Enzymes

Part 2

M. Zaharna Clin. Chem. 2009

 Alanine Aminotransferase (ALT)
• A transferase with enzymatic activity similar to AST
• Converts alanine + α-ketoglutarate to pyruvate
and glutamate

M. Zaharna Clin. Chem. 2009

 Alanine Aminotransferase
• Pyridoxal phosphate is coenzyme
• Source is heart, liver, and skeletal muscle
• Primarily associated with liver disease
• Often higher than AST with liver damage
and tend to remain elevated longer
• Remains normal in AMI

M. Zaharna Clin. Chem. 2009

 Assay for enzyme activity
• The typical assay procedure consists of a
coupled enzymatic reaction using LD in the
second reaction
• NADH is oxidized in the second reaction
• The change in absorbance is at 340 nm is
measured
ALT
Alanine + -Ketoglutarate Pyruvate + Glutamate
LD
Pyruvate + NADH + H Lactate + NAD

M. Zaharna Clin. Chem. 2009

 Alkaline Phosphatase (ALP)
• Belongs to a group of enzymes that catalyze
hydrolysis of phosphate monoesters at alkaline
pH (9-10)
• Removes inorganic phosphate from organic
phosphate ester & generates alcohol
• Requires Mg as an activator

M. Zaharna Clin. Chem. 2009

 Alkaline Phosphatase (ALP)
• Present on all cell surfaces
• Highest in intestine, liver, bone
(osteoblasts), spleen, placenta, and kidney
• In Liver enzyme is located on both
sinusoidal and bile canalicular membranes
• ALP are most diagnostic significance in
the evaluation of hepatobiliary and bone
disorders

M. Zaharna Clin. Chem. 2009

M. Zaharna Clin. Chem. 2009
 Isoenzymes
• ALP exists as a number of isoenzymes
• Major are those found in Liver, bone,
placenta, and then intestinal fraction
• Electrophoresis for isoenzyme analysis
• Liver isoenzyme (fastest)
• Bone isoenzyme
• Placental isoenzyme
• Intestinal isoenzyme (slowest)
• Immunochemical methods now available

M. Zaharna Clin. Chem. 2009

 Clinical Significance
• Liver
• Obstructive 3-10 x ULN
– elevation is due to increased synthesis of the enzyme induced
by cholestasis.
• Hepatocellular < 3 x ULN

• Bone
• Highest Paget’s Disease
• Osteomalacia, rickets, hyperparathyroidism,
• Healing fractures

• 1.5 x normal during late pregnancy

• Low in hypophosphatasia
• Inherited inadequate bone calcification

M. Zaharna Clin. Chem. 2009

 Assay for enzyme activity
• Different methods due to non-specificity of the enzyme
group
• Bowers and Combs method based on absorption of p-
nitrophenol at 405 nm

yellow

colorless
M. Zaharna Clin. Chem. 2009
 Acid Phosphatase (ACP)
• Belong to the same group of phosphatase enzymes as
ALP
• Catalyze hydrolysis of phosphate monoesters at acid
(5.0) pH

• High concentrations in the prostate gland,

• Found also in bone, liver spleen, kidney, RBC and
platelets

M. Zaharna Clin. Chem. 2009

 Clinical Significance
• ACP used as an aid in the detection of prostatic
carcinoma particularly metastatic carcinoma of
the prostate
– Prostatic specific antigen (PSA) now better
• Total ACP determination relatively insensitive
technique
• Chemical inhibition methods to differentiate the
prostatic portion
• Serum incubated with and without addition of
tartarate (does inhibit P. ACP)

M. Zaharna Clin. Chem. 2009

• Total ACP – ACP after tartrate inhibition =
prostatic ACP

• Other methods
• Immunological test

• Other diagnostics
• Vaginal washings for rape cases
• Serum levels in bone disease (osteoclasts)
• ITP – platelet destruction

M. Zaharna Clin. Chem. 2009

 Assay for enzyme activity
• Same as ALP but at acidic pH
ACP
P-Nitrophenolphosphate p-nitrophenol + phosphate ion
pH 5

• Products are colorless at acidic pH

• Addition of alkali stops the reaction and
transforms the products into chromogens, can
be measured spectrophotometery

M. Zaharna Clin. Chem. 2009

 γ-Glutamyltransferase (GGT)
• Transfers γ-glutamyl residue from γ-
glutamyl peptides (usually glutathione) to
amino acids, H2O and other small peptides
GGT
Glutathione + Amino Acid
glutamyl-peptide + L-cysteinylglycine

• High concentrations in liver tissue

• Also in pancreas and kidney

M. Zaharna Clin. Chem. 2009

 Clinical Significance
• Increased plasma GGT is associated with
– Hepatobiliary disease
• Highest seen in biliary obstruction
– Alcoholic cirrhosis
• Used with ALP to differentiate between liver and
bone diseases

M. Zaharna Clin. Chem. 2009

 Assay for enzyme activity
• γ- glutamyl residue of γ glutamyl-p-nitroanilide is
transferred to glycylglycine, releasing p-nitroaniline

GGT
L-γ-glutamyl-p-nitroanilide + glycylglycine
p-nitroaniline + y-glutamyl glycylglycine

• The rate of liberation of p-nitroaniline is directly related to

the GGT activity in the sample and is quantitated by
measuring the increase in absorbance at 405 nm.

