● Gaucher's disease: caused by a

deficiency of the enzyme

ACID PHOSPHATASE (ACP)
glucocerebrosidase
E.C. 3.1.3.2 ● Platelet destruction
Orthophosphoric Monoester - Thrombocytopenia
Phosphohydrolase (Acid Optimum) - Resulting from excessive
platelet destruction from
Idiopathic Thrombocytopenic
Purpura (ITP)

Tissue Sources
- Thymolphthalein monophosphate
- Prostate - richest source
● Substrate of choice for quantitative
- Bone
endpoint reactions
- Liver
- Alpha-naphthyl phosphate
- Spleen
● Substrate of choice for continuous
- Kidney
monitoring methods
- RBC
- Immunochemical Techniques
- Platelets
● RIA
● Counterimmunoelectrophoresis
Diagnostic Significance ● Immunoprecipitation
● Immunoenzymatic assay (Tandem E)
- has been used as an aid in the detection of
- Incubation with an antibody to
prostatic carcinoma, particularly metastatic CA
prostatic ACP followed by
of the prostate
washing and incubation with
- Prostate Specific Antigen
p-NPP
● more specific than ACP
- p-NP formed is proportional to
● may elevate in benign prostatic
the prostatic ACP in the sample
hypertrophy and prostatitis

Sources of Error
- Thymolphthalein Monophosphate
● Substrate
- Tartrate
● Chemical inhibitor of prostatic ACP
● Not entirely specific for prostatic ACP
- Proven useful in forensic clinical chemistry,
particularly in the investigation of rape
● Vaginal washings are examined for
seminal fluid-ACP activity, which can
persist for up to 4 days
● Presumptive evidence of rape
- Increase ACP
● Bone disease: associated with the
osteoclasts
● Paget's disease: chronic bone disorder
that is due to irregular breakdown and
formation of bone tissue
● Breast cancer with bone metastases
- Serum should be separated from the red cells
ASAP to prevent leakage of RBC and platelet
ACP
- Activity decreases within 1-2 hours if the sample
is left at RT
- Decrease activity is a result of a loss of CO2
from the serum (increase pH)
- If not assayed immediately, serum should be
frozen or acidified to a pH lower than 6.5
- With acidification, ACP is stable for 2 days at
room temperature
- Hemolysis should be avoided because of
contamination from erythrocyte ACP
- RIA procedures for measurement of prostatic
ACP require non acidified serum samples
● Stable for 2 days at 4 degrees Celsius
 CREATINE KINASE (CK) CK-MB Heart Myocardial infarction
Skeletal muscle Myocardial injury Ischemia
E.C.2.7.3.2
Angina
ATP: Creatine N-phosphotransferase
Inflammatory heart disease
- Associated with ATP regeneration in contractile
Cardiac surgery
or transport system
Duchenne-type muscular
- Predominant in muscle cells
dystrophy
Polymyositis
Malignant hyperthermia
Reye's syndrome
Tissue Sources Rocky Mountain spotted
- Skeletal muscles fever
- Heart muscle Carbon monoxide Poisoning
- Brain tissues
CK-BB Brain Central nervous system
Others: Bladder, placenta, GIT, thyroid,
shock
uterus, kidney, lung, prostate, spleen,
liver, and pancreas Bladder Anoxic encephalopathy

Isoenzymes Lung Cerebrovascular accident

- CK-MM Prostate Seizure

● Skeletal muscles
● CK-3 Uterus Placental or uterine trauma
- CK-MB
● Heart muscle Colon Carcinoma
● CK-2
- CK-BB Stomach Reye's syndrome
● Brain tissues
Thyroid Carbon monoxide poisoning
● CK-1
Malignant hyperthermia
Acute and chronic renal
failure
Note:
- CK-MB: Myocardial Infarction
- CK-Mi - bound to exterior surface of the inner ● Rise: within 4-8 hours after the onset
mitochondrial membranes of muscle, brain, and ● Peak: 12-24 hours
liver ● Return to normal: within 48-72 hours
- Macro CK
● largely comprises CK-BB complexed Assays
with Ig (Ig) - Tanzer-Gilvarg: forward method
● CK-MM + lipoproteins

ISOENZYME TISSUE CONDITION

CK-MM Heart Myocardial infarction

Skeletal muscle Skeletal muscle disorder - Oliver-Rosalki Method: backward method
Muscular dystrophy
Polymyositis
Hypothyroidism
Malignant hyperthermia
Physical activity
Intramuscular injection
- Most commonly performed method
 - Proceeds 2-6 times faster than the forward
Pancreas Lymphocytosis
reaction
Acute pancreatitis
- Optimal pH (backward): 6.8
Carcinoma
- Optimal pH (forward): 9.0
LD-4 (HMMM) Liver Hepatic injury or
Sources of Error LD-5 (MMMM) Skeletal muscle inflammation
Skeletal muscle injury
- Hemolyzed samples elevate CK activity
- Serum should be stored in dark place
● Activity can be restored after storage in DISTRIBUTION OF ISOENZYMES
the dark at 4 degrees Celsius for 7 days ISOENZYME PERCENTAGE
or at -20 degrees Celsius for 1 month
when the assay is conducted using a LD-1 14-26%
sulfhydryl activator
- Muscle activity prior the blood collection affects LD-2 29-39%
CK activity
LD-3 20-26%

LACTATE DEHYDROGENASE (LD) LD-4 8-16%

E.C. 1.1.1.27 LD-5 6-16%

L-Lactate: NAD+ Oxidoreductase Myocardial Infarction
- Rise: within 12-24 hours after the onset
- Peak: 48-72 hours
- Remain elevated: 10 days
- LD 1 > LD 2
● LD flipped pattern
- Catalyzes the interconversion of lactic and ● Suggestive of AMI
pyruvic acids
- Hydrogen-transfer enzyme that uses the
coenzyme NAD+ Analysis of LD
- Electrophoresis
Tissue Sources ● Isoenzymes can be detected either by
fluorometry or colorimetry
- Widely distributed in the body ● Substrate: alpha-hydroxybutyrate (with
● Heart increase affinity with H subunit than M
● Liver subunit)
● Skeletal muscle - For LD-1 activity
● Kidney ● Rate of the reverse reaction is approx. 3
● Erythrocytes times faster but more susceptible to
● Lung substrate exhaustion and loss of
● Smooth muscle linearity
● Brain ● Optimal pH (forward): 8.3 - 8.9
● Optimal pH (backward): 7.1 -7.4
ISOENZYME TISSUE DISORDER

LD-1 (HHHH) Heart Myocardial infarct

LD-2 (HHHM) Red blood cells Hemolytic anemia
Renal cortex Megaloblastic anemia
Acute renal infarct
Hemolyzed specimen

LD-3 (HHMM) Lung Pulmonary embolism

Lymphocytes Extensive pulmonary
Spleen pneumonia
 - Within the hepatic parenchyma, GGT exists to a
Sources of Error
large extent in the smooth ER, subject to hepatic
- RBC contains LD concentration 100-150 times microsomal induction
than found in serum ● GGT is increased in patients receiving
- LD activity is unstable in serum regardless of the enzyme-inducing drugs (warfarin,
temperature phenobarbital, phenytoin)
● Should be stored at RT and analyzed - Chronic Alcoholism
within 48 hours ● Unknown etiology
● LD-5 is the most labile (loss of activity ● Acute pancreatitis
more quickly at 4 degrees Celsius at ● DM and MI
RT)
- LD isoenzyme analysis should be stored at RT
Hepatocellular Biliary
and analyzed within 24 hours of collection
Involvement Obstruction
- Assay:
GGT Normal Increase

ALP (if bone ALP High Increase

GAMMA-GLUTAMYLTRANSFERASE (GGT) is normal)
E.C. 2.3.2.2
(G-Glutamyl) Peptide: Amino Assay:
Acid-5-Glutamyl-Transferase

- Involved in the transfer of the gamma-glutamyl

residue from gamma-glutamyl peptides to amino
acids, H2O, and other small peptides
- Glutathione serve as the glutamyl donor

Physiology
- Has not been clearly established
- Involved in peptide and protein synthesis Sources of Error
- Regulation of tissue glutathione levels
- Transport of amino acids across cell membranes - GGT activity is stable, with no loss activity for 1
week at 4 degrees Celsius
- Hemolysis does not interfere with GGT levels
Tissue Sources
- Kidney GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G-6-PD)
- Brain
- Prostate E.C. 1.1.1.49
- Pancreas D-Glucose-6-phosphate: NADP+ 1- Oxidoreductase
- Liver
● Clinical applications are confined mainly - Catalyzes the oxidation of glucose-6- phosphate
to evaluation of liver and biliary system to 6-phosphogluconate or the corresponding
disorders lactone
- Reaction is important in the pentose- phosphate
shunt of glucose metabolism with the ultimate
Diagnostic Significance production of NADPH
- Located in the canaliculi of the hepatic cells and
particularly in the epithelial cells lining the biliary
ductules
● Elevated in all hepatobiliary disorders
● Biliary tract obstruction
 Tissue Sources
- Adrenal cortex
- Spleen
- Thymus
- Lymph nodes
● Lactating mammary gland Erythrocytes

Diagnostic Significance
- RBC
● Maintain NADPH in reduced form
- Required to regenerate sulfhydryl-containing
proteins (e.g. glutathione) from the oxidized to
the reduced state
- Glutathione in the reduced form protects Hb
from oxidation
- Inherited sex-linked trait
- Can cause hemolytic anemia
Assay
Measurement
- Modulates peripheral vascular resistance as well
as renal and cardiovascular function
● Renal and cardiovascular function
● Zinc-metalloprotease
- Tissue-bound, with much lower levels circulating
in plasma
● Predominantly found in endothelial cell
membranes throughout the body
● Lungs and Testes - rich in ACE
- Measured by its ability to cleave synthetic
peptides, releasing hippuric acid

ANGIOTENSIN-CONVERTING ENZYME (ACE)

Causes of Abnormal Results
E.C. 3.4.15.1
Angiotensin I-Converting Enzyme
- Kininase II
- Peptidyl-Dipeptidase A
- Hydrolysis of peptide bonds at a free C-terminus
- Releasing a dipeptide in the reaction
- Act as endopeptidase or an aminopeptidase
- Conversion of angiotensin I to angiotensin II
(RAAS)
- Inactivation of bradykinin (Kallikrein-Kinin
System)
- The most common reason for ordering ACE
levels is in diagnosis and monitoring of
sarcoidosis
- Sarcoidosis
● commonly referred to simply as
'sarcoid', areas of inflammation called
granulomas may appear on the body.
● Any part of the body can be affected but
the most commonly affected areas are
the lungs, skin, eyes and lymph nodes

CHOLINESTERASE (ChE)
E.C. 3.1.1.7: Acetylcholinesterase
E.C. 3.1.1.8: Cholinesterase

- E.C. 3.1.1.7
 ● Acetylcholinesterate - To function in extracellular adenosine
● Acetylcholine Acylhydrolase production, nutrient absorption, and cell
- "True" AChE proliferation
"True" AChE - A metalloenzyme (zinc)
- Found in the synapse of the nerve and RBC - Widely distributed in the body, predominantly
- Not normally found in amniotic fluid attached to cell membranes (similarly to ALP
- Hydrolyzes acetyl-beta-methylcholine and GGT)
- Predominantly derived from the liver

Pseudo-acetylcholinesterase
- Found in the serum
- Production occurs primarily in the liver
- For cleavage of succinylcholine and mivacurium
- Hydrolyzes butyryl- and benzoylcholine
Measurement
Measurement
- Made difficult because other phosphatases are
capable of cleaving the substrate
- Uses ALP inhibitors
- Chelating agents inhibit activity

- Ellman's Method
● Substrate: acylthiocholine ester ▸ Clinical Significance
Released thiocholine react with - Commonly used to determine if the source of an
dithiobisnitrobenzoic acid elevated ALP is from liver or bone
● Product: 5-mercapto-2-nitro benzoic - Cholestatic disorders
acid - Ovarian CA, rheumatoid arthritis
- PseudoChE: Serum
- True ChE: Hemolysate of washed RBC
- PseudoChE
● To monitor exposure to cholinesterase
inhibitors
● As a liver function
● For diagnosis of genetic variants
- PChE
● Organophosphate Insecticides
● Irreversible inhibitors of both AChE and
PCHE
● PChE activity (serum) falls before AChE
activity (RBC)
- AChE
● Useful in organophosphate exposure
and poisoning
● Qualitative analysis in amniotic fluid may
be useful in the diagnosis of neural tube
defects

5'-NUCLEOTIDASE (5'N)
E.C. 3.1.3.5
5'-Ribonucleotide Phosphohydrolase
- Is a cytoplasmic membrane-bound phosphatase
- Acts only on nucleotides