This slide briefly imparts the knowledge of Amylase and Lipase enzymes. The clinical importance, calculation, concentration, sources and principle of amylase estimation are the major components of uploaded slide.

1. ESTIMATION OF
AMYLASE AND LIPASE
Binaya Tamang
UCMS-TH

3. AMYLASE
• Class of hydrolases  catalyze the breakdown of starch and
glycogen.
• An endoglycosidase that acts randomly on internal α 1-4 glycosidic
linkages
• Starch consists of both amylose and amylopectin.
• Amylose unbranched  [ maltose, maltotriose and glucose]
• Amylopectin  branched chain [ maltose , maltotriose, glucose and
limit dextrin]

4. TWO types : α and β. ( somewhere γ )
• α : Pancreatic and salivary amylase.
• β : mostly of plant origin, an exopeptidase active on terminal
linkages.
• γ: fungi
Relation with elements [Ca- metalloenzymes].
• Calcium absolutely required for functional integrity.
• full activity & to increase efficiency  in the presence of various
anions
• Chloride and bromide  most effective activators

5. Optimal pH required :- pH 6.9 to 7.0.
 Tissue sources:
• Major : acinar cells of the pancreas and the salivary glands
• Lesser concentrations are found in (skeletal muscle, small intestine,
fallopian tubes and other organs).
 To know:
• 54,000 to 62,000 ( small mol.wt)
• Small enough to pass through the glomeruli of the kidney.
• Only plasma enzyme normally found in urine.

6.  Role:
salivary AMS begins digestion in mouth
⇓
short duration (inactivated by the gastric acid)
⇓
In intestine , role is taken by pancreatic AMS

7. Isoenzymes : P-type and S-type
• P-type: (pancreatic ) and S-type : (main: salivary gland , minor: fallopian
tube and lung).
• Again, post translational modification  deamidation, glycosylation and
deglycosylation  no. of isoforms.
• Separation: electrophoresis, chromatography and isoelectric focusing (
s-type migrate faster than p-type, corresponding to β to α globulin
regions))
• Wheat germ protein ( lectin) inhibits S- type treating with it
measuring enzyme activity  only of P-type.
• Normally ,serum AMY ( 2/3 S-AMY and 1/3 P-AMY)= Total AMY

8. Urinary amylase
• Increase in serum AMY increase in clearance via renal increase in urinary
AMY. Therefore, increased in pancreatitis.
• urinary amylase increases and persists a little longer than serum activity.
Helps in late diagnosis.
• Increases on 1st day and remains elevated for 7-10 days.
• Diagnostic role in acute pancreatitis and macroamylasemia (?????)

9. Amylase Creatinine clearance Rate
(ACCR)
• Diagnosis of acute pancreatitis, differentiation also
• Normal value : 1-4%
• Acute pancreatitis: 7-15%
• Macroamylasemia: < 1 %

10. Macroamylasemia
• These are the complexes between both ordinary amylase ( usually s-
type) and IgA or IgG.
• Cannot be filtered through the glomeruli because of their large size
(> MW 200,000) and are thus retained in the plasma .
• ⇑ es AMY activity (2-8 above the URL).
• NO clinical symptoms are associated with this disorders.
• Differentiated by measuring urinary and serum amylase.
• Acute pancreatitis: both increases
• Macroamylasemia: only in serum ( AMY is increased)

11. Clinical significance
serum and urine AMS
• Mainly, acute pancreatitis and salivary gland inflammation.
• Disorders of other tissue can also produce elevations in AMS levels (
20-60%).
• Therefore, an elevated AMS level is a nonspecific finding.
• Lack of specificity of total AMY can be replaced by directly measuring
P-type AMY ( > 90% for acute pancreatitis ) for differential diagnosis
of abdominal pain.

12. Clinical significance
Pancreatic disease Salivary gland
disease
Other condition (
small increase)
Acute pancreatitis,
pancreatic tumors,
carcinoma of head of
pancreas,
Pancreatic pseudocyst.
Acute parotitis (
mumps),
salivary calculi,
tumors of salivary
glands,
salivary gland
disorders
Intestinal obstruction,
perforated peptic
ulcers,
acute appendicitis,
abdominal trauma,
acute peritonitis,
ruptured ectopic
pregnancy,
cancers of lungs and
ovary, renal failure,
mesenteric infarction
Drugs ( morphine)
• Acute pancreatistis usually > 500 Somogyi units per dL.
• Other condition usually < 500 Somogyi units per dL.

14. Acute pancreatitis.
Time table
• Start to rise :- 5 to 8 hours after onset of symptoms
• Maximum at 12-72 hrs ( 24 hrs)
• return to normal levels within 3 to 5 days. ( persistent elevation longer than
this suggests continuing necrosis or pseudo cyst formation)
• Rise may be: 4-8 folds of URL
• Increase in p-type more than s-type

15. • Magnitude of elevation of Serum enzymes : not related to the severity
of pancreatic involvement due to normal loss in urine.
• Mild severity may show normal serum AMY due to its clearance in
urine.
• However, greater the rise, greater the probability of acute
pancreatitis.
• Usually > 500 somogyi units/dl

16. Methods of estimation
• AMS can be assayed by a variety of different methods
• Two main approaches
• Amyloclastic and saccharogenic (Both are starch based methods).
• In amyloclastic, AMS is allowed to act on a starch substrate to which
iodine has been attached.
• As AMS hydrolyzes the starch  the iodine is released and a decrease
occurs in the initial dark-blue color intensity of the starch–iodine
complex.
• The decrease in color is proportional to the AMS concentration.

17. • The saccharogenic method uses a starch substrate that is hydrolyzed by
the action of AMS  constituent carbohydrate that have reducing
properties.
• The amount of reducing sugars is then measured where the
concentration is proportional to AMS activity.
• Somogyi units are an expression of the number of milligrams of glucose
released in 30 minutes at 37°C under specific assay conditions.
OR
• Amylase unit is define as the amount of enzyme necessary to convert
5mg of starch in to product within 15 mins at 37 c and other specific
condition.

18. Principle of Amyloclastic method
• based on the hydrolysis of starch by serum amylase.
• starch reacts with the iodine to give blue color. On adding amylase,
starch is hydrolysed and hence the color intensity decreases.
• Difference of blue color produced by the original starch solution and
after hydrolysis is a measure of enzyme activity.
Or,
• this decrease is proportional to the amylase activity and is measured
at 660 nm.
Normal value : 80-180 SO/dl, 25-130 IU/L.

19. Procedure
• Dry clean test tubes : control (C) and test (T)
• MIX well and compare the OD of C and T at 660 nm against H2O
Reagents Control Test
Buffered starch solution 0.9 ml 0.9 ml
Incubate at 37 c for 3 min
Diluted serum ( 1: 10) - 100μl
Normal saline 100 μl -
Incubate at 37 c for 15 min
0.01 N iodine solution 0.4 ml 0.4 ml
De-ionized water 8.6 ml 8.6ml

20. SHORT TRIP
TO
“LIPASE”

21. Lipase.
• Lipase (LPS)  hydrolyzes the ester linkages of fats (TAG)  Attack on
C1 and 3  2 mol. of fatty acids and 1 mol of 2-acyl glycerol
• MW 48000 : [Urinary Lipase is absent because of its complete
reabsorption by renal tubules].
• Increased reaction rate : cofactor colipase and a bile salt.
• Tissue Source: primarily in the pancreas ( minor: stomach and small
intestine).
• almost 5000 times greater than other tissue and conc gradient between
pancreas and serum is aprox 20000 folds.

22. Clinical significance
• Used to diagnose acute pancreatitis ( Time table is almost same as
AMY)
• Start: 4-8 hrs
• Peaks about 24 hrs
• Normal : 8-14 days.
• Rise may be 2-50 folds
• considered more specific for pancreatic disorders than AMS ( > 5
time of URL is more specific).
• Both arises quickly but LPS persists for longer duration than AMS,
extra advantage.
• LPS: 0–1.0 U/mL

23. • may be found in other intra-abdominal conditions but with less
frequency than elevations of serum AMS.
• In contrast to AMS levels, LPS levels are normal in conditions of
salivary gland involvement.
• Differentiate Pancreatic vs salivary gland involvement.