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Cchm-lab-midterms - Lecture notes
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Clinical Chemistry 2 (MDT 3122L)
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Introduction (LAB)- Amylase and Lipase Determination ( midterms )
2
nd
SEM 2022
Amylase Determination
● Reagent Manufacturer:
○ STANBIO LABORATORY
Principles
● Amylase catalyzes the hydrolysis of
starch into simpler molecules with
maltose as the end product. Amylase
is formed in the pancreas and is found
in increased amounts in the serum and
urine in varying degrees of pancreatic
disturbance. Normally small amounts
of amylase are found in the blood.
Increased amounts of pancreatic
amylase is found in the blood during
the early stage of acute pancreatitis.
● The greatest concentration of amylase
is in the pancreas where the enzymes
is synthesized and secreted into the
intestinal tract for digestion of starch.
Amylase is also secreted by the
salivary glands and is present in
saliva, where it initiates hydrolysis of
starch while the food still in the mouth
and esophagus.
● Amylase is increased in acute
pancreatitis, carcinoma of head of
pancreas, duodenal ulcer, perforation
of gastric ulcer, hyperthyroidism,
mumps, acute injury of spleen and
obstruction. It is decreased in cirrhosis
and carcinoma of the liver, liver
abscess, hepatitis, acute alcoholism
and toxemia of pregnancy.
● The enzymatic procedure presented is
based on modifications of Wallenfels,
using as substrate
p-Nitrophenyl-D-maltohepatoside (
PNG7 ) with the terminal glucose
blocked to reduce spontaneous
degradation of the substrate by
glucosidase and glucoamylase. The
test is performed in a kinetic mode with
a very short lag time and offers much
greater stability than previous amylase
methodologies.
● Amylase hydrolyzes
p-Nitrophenyl-D-maltohepatoside (
PNG7 ) to
p-Nitrophenyl-D-maltostriose ( PNPG
) and maltotetraose. Glucoamylase
hydrolyzes PNPG3 to
p-Nitrophenylglycoside ( PNPG1 ) and
glucose. Then is hydrolyzed by
glucosidase to glucose and
p-Nitrophenol, which produce a yellow
color, the rate of increase in
absorbance is measured at 405 nm is
proportional to the amylase activity in
the sample.
ADDED NOTES:
● Amylase and Lipase are pancreatic
markers.
● Amylase also called alpha
glucan-glucanohydrolase
● Amylase is under the hydrolase group
● Amylase is considered as the smallest
enzyme, it is filtered by the kidneys
and appears into the urine.
● Two major isoenzymes: S-type (
ptyalin ) and P-type ( amyloid )
● Amylase earliest pancreatic marker:
rise between 2-12 hours, peak 24
hours, normalize 2-5 days
● Multiple measurement, kinetic method
● Amylase aids in the digestion of
carbohydrates ( strach and glycogen )
● Enzymes glucosidase and
glucoamylase came from microbial
source
● Amylase tissue source are pancreas
and salivary gland
Materials
● Spectrophotometer
● Accurate pipetting devices
● Cuvets
● Interval timer
● Test tubes
Reagents
● Amylase reagent ( powder )
○ Composition when reconstituted
_____________________________________________________________________________________ 1
Introduction (LAB)- Amylase and Lipase Determination ( midterms )
2
nd
SEM 2022
■ p-Nitrophenyl-D-maltohe
patoside
■ Glucosidase (microbial)
■ Glucoamylase (microbial)
■ Sodium chloride
■ Calcium chloride
■ Buffer , pH 6 +/- 0.
ADDED NOTES:
● Actual amylase came from the sample
● Reagent for amylase is ready to use
● The reagent for amylase can last until
its expiration date when it is stored at
refrigerator temperature between
2-8C. It must be protected from light
and check if there is discoloration,
cloudiness, and particulate found in
the reagent.
Specimen Collection and Preparation:
● Unhemolyzed serum is the specimen
of choice. Plasma from heparin tubes
may be used. Other anticoagulants,
such as citrate and EDTA, bind
calcium, an ion needed for amylase
activity. Therefore, plasma with any
anticoagulant other than heparin
should not be used.
ADDED NOTES:
● Heparin use in plasma
● Red top tube or plain tube
Procedures
● Wavelength: 405 nm
● Temperature: 37 degrees celsius
1. For each sample add 1 ml
reconstituted reagent to cuvette or test
tube and pre-warm at 37C for atleast 3
minutes.
2. Zero spectrophotometer with distilled
water at 405 nm..
3. Add 0 mL( 25 ul ) serum to its
respective tube and read immediately.
4. Read increase in absorbance at 30
second interval for 2 minutes.
5. Determine the mean absorbance
difference per minute (ΔAbs/minute)
6. Multiply the ΔAbs/minute by 4824 to
obtain the result in U/L
ADDED NOTES:
● Total of 5 readings for amylase
Computation
● Values are determined based on the
milimolar absorptivity p-Nitrophenol
which is 8 at 405 nm under the test
conditions described.
● Formula:
○ U/L=(ΔA/minute x total volume x
1000 ( U/mL to U/L) /
absorptivity x sample volume x
light path
○ U/L=ΔA/minute x 1 x 1000
/ 8 x 0 x 1.
○ U/L = ΔA/minute x 4824
Reference Values
● Normal Range:
○ Serum - 25-125 U/L
○ Urine - 1-17 U/Hour
_____________________________________________________________________________________ 2
Introduction (LAB)- Amylase and Lipase Determination ( midterms )
2
nd
SEM 2022
ADDED NOTES:
● 2 tubes - blank tube and test tube
Computation
● Formula:
○ U/L=Corrected ΔAbs/minute/
5min / Initial blank absorbance x
1953
○ Corrected ΔAbs/minute/ 5min =
ΔAbs Test - ΔAbs Blank
Reference Values
● Normal Range:
○ Adults : 10 - 150 U/L ( age more
than 60 years old: 18 - 180 U/L)
_____________________________________________________________________________________ 4
Introduction (LAB)- Sodium and Potassium Determination ( midterms )
2
nd
SEM, 2022
Sodium Determination
ADDED NOTES:
● Sodium is an electrolyte.
● Electrolytes are defined as ions which
are capable of carrying an electric
charge.
● The electrolytes could be anion or
cation. Cation positively charge. Anion
negatively charge.
Principle
● Kolthoff
○ 1927 —> Precipitation of
sodium of as triple salt —>
Sodium uranyl zinc acetate (
salt is measured )
● Albanese and Lein
○ Colorimetric measurement of
the solubilized residue.
● Bradbury
○ Monitoring the color fade of the
yellow supernate after
precipitation.
● Stanbio
○ Sodium is precipitated from a
protein-free supernate as the
triple salt —> decrease in
absorbance of the
supernate-color reagent mixture
—> sodium content of the
specimen.
ADDED NOTES:
● Sodium is abbreviated as NA or
natrium. Most abundant cation into the
ECF
● Sodium is a major osmotic particle
outside of the cell. It largely
determines the osmolality of the blood
or the plasma.
● Importance of electrolytes in our body
○ for the blood volume and
osmotic regulation, myocardial
rhythm and contractility
○