M. Zaharna Clin. Chem. 2009

 Amylase (AMS)
• Hydrolyzes starch and glycogen
– Starch = amylose & amylopectin
• Amylose = long, unbranched α,1-4 linkages
• Amylopectin = branched chain α,1-6 linkages
– Glycogen similar to amylopectin but more
branches
• α-Amylase attacks only α-1-4 glycosidic bonds
• Generates glucose, maltose, and dextrins (branch
points)
• Requires Ca+ and Cl- for activation
• Cellulose is not attacked by α-Amylase due to α-1-6
bonds
M. Zaharna Clin. Chem. 2009
• Acinar cells of pancreas and
salivary glands major tissue sources
• AMS is smallest enzyme – 50 - 55,000
daltons
• Readily filtered into urine
• Digestion of starches begins in the mouth
• Inactivated by stomach acid Pancreatic
AMS takes over in intestine

M. Zaharna Clin. Chem. 2009

 Isoenzymes
• Two isoenzymes are found in normal
serum;
–P isoamylase … derived from pancreas
–S isoamylase … derived from salivary
glands
• They are identified using electrophoresis
chromatography or isoelectric focusing.

M. Zaharna Clin. Chem. 2009

 Clinical Significance
• In Acute pancreatitis
– Amylase begin to rise 2-12 hours after onset
of attack
– Peak 24 hours, return to normal 3-5 days
• 2.5 x ULN
• Also elevated in salivary gland disorders
and Intraabdominal disease (e.g.
Perforated peptic ulcer)

M. Zaharna Clin. Chem. 2009

 Assay for enzyme activity
Measurement – 4 different methods
• Amyloclastic – disappearance of starch substrate
– Starch substrate with Iodine attached
– As hydrolyzed Iodine is released = decrease in dark
blue color
• Saccharogenic – appearance of product
– Classic Somogyi method
– Starch substrate is hydrolyzed to CHO molecules that
have reducing ability
– Amount of reducing sugars present = amount of AMS
present

M. Zaharna Clin. Chem. 2009

 Assay for enzyme activity
• Chromogenic – increase in product coupled to
chromogenic dye
• Continuous – coupling enzymes to monitor AMS
activity (NADH produced)

• Somogyi units
– A measure of the level of activity of amylase in blood
serum.
– One Somogyi Unit is defined as the amount of
amylase required to produce the equivalent of 1
milligram of glucose when acting on a standard starch
solution in 30 minutes incubation time at 37°C.

M. Zaharna Clin. Chem. 2009

 Lipase (LPS)
• Lipase is an enzyme that hydrolyzes the
ester linkage of fats to produce alcohols
and fatty acids
• LPS catalyses the partial hydrolysis of
dietary triglycerides in the intestine
• Pancreatic LPS specific for fatty acid
residues at position 1 and 3 of triglyceride
• Accelerated by presence of bile salts &
colipase
M. Zaharna Clin. Chem. 2009
Lipase (LPS)

M. Zaharna Clin. Chem. 2009

 Clinical Significance
• Primarily found in pancreas
• Some from stomach and small intestine
• Used in diagnosis of acute pancreatitis
• Similar pattern to AMS but lasts longer
• May be elevated in other abdominal
conditions
• Normal in salivary gland conditions
(AMS↑)

M. Zaharna Clin. Chem. 2009

 Assay for enzyme activity
• Cherry-Crandall method – classic
• Triolein as substrate
• Titrate amount of fatty acids generated
LPS
Triglyceride + 2 H2O pH 8.6 - 9 2-monoglyceride + 2 fatty acids

M. Zaharna Clin. Chem. 2009

 Assay for enzyme activity

• Turbidimetric methods
– Fats in solution = cloudy
– LPS hydrolysis causes clearing of solution
• Colorimetric
– Coupled reactions with peroxidase or glycerol
kinase

M. Zaharna Clin. Chem. 2009

 Glucose-6-Phosphate
Dehydrogenase (G6PD)
• Oxidoreductase catalyzes oxidation of G6P to 6-
phosphogluconate or 6-phosphogluconolactone
• First step in pentose-phosphate shunt of glucose
metabolism

M. Zaharna Clin. Chem. 2009

• Adrenal cortex, spleen, thymus, lymph
nodes, lactating mammary gland, and
RBC
• Little activity is found in normal serum
• Primarily focus of RBC Keeps
– NADPH in reduced form
– Helps to generate glutathione
– Which in turn protect Hb from oxidation
– Cells hemolyze without it

M. Zaharna Clin. Chem. 2009

 Clinical Significance
• Deficiency of G6PD inherited sex-linked
trait
• Drug-induced hemolytic anemia
(primaquine)
• Increased levels reported in AMI and
megaloblastic anemia

M. Zaharna Clin. Chem. 2009

 Assay for enzyme activity
G6PD
G-6-P + NADP+ 6-phosphogluconate + NADPH + H+

• A red cell hemolysate is used to assay for deficiency of

the enzyme
• Serum is used for evaluation of enzyme elevations

M. Zaharna Clin. Chem. 2009