CC2

MIDTERMS DAY 4
PRFBREMNER
Amylase
EC. 3.2.1.1 ( alpha – Amylase)
•Belongs to Hydrolases that catalyze breakdown of
starch and glycogen.
•Smallest enzyme; Filtered by the glomerulus.
Tissue location: Acinar cells of the pancreas and the
salivary glands. Adipose tissues, fallopian tubes, small
intestine and skeletal muscles.
Isoenzyme/ Forms/ Sources
P form( Amylopsin)- from the acinar cells of the
pancreas
S form( Ptyalin)- salivary glands
Amylase
With salivary Amylase – MOUTH digests starch.
Pancreatic Amylase – performs the major digestive
action of starches

Sample Requirement:
•With the exception of heparin, all common
anticoagulants inhibit AMY activity because they chelate
Ca++
•USE ONLY serum or heparinized plasma.
Amylase
Amylase Assays
1.Amyloclastic- measures disappearance of starch and
measuring the amount of polysaccharide remaining.
•a starch-iodine reaction producing a blue iodine
inclusion compound with amylose/amylase.
•DECREASE in color is proportional to AMS concentration.
2. Saccharogenic- measures the appearance of the
product ( reducing sugar) in a fixed time.
•Not satisfactory
•The amount od Reducing sugars is measured,
proportional to AMS concentration.
Amylase
Reported in Somogyi Unit-

3. Chromogenic – measures increasing color from

production of product coupled w/ chromogenic dye.
•AMS hydrolyzes starch substrate , smaller dye substrate
solution is proportional to AMS concentration.

4. Enzymatic: Defined substrate used in coupled-

enzymatic reactions
Enzymatic methods
• Coupled enzyme systems have been used to
determine AMS activity by continous monitoring
technique in which a change in absorbance od
AND is measured.
1.Substrate, ethylidene-pNP-G7, cleaved by the
amylase.
• One unit is the amount of amylase that cleaves
ethylidene-pNP-G7 to generate 1.0 µmole of p-
nitrophenol per minute at 25 °C.
Amylase
Substrates:
2.Malto pentaose
3. Maltopentaose
Substrate :
4. 4 NP glycoside

5. 2-chloro-p-nitrophenyl alpha d maltorioside

(CNP-G3)- a substrate
•This assay does not require glucosidase and is
considered a "direct" assay”
Amylase
• AMYLASE ISOENZYME MEASUREMENT:
Salivary amylase( S1,S2,S3) migrate More quickly
Than Pancreatic isoenzymes (p1,p2,p3)
(1) electrophoresis
(2 ) ion exchange chromatography
(3) isoelectric focusing
(4) selective inhibition of the S-AMY by a wheat germ
inhibitor
(5) immunoprecipitation by a monoclonal antibody
(6) immunoinhibition
 Amylase

Clinical Significance:
1.Acute Pancreatitis (Rise: 2-12 hours, Peak: 24 hours, Normal: 3-
5 days).
P3 isoenzyme is predominantly increased
1.Parotitis due to mumps
2.Ectopic Pregnancy
3.Peptic ulcers
4.Alcoholism
5.Acute appendicitis
6.Mesenteric infarction
7.*Macroamylasemia – Amylase bound to Immunoglobulins
Amylase
2. Biliary tract diseases, such as cholecystitis cause up
to four fold elevations of the serum AMY

3. Hyperamylasemia- when Amylase molecules combine

w/ Immunoglobulins to form a complex TOO large for
glomerulus to filter.

** Salivary amylase INCREASE in acute parotitis.

Lipase ( EC. 3.1.3.3) LPS
- An enzyme that hydrolyses the ester linkages of
FATS to produce alcohols and fatty acids.
- Hydrolyses TRIGLYCERIDES

Tissue Source: Pancreas,( most specific pancreatic

marker) small Intestine and adipose tissues
.
Methodology
Assay: by Cherry Crandall Method
hydrolyzed by lipase
Triglyceride (Triolein)+ H2O ------- mono /di glyceride+
free fatty acids ( dissociated H ions)
1.Titrimetric- measures DIRECTLY free fatty acids by
Titrating with dilute alkali.

•The amount of alkali used is recorded as a function of

time and serves as a measure of fatty acid produced
during the reaction.
Lipase

2. Turbidimetric- measures the decrease of light

scattering after hydrolysis to mono/di esters.
Oleic acid emulsion( 0.8%) + H2O fatty acid
(Decrease turbidity)

+ 2 monoglyceride
Lipase
3. Spectrophotometric
Use of a substrate - 1,2 - O-dilauryl-rac-glycero—3
glutaric acid ( 4-methyl-resorufin) - ester has been
proposed to measure LPS activity.
•LPS hydrolyzes the substrate in an alkaline medium
to an unstable dicarbonic acid ester that
spontaneously hydrolyzes to yield
•glutaric acid and methylresorufin, a bluish-purple
chromophore with peak absorption at 580 nm.
Lipase
• The rate of methyl resorufin formation is directly
proportional to the LPS activity of the sample.

