long

chain fatty acids at the C1 and C3 bonds, producing 2 mole of fatty
acids and 1 mole 2-monoglyceride
Triacylglycerol + H2O –-LPS, Ca--> 2-monoglyceride + fatty acids
Substrate should be emulsified for activity to occur
Reaction is accelerated by the presence of colipase and a bile salt
Calcium is required for activation, but a high level is inhibitory due to
interference with bile salt action
Heavy metals and quinine inhibit lipase activity
 Lipase
Isoenzyme Tissue Notes
Pancreatic tissue and secretions
GI mucosa (Stomach, Intestine) L2 is the most sensitive and
L – L1, L2, L3
RBCs and WBCs specific isoenzyme
Liver, fat cells, milk

Specimen Considerations
1. Non-hemolyzed serum; hemolysis causes false decrease due to inhibitory action of
hemoglobin
2. Stability: RT – 1 week; 4oC – 3 weeks; Frozen – 2 months
3. Optimal reaction temperature: 40oC; Optimal pH: 8.8, reactions may still occur at
pH 7.0-9.0
 Lipase
Laboratory Methods
1.Cherry-Crandall: classic method
-uses olive oil as substrate-->24-hour incubation-->measured by titration
-Modification using triolein as a substrate for a purer form of TAG
Triacylglycerol + H2O –-LPS, Ca--> 2-monoglyceride + fatty acids

2.Turbidimetry: simpler and more rapid than titration

-Presence of fats causes a cloudy emulsion -->action of LPS-->particles
disperse -->rate of clearing is measured = LPS activity

3. Coupled enzyme assays: uses peroxidase or glycerol kinase

Reference Ranges: 0–1.0 U/mL

 Lipase
Clinical Significance
1.Pancreatitis: more specific than AMS
Acute pancreatitis: AMS activity rises 12-24 hours after onset, peaks at 2-4
days, remains elevated for 5 days or more
*Level of elevation does NOT correlate with severity of disease
Chronic pancreatitis
2. Intraabdominal conditions and other conditions also associated with AMS:
increases are less frequent than that of AMS – perforated/penetrating peptic
ulcer, duodenal ulcer, intestinal obstruction, acute cholecystitis
3. Drugs inducing spasm of the sphincter of Oddi: opiates/morphine
 Cholinesterases
• catalyzes esters with choline groups
Types Acetylcholinesterase (AChE) Pseudocholinesterase (PChE)
RBC cholinesterase *Serum cholinesterase (SChE)
Choline esterase I Choline esterase II
Names
Acetylcholine acylhydrolase Acylcholine acylhydrolase
True cholinesterase Butyrylcholinesterase
Reaction Acetylcholine + waterà acetate + Acylcholine + waterà Carboxylate
Catalyzed choline + choline
Butyrylcholine
Specificity Acetyl-B-methylcholine
Benzoylcholine
Tissue RBC, nerve synapse, gray matter Serum, liver, heart, pancreas,
Sources of the brain, lungs, spleen white matter of the brain
Unknown normal function
Physiological Produces rapid termination of
Cleaves muscle relaxants used
Role neurotransmission
during surgery
 Cholinesterases
Specimen Considerations
1. AChE: RBC hemolysate
2. PChE: Non-hemolyzed serum or heparinized plasma
3. PChE specimens for patients experiencing complications due to muscle relaxants
should be drawn when the paralysis has passed
4. Reference values are dependent to age, gender and body mass:
• Females have ~75% PChE activity that of males
• Pregnant females have even lower activities than nonpregnant females
• Oral contraceptives cause a 15% decrease
• Infants up to 6 months old have 40-50% PChE activity that of adults
• Increased BMI associated with ncreased PChE; Low protein intake leads to
decreased PChE
 Cholinesterases
Laboratory Methods
1. Ellman: uses acylthiocholine ester as substrate and dithiobisnitrobenzoic acid as
reagent
Acylthiocholine --SChE--> Carboxylic acid + Thiocholine
Thiocholine + Ellman's reagent-->5-mercapto-2-nitrobenzoic acid
2. Michel: uses benzoylcholine as substrate; measures the change in UV absorption
3. Gel electrophoresis: AChE detected in amniotic fluid

Reference Ranges: 6,000-12,000 U/L (adults, serum)

 Cholinesterases
Clinical Significance
1. Marker for poisoning by organic phosphorus insecticides which causes irreversible
inhibition of PChE (acute toxicity) and AChE (chronic exposure)
2. Diagnosis of genetic variants of ChE
3. Indicator of synthetic capacity of the liver: PChE low in acute hepatitis, cirrhosis,
liver CA, malnutrition
4. Nephrotic syndrome: PChE may be normal to increased
5. Neural tube defects: AChE is present in amniotic fluid
 Clinical Enzymology

Enzymes of Clinical Significance:

LYASES
 Aldolase
• catalyst of one of the reactions in glycolysis; seen in all body cells

Reaction Catalyzed:
D-fructose-1,6-diphosphate <–-ALD--> dihydroxyacetone
phosphate + D-glyceraldehyde-3-phosphate

Clinical Significance
1. Muscle diseases: highest in progressive muscular dystrophy
2. Myocardial infarction: rises in 6-8 hours, peaks at 24-48 hours and stays
for up to 3-4 days
 Clinical Enzymology

Enzymes of Clinical Significance:

Summary of Organ Markers
 Cardiac Markers
MARKER RISE FROM ONSET PEAK OF ACTIVITY BACK TO NORMAL
OF CHEST PAIN LEVELS
ALD 6-8 hours 24-48 hours 3-4 days
AST 6-8 hours ~24 hours ~5 days
LDH 12-24 hours *flipped 48-72 hours ~10 days
CK 2-4 hours 12-36 hours 2-4 days
CK-MB 4-6 hours 12-24 hours 2-3 days
Myoglobin 1-4 hours 6-9 hours 18-24 hours
Total 4-10 hours 12-48 hours 4-10 days
troponins
Troponin I 4-6 hours 12-18 hours ~6 days

Troponin T ~4 hours ~48 hours 7-10 days

Other Organ Function Markers
ORGAN MARKER/S
Liver: Hepatocellular ALT, AST, GGT, LD, LD-4, LD-5, ALD,
ICD
Liver: Obstructive ALP, GGT, 5NT, LAP
Skeletal muscle CK, CK-MM, AST, LD, LD-4, LD-5,
ALD
Pancreas LPS, AMS
Bone ALP
Prostate ACP
Lungs LD, LD-3
Brain CK-BB*