MLS 418: CLINICAL CHEMISTRY LABORATORY Pancreas

PRELIM WEEK 2 – LESSON 2 - Involved in the secretion of amylase

Amylase and Lipase - Glandular organ (behind the stomach)
- Produce digestive juices, that empty into small
intestine
ENZYMES OF CLINICAL SIGNIFICANCE - Two main function:
● Amylase - Exocrine: helps in digestion
● Lipase - Endocrine: regulates the blood sugar
● Alanine Aminotransferase (ALT) & Aspartate
Aminotransferase (AST) ❏ Tissue Source
○ ALT formerly known as Serum Glutamic ❏ Isoenzyme
Pyruvic transaminase (SGPT): ❏ Properties
- widely distributed in the body but ❏ Diagnostic significance
the highest concentration is in the ❏ Assays
liver (Liver-specific) ❏ Sources of Error
- Liver disease
○ AST also called as Serum Glutamic AMYLASE (AMS)
oxaloacetic Transaminase (SGOT): ● E.C. 3.2.1.1
- also found in the liver but its ○ Hydrolase: bond breakage
highest concentration is found in ● 1,4-D-Glucan Glucanohydrolase
cardiac tissue ● Breakdown of starch and glycogen to
- Heart disease monosaccharides
○ These are liver enzymes. ○ Starch: breaks down into maltose and
● Acid Phosphatase (ACP) & Alkaline glucose unit
Phosphatase (ALP) ● Activators: Calcium ions and Chloride
○ ACP:
- rich in prostate
- Used in detection of prostatic
carcinoma or prostate cancer
○ ALP
- Found in hepatobiliary conditions,
particularly Liver
- Test requested for those patients
with bone disease
● Creatine Kinase (CK)
- Test for heart disease (myocardial infection)
● Lactic Dehydrogenase (LDH)
● Gamma Glutamine Transferase (GGT)
- Regulate the transport of amino acids
across the cell membranes, by catalyzing ● Starch is composed of Amylose (20%) and
the transfer of the glutamine group from the Amylopectin (80%)
glutathione to a free amino acid.
● MISC ENZYMES (ACE: Angiotensin Converting
Enzyme, CHE: Cholinesterase AND
5’NUCLEOTIDAE)

PANCREATIC ENZYMES
● Amylase
● Lipase

- A branched chain polysaccharides.

- A polymer which consists of branching

** see appendix

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 ISOENZYME

P-type isoenzyme S-type isoamylase

Pancreas Salivary gland

Lungs

Fallopian tubes
● Both are metalloenzymes- containing calcium ion
● Bromide and iodide serve as activators (Henry’s)