Reference Range: 0-1 U/mL

The upper reference limit is 38 U/L at370C

Source of Error :
NO Hemolysis – Hgb INHIBITS LIPASE activity in
serum
 Lipase
Clinical Significance:
1.Acute Pancreatitis (Rise: 6 hours, Peak:24 hours, Normal:after
7 days)
Concentrations often remain elevated longer than those
of AMY.
2. Perforated Ulcers
3. Duodenal Ulcers
4. Intestinal Obstruction
5. Cholecystitis
Gamma Glutamyl transferase
• EC 2.3.2.1
• regulates the transport of amino acids across cell
membranes by catalyzing the transfer of a
glutamyl group from glutathione to a free amino
acid.
Tissue location: Canaliculi of hepatic cells,
particularly epithelial cells of the biliary truct,
kidney, prostate and pancreas
.
Methodology by Szasz
GGT
y-glutamyl-p-nitroanilide(gamma GPNA) 
glycylglycine ( glutamyl receptor) + p-nitroaniline ,
(produced in the reaction is determined by its yellow
color) at 405 nm.

•L-y-glutamyl-3-carboxy-4-nitroanilide- IFCC substrate

presently used measured at 410 nm

NV: 0-38 U/L for females and 0- 55 U/L for males

Gamma Glutamyl transferase
Clinical Significance:
Renal tissue – highest GGT BUT serum has liver GGT
•Elevated activities of GGT are found in the sera of
patients with alcoholic hepatitis and in the majority
of sera from people who are heavy drinkers.
•Indicator of hepatobiliary disease
•Primary / metastatic liver neoplasms
•Moderate elevations in Infectious hepatitis.
•Differentiates INCREASE in ALP.
Glucose 6 phoshate de hydrogenase
EC 1.1.1.49
•is expressed in all cells and catalyzes the first step
in the hexose monophosphate pathway, the
conversion of glucose-6-phosphate to 6-
phosphogluconate, generaring NADPH.

•It is important in the pathway that supplies reducing

energy to cells by maintaining the NADPH .NADPH is
required to synthesize GLUTATHIONE.
G6pD
• Gluthathione in reduced form protects
Hemoglobin from oxidation by agents present in
the cell.
• G6pd deficiency is an inherited sex linked trait
G6pD
Tissue sources:
Adrenal cortex
Spleen
Thymus
Lymph Nodes
Lactating mammary gland
RBC
Methodology

g6pd
Glucose 6 phosphate + NADP-
6 phosphogluconate + NADPH + H

Sample : Red cell hemolysate

Ref Ranges: 10-15 U/g Hgb

Clinical Significance
Deficiency of G6pD
•Drug induced hemolysis
•Infection induced hemolysis
•Favism
•Neonatal jaundice
•Chronic Non spherocytic hemolytic Leukemia
 Cholinesterase
Two related enzymes:
Acetylcholinesterase (AChE)/true cholinesterase
Acylcholine acylhydrolase (PChE)/pseudocholinesterase

Tissue location
1) True cholinesterase found in red blood cells, lungs,
spleen, nerve endings, gray matter of brain

2) Pseudocholinesterase found in liver, pancreas, heart,

white matter of brain, serum
Methodology
• acylthiocholine esters such as butyrylthiocholine as substrate
Is Hydrolysed to butyrate and thiocholine.
• Thiocholine +m5’dithiobis or 2-nirrobenzoate(DTNB)-
colored 5-mercapto- 2-nitrobenzoic acid, which is measured
spectrophotometrically at 410 nm.

• Serum is the prefered sample

NV: 33-76 U/L

 Clinical Significance
• as a test of liver function
• as an indicator of possible insecticide poisoning
• for the detection of patients with atypical
forms of the enzyme who are at risk of prolonged
responses to certain muscle relaxants used in
surgical procedures.
5 Nucleotidase
• Biliary epithelium is main source of 5’-
nucleotidase, and levels are highest cholestatic
conditions.
• Due to its specificity, it is another test to confirm
that an elevated ALP is due to hepatobiliary
disease
• Uses adenosine 5’ monophosphate as substrate
(5’AMP) - converted to adenosine and inorganic
phosphorus