ACID PHOSPHATASE  Thrombocytopenia

E.C. 3.1.3.2 - Resulting from excessive platelet destruction

 Orthophosphoric Monoester
 Phosphohydrolase (Acid Optimum)
- pH: below 7.0
- belongs to hydrolase
- Plasma ACP is stabilized by acidification to pH
below 6.5 with an optimal pH of approximately
5.0.
Tissue Sources
- Prostate - richest source
- Bone
- Liver
- Spleen
- Kidney
- RBC
- Platelets
Diagnostic Significance
 has been used as an aid in the detection of
prostatic carcinoma, particularly metastatic CA
of the prostate
 Prostate Specific Antigen (new markers)
 more specific than ACP
 most useful diagnostic tool
 may elevate in benign prostatic hypertrophy
and prostatitis
 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
 Gaucher's disease:
- caused by a Deficiency of the enzyme
glucocerebrosidase
 Platelet destruction
from Idiopathic Thrombocytopenic Purpura
(ITP)
 Thymolphthalein monophosphate
- Substrate of choice for quantitative
endpoint reactions
 Alpha-naphthyl phosphate
- Substrate of choice for continuous
monitoring methods
 Immunochemical Techniques
 RIA
 Counterimmunoelectrophoresis
 Immunoprecipitation
 Immunoenzymatic assay (Tandem E)
 Incubation with an antibody to prostatic ACP
followed by washing and incubation with p-NPP
 p-Nitrophenolphosphate formed is proportional
to the prostatic ACP in the sample
Sources of Error
- 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
REFERENCE RANGE
Prostatic ACP 0-3.5 ng/mL
Total ACP
ADULTS 1.5-4.5 U/L (34C)
CHILDREN 3.5-9.0 U/L (37C)
 CREATINE KINASE Plasma CK levels and CK/progesterone ratio
E.C.2.7.3.2 - Useful in the diagnosis of ectopic
ATP: Creatine N-phosphotransferase pregnancies
- Associated with ATP regeneration in contractile Total plasma CK
or transport system - Early diagnostic tool to identify px w/
- Predominant in muscle cells Vibrio vulnificus infxns.
- 82,000 MW
- Involve in the storage of high-energy creatine CK occurs as DIMER with 2 subunits:
phosphate  B type
 M type
3 isozymes:
1. CK-BB
Tissue Sources - Brain type
- Skeletal muscles - Migrate fastest toward anode (CK-1)
- Heart muscle 2. CK-MB (CK-2)
- Brain tissues - CK-MB 1- modified by plasma
Others: Bladder, placenta, GIT, thyroid, Carboxypeptidase
uterus, kidney, lung, prostate, spleen, - CK-MB 2- tissue isoform and predominant
liver, and pancreas isoform in plasma.
Isoenzymes - CK-MB 1 AND CK-MB2 ratio  </-1
 CK-MM 3. CK-MM
 Skeletal muscles - Slowest mobility (CK-3)
 CK-3 - Plasma of healthy people
 CK-MB - Major isozyme fraction found in striated muscle
 Heart muscle with a small amount of CK-MB
 CK-2
 CK-BB
 Brain tissues
 CK-1

 CK-Mi (mitochondrial CK) - bound to exterior

surface of the inner mitochondrial membranes
of muscle, brain, and liver
- It migrates to a point cathodal to CK-MM and
exists as a dimeric molecules of two identical
subunits
 Macro CK
 largely comprises CK-BB complexed with Ig
(IgG)
 migrate to a position midway between CK-
MM and CK-MB
 CK-MM + lipoproteins
DIAGNOSTIC SIGNIFICANCE
- Plasma CK levels are frequently
elevated in disorders of cardiac and skeletal
muscle
- The CK level is considered a sensitive
indicator of acute myocardial infarction and
muscular dystrophy  DUCHENNE TYPE
DUCHENNE TYPE:
- Extreme elevations of CK occurs
- Values reaching 50-100 times ULN
 LACTATE DEHYDROGENASE
Note: E.C. 1.1.1.27
 CK-MB: Myocardial Infarction L-Lactate: NAD+ Oxidoreductase
 Rise: within 4-8 hours after the onset
 Peak: 12-24 hours
 Return to normal: within 48-72 hours
ELECTROPHORESIS
- Commonly used as reference method
for the distinction of the different forms.  Catalyzes the interconversion of
lactic and pyruvic acids
Adenylate kinase: AK may interfere with chemical or
 Hydrogen-transfer enzyme that uses the
immu-noinhibition methods, causing a falsely elevated
coenzyme NAD+
CKor CK-MB value
Tissue Sources
ION EXCHANGE CHROMATOGRAPHY
- Has the potential for being more sensitive and
precise than electrophoretic procedures performed
with good technique
Assays
1. Tanzer-Gilvarg: forward method

2. Oliver-Rosalki Method: backward method

- Most commonly performed method
- Proceeds 2-6 times faster than
the forward reaction
- Optimal pH (backward): 6.8 Widely distributed in the body
- Optimal pH (forward): 9.0  Heart
Sources of Error  Liver
 Hemolyzed samples elevate CK activity  Skeletal muscle
 Serum should be stored in dark place  Kidney
 Activity can be restored after storage in the  Erythrocytes
dark at 4 degrees Celsius for 7 days or at -20  Lung
degrees Celsius for 1 month when the assay  Smooth muscle
is conducted using a  Brain
sulfhydryl activator DISTRIBUTION OF ISOENZYMES
 Muscle activity prior the blood collection ISOENZYME PERCENTAGE
affects CK activity LD-1 14-26%
Reference Range LD-2 29-39%
TOTAL CK: LD-3 20-26%
MALE 46-171 U/L (37C) LD-4 8-16%
LD-5 6-16%
FEMALE 34-145 U/L (37C)
Myocardial Infarction
CK-MB <5% TOTAL CK
 Rise: within 12-24 hours after the onset
 Peak: 48-72 hours
 Remain elevated: 10 days
 LD 1 > LD 2
 LD flipped pattern
 Suggestive of AMI
 Diagnostic Significance GAMMA-GLUTAMYLTRANSFERASE (GGT)
 Pernicious anemia and Hemolytic Disorders E.C. 2.3.2.2
- Highest plasma levels of total LD. (G-Glutamyl) Peptide:
 ALP and Transaminases Amino Acid-5-Glutamyl Transferase
- Are better plasma makers for liver - Involved in the transfer of the gamma-glutamyl
damage. residue from gamma-glutamyl peptides to
LD isozymes: amino acids, H2O, and other small peptides
1. LD 1- higher in cardiac tissue and RBCs - Glutathione: serve as the glutamyl donor
2. LD 2- the major isozyme fraction
3. LD 3- elevation occurs most frequently with
pulmonary involvement and in px having
carcinomas Physiology
4. LD 4- found primarily in the liver and skeletal Has not been clearly established
-
muscle tissue Involved in peptide and protein synthesis
-
5. LD 5- found primarily in the liver and skeletal Regulation of tissue glutathione levels
-
muscle tissue Transport of amino acids across cell
-
- Levels have greatest clinical membranes
significance in the detection of hepatic Tissue Sources
disorders - Kidney – highest conc.
Analysis of LD - Brain
 Electrophoresis - Prostate
 Isoenzymes can be detected either by - Pancreas
fluorometry or colorimetry - Liver
 Substrate: alpha-hydroxybutyrate (with ● Clinical applications are confined mainly to
increase affinity with H subunit than M subunit) evaluation of liver and biliary system disorders
 For LD-1 activity Diagnostic Significance
 Rate of the reverse reaction is approx. 3 times  Located in the canaliculi of the hepatic cells and
faster but more susceptible to substrate particularly in the epithelial cells lining the biliary
exhaustion and loss of linearity
ductules
● Optimal pH (forward): 8.3 - 8.9
 Elevated in all hepatobiliary disorders
● Optimal pH (backward): 7.1 -7.4
 Biliary tract obstruction (5-30 times ULN)
 Within the hepatic parenchyma, GGT exists to a
large extent in the smooth ER, subject to hepatic
microsomal induction
 GGT is increased in patients receiving enzyme-
inducing drugs (warfarin, phenobarbital,
phenytoin)

 Chronic Alcoholism – high alcohol consumption

Sources of Error
● Unknown etiology
- RBC contains LD concentration 100-150 times
● Acute pancreatitis
than found in serum
● DM and MI
- LD activity is unstable in serum regardless of the
Hepatocellular Biliary
temperature
Involvement Obstruction
● Should be stored at RT and analyzed
GGT Normal Increase
within 48 hours
ALP (if bone High Increase
● LD-5 is the most labile (loss of activity
ALP is normal)
more quickly at 4 degrees Celsius at RT)
- LD isoenzyme analysis should be stored at RT
and analyzed within 24 hours of collection
Assay

Reference range:
LD: 125-220 u/l (37C)
 ASSAY Tissue Sources
 Gamma (y)-glutamyl-p-nitroanilide  Adrenal cortex
- The most widely accepted substrate for use in  Spleen
GGTanalysis.  Thymus
- The y-glutamylresidue is transferred to  Lymph nodes
glycylglycine, releasing p-nitroaniline. - Lactating mammary gland erythrocytes
 p-nitroaniline- a yellow chromogenic product Diagnostic Significance
with astrong absorbance at 405 to 420 nm.  RBC - Maintain NADPH in reduced form
- the reaction,which can be used as a  Required to regenerate sulfhydryl-containing
continuous monitoring orfixed-point method. 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
Red cell hemolysate- used to assay for deficiency of
the enzyme.
REFERENCE RANGE
7.9-16.3 U/g Hgb

ANGIOTENSIN-CONVERTING ENZYME
SOURCES OF ERROR
E.C. 3.4.15.1
- GGT activity is stable, with no loss activity for
Angiotensin I -Converting Enzyme
1 week at 4 degrees Celsius
- Kininase II
- Hemolysis does not interfere with GGT levels
- Peptidyl-Dipeptidase A
REFERENCE RANGE
- Hydrolysis of peptide bonds at a free C-terminus
GGT:
- Releasing a dipeptide in the reaction
 male, 6-55 U/L (37°C)
- Act as endopeptidase or an aminopeptidase
 female, 5-38 U/L(37°C)
- Conversion of angiotensin I to angiotensin II (RAAS)
Values are lower in females, presumably becauseof
- Inactivation of bradykinin (Kallikrein-Kinin System)
suppression of enzyme activity resulting from estro-
genic or progestational hormones.
GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G-6-PD)
E.C. 1.1.1.49
- An oxidoreductase that catalyzes the
oxidation of glucose-6- phosphate to 6-
phosphogluconate or the corresponding
lactone
- Reaction is important in the pentose-
phosphate shunt of glucose metabolism with
the ultimate production of NADPH
-

- Modulates peripheral vascular resistance as

well as renal and cardiovascular function
● Renal and cardiovascular function
● Zinc-metalloprotease
Measurement - Tissue-bound, with much lower levels circulating
- Measured by its ability to cleave synthetic peptides, in plasma
releasing hippuric acid ● Predominantly found in endothelial cell
membranes throughout the body
DIAGNOSTIC SIGNIFICANCE ● Lungs and Testes - rich in ACE
- Most of the interest of G-6-PD focuses on its rolein the
erythrocyte.
- It functions to maintain NADPH in reduced form.  PChE
- An adequate concentration of NADPH is required for  Organophosphate Insecticides
anabolic pathways of metabolism and the  Irreversible inhibitors of both AChE and PCHE
maintenance of glutathione levels from the oxidized to  PChE activity (serum) falls before AChE activity
the reduced state. (RBC)
- G-6-PD deficiency is an inherited sex-linked trait. The  AChE
disorder can result in several different clinical  Useful in organophosphate exposure and
manifestations, including drug-induced hemolytic poisoning
anemia.  Qualitative analysis in amniotic fluid may be
Causes of Abnormal Results useful in the diagnosis of neural tube defects
- The most common reason for ordering ACE levels is in 5'-NUCLEOTIDASE (5'N)
diagnosis and monitoring of sarcoidosis E.C. 3.1.3.5
- Sarcoidosis : 5'-Ribonucleotide Phosphohydrolase
 commonly referred to simply as 'sarcoid', areas - Is a cytoplasmic membrane-bound
of inflammation called granulomas may phosphatase
appear on the body. - Acts only on nucleotides
 Any part of the body can be affected but the - To function in extracellular adenosine
most commonly affected areas are the lungs, production, nutrient absorption, and cell
skin, eyes and lymph nodes proliferation
CHOLINESTERASE (ChE) - A metalloenzyme (zinc)
E.C. 3.1.1.7: Acetylcholinesterase - Widely distributed in the body, predominantly
E.C. 3.1.1.8: Cholinesterase attached to cell membranes (similarly to ALP
E.C. 3.1.1.7 and GGT)
● Acetylcholinesterate - Predominantly derived from the liver
● Acetylcholine Acylhydrolase

"True" AChE
- Found in the synapse of the nerve and RBC
- Not normally found in amniotic fluid Measurement
- Hydrolyzes acetyl-beta-methylcholine - Made difficult because other phosphatases are
capable of cleaving the substrate
- Uses ALP inhibitors
- Chelating agents inhibit activity
Clinical Significance
- Commonly used to determine if the source of an
elevated ALP is from liver or bone
Pseudo-acetylcholinesterase - Cholestatic disorders
- Found in the serum - Ovarian CA, rheumatoid arthritis
- Production occurs primarily in the liver
- For cleavage of succinylcholine and
mivacurium
- Hydrolyzes butyryl- and benzoylcholine
MEASUREMENT
 Ellman's Method
 Substrate: acylthiocholine ester
- Released thiocholine react with
dithiobisnitrobenzoic acid
 Product: 5-mercapto-2-nitro benzoic acid
 PseudoChE: Serum
 True ChE: Hemolysate of washed RBC
 PseudoChE
 To monitor exposure to cholinesterase inhibitors
 As a liver function
 For diagnosis of genetic variants