** see appendix
● Alpha-Amylase:
- Major digestive enzyme
- Calcium metalloenzyme (not capable of
functioning in a absence of calcium)
● Beta-Amylase:
- enzyme present in seed germination or fruit
ripening
● Both used in fermentation processes (brewing beer
and liquor)
TISSUE SOURCE
● Acinar cells of the pancreas
○ Pancreatic AMS
- Luminal digestion of carbohydrates
● Salivary glands
○ Salivary AMS
- Initiate the carbohydrate digestion
- Important in initiating starch
digestion, depending on the time
spent in chewing
○ Ptyalin
● There are other tissue sources (e.g. Fallopian tube)
● Amylase in the stomach inactivates because of it
acidic environment (gastric lumen)
● Human Salivary Amylase is 94% identical with the
pancreatic amylase, differ gene types.
➔ Initial digestion (mouth)
➔ Small Intestine: Majority of Carbohydrates digestion
takes place
➔ The presence of food stimulates the SI to secrete
CCK
➔ CCK signals the pancreas to secrete amylase
➔ Pancreatic Amylase, released into SI through the
pancreatic duct. Where it degradates starch and
other polysaccharides
PROPERTIES
● MW 50,000-55,000
○ Smallest enzyme
● Readily filtered by the renal glomerulus
○ Only plasma enzyme normally found in the
urine
● Mouth - starch initial digestion by salivary AMS
○ Inactivated in the stomach
● Small Intestine - final digestion by pancreatic AMS
DIAGNOSTIC SIGNIFICANCE
● Acute Pancreatitis
○ Sudden inflammation of pancreas
○ Rise:
- 6-48 hours after onset of an attack
(Henry)
- 2-12 hours (Bishop)
○ Peak: 24 hours (Bishop)
○ Normalize (stable): 3-5 days (Bishop and
Henry’s)
○ It is important to test amylase: useful to
determine whether the condition is caused
by pancreas or by the salivary glands
○ Diagnosis of acute pancreatitis is
sometimes difficult because it must be
differentiated from other acute intra
abdominal disorder and an increase in
serum amylase is not necessarily due to
pancreas
○ Elevated serum amylase = non-specific
finding
○ Leading cause: gallstone (stone get stuck
in a bile or pancreatic duct) and alcohol
○ Major symptoms: pain in abdomen then
spread at the back
● Salivary gland involvement
● Diseases (elevated amylase):
○ Mumps
○ Parotitis Parotiditis
○ Perforated peptic ulcer
○ Intestinal obstruction
○ Cholecystitis
○ Ruptured ectopic pregnancy
○ Mesenteric infarction
○ Acute appendicitis
● Macroamylasemia
○ persistent increase in serum amylase is
seen without clinical symptoms
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 ○ Not a disease but a acquired benign
condition
○ AMS molecule combines with IgG or igA
- Normal heterogeneous complexes
of normal amylase (salivary
isoenzyme with Ig)
○ Because of it large size, it cannot be filtered
through the glomerulus and are retained in
the plasma
- Not seen in the urine
○ Plasm enzyme activity, increase → 2-8folds
ASSAY FOR ENZYME ACTIVITY
● Amyloclastic
○ As AMS hydrolyzes the starch, the iodine is
released and a decrease in the initial
dark-blue color intensity of the starch-iodine
complex occurs
○ THE DECREASE IN COLOR IS
PROPORTIONAL TO THE AMS
CONCENTRATION
● Saccharogenic
○ THE AMOUNT OF REDUCING SUGARS
IS THEN MEASURED WHERE THE
CONCENTRATION IS PROPORTIONAL
TO AMS ACTIVITY
○ Classic reference method in Somogyi
units
- Amount of amylase to produce 1mg
of glucose when acting on a
standard starch solution in 30
minutes incubation time at 40ºC
- 1 ssu = 1.85 unit per liter
- >200 ssu or <40 ssu = clinical
concern
● Chromogenic
○ As AMS hydrolyzes the starch substrate,
smaller dye-substrate fragments are
produced, and these are water-soluble
○ Uses starch substrate (chromogenic dye)
→ insoluble substrate complex
○ THE INCREASE IN COLOR INTENSITY
OF THE SOLUBLE DYE-SUBSTRATE
SOLUTION IS PROPORTIONAL TO AMS
ACTIVITY
● Coupled Enzyme
○ Have been used to determine AMS activity
by a continuous monitoring technique in
which the change in absorbance of NAD+
at 340 nm is measured
○ Optimum pH: 6.9-7.0
● Wheat germ lectin
○ Inhibits salivary amylase
○ salivary and pancreatic AMS can be
estimated by measuring total AMS in the
presence and absence of lectin
SOURCE OF ERROR
● AMS in serum and urine is stable
● Little loss of activity occurs at RT for 1 week or at 4
ºC for 2 months or 6 months (Henry)
● AMS values may be normal in acute pancreatitis
with hyperlipemia
○ Plasma triglycerides inhibits serum amylase
● Morphine and other opiates
○ Falsely elevate serum AMS levels
○ Can cause constriction of Oddi’s sphincter
and pancreatic ducts
○ With consequent elevation of inarticulate
pressure causing regurgitation of AMS into
the serum
● Citrate or oxalate as anticoagulant
○ Falsely low activity
○ Calcium chelated by citrate
○ Should use: HEPARIN
● Contamination of saliva
○ Approx. 700 times that of serum
● Red cells contain no AMS
○ so hemolysis generally presents no
problem with most methods except those
coupled-enzyme methods
- The released peroxide is
determined by a copper peroxidase
reaction
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 ● Absorbance at 400 nm, using spectrophotometer
● The resulting absorbance increase per minute is
directly proportional to the AMS activity of the
sample.
● Available commercially worldwide; ready to use
liquid form
● The assay is calibrated using the Molar Absorptivity
of CNP
● Short log phase, with wide measurement range, no
interference from any endogenous glucose

**read the package insert

● Storage stability of the reagent come up after

opening
- Stable for 12 weeks at Light-protected at
2-8ºC
- 4 weeks at 15-25ºC
● Specimen: Serum, Heparinized plasma, urine
● No loss of activity within 5 days at 4-25ºC

● Based on the International Federation of Clinical

Chemistry (IFCC)
● This procedure is a direct assay using 2
chloro-4-nitrophenol maltotriose (CNPG3) as a
substrate
- Hydrolyzed by the AMS without any
auxiliary enzyme to yield → 3 CNP + 2
CNPG2 + 3 G3 + 2 G
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LIPASE (LPS)
● E.C. 3.1.1.3
● Triacylglycerol Acylhydrolase
● Hydrolyzes the (glycerol) ester linkages of fats (long
chain fatty acid) at C’1,3 ester bond to produce
alcohols and fatty acids (2 moles of fatty acids and
1 mole of monoglyceride)
● Catalyzes the partial hydrolysis of dietary TAG in
the intestine to the 2-monoglyceride and fatty acids

● Has low molecular weight

○ Filtered by the glomerulus
○ Normally completely reabsorbed by the
PCT, thus absent from the normal urine
● MW = 45,000
- Walay daw niya gispicifically write the MW kay varied daw.
Pero ang 45,000 MW kay gikan sa iyang ppt handout :)
● Enzymatic activity is specific for the fatty acid
residues at positions 1 and 3 of the TAG molecules
● Substrate must be an emulsion for activity to occur
● Reaction rate is accelerated by the presence of
colipase and bile salt
- Bile salt prevent the denaturation of lipase
● Proteins, bile acids, and phospholipids
○ Inhibit serum lipase
● Colipases
○ Reverse this inhibition of bile salts
- Inhibitory effect of bile salts or
bile-acid water on the LPS
catalyzed into the hydrolysis of
dietary long TAG
○ A protein enzyme required for optimal
enzyme activity of pancreatic LPS
○ It is secreted by the pancreas in an inactive
form called Pro-colipase (activated in the
intestinal lumen in the Intestine)
● Calcium is necessary for maximal lipase activity
- Higher concentration has inhibitory effect
● Heavy metals and quinine inhibit LPS activity

TISSUE SOURCE
● Primarily in the pancreas
● Also present in lesser concentration → liver,
stomach , small intestine , white blood cells, fat
cells and milk (Henry)

DIAGNOSTIC SIGNIFICANCE
● Almost exclusively to the diagnosis of acute
pancreatitis
○ Rise: 2-12 hours after onset of an attack
○ Peak: 24 hours
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 ○ Persist for approximately 5 days (Bishop);
for about 7-10 days (HENRY’S)
● Found also in other intra-abdominal conditions
○ Duodenal ulcers, perforated peptic ulcers,
intestinal obstruction, acute cholecystitis
● Normal in conditions of salivary gland involvement
● Acute pancreatitis:
- LPS persists for 8 days
- AMS for 2-3 days
- Life extent elevation does not correlate with
the severity of the condition.
● Among the 3 isoenzyme of LPS, L2 is the most
clinically specific and sensitive
ASSAY FOR ENZYME ACTIVITY
● Includes estimation of liberated fatty acids and
turbidimetric methods
● Optimal temperature: 40˚C
● Optimum pH: 8.8
○ It has been reported that optimum ph of
LPS could range from 7-9
● If AMS and LPS are higher that normal =
Pancreatic injury
- Levels of greater than 3 times the upper
limit of the normal usually lead to diagnosis
of pancreatic
● LPS levels alone cannot determine the severity of
an acute pancreatitis
● Abnormal result → need other test to diagnose
pancreatitis
- Other test: Ultrasound, Ct Scan, MRI or
Endoscopy
● Abnormal AMS, not necessarily involve pancreas
● LPS: more specific for pancreatic disorder

- DM US IF MAY CORRECTIONS PLS :) TY

● Classic Cherry Crandall Method

○ Substrate: Olive Oil
○ Measured the liberated fatty acids by
titration after a 24-hour incubation
○ Modified: Triolein (one substrate used as
more pure form of TAG)
○ CLASSIC REFERENCE METHOD
● Turbidimetric Methods
○ As the fats are hydrolyzed by LPS, the
particles disperse, and the rate of clearing
can be measured as an estimation of LPS
activity
○ Icterus , lipemia , and hemolysis do not
interfere
○ More rapid than titrimetric assys
● Colorimetric Methods
○ Based on coupled reactions with enzymes
such as peroxidase or glycerol kinase

SOURCE OF ERROR
● LPS is stable in serum, with negligible loss in
activity at RT for 1 week or for 3 weeks at 4
degrees Celsius
● Hemolysis should be avoided because hemoglobin
inhibits serum LPS

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APPENDIX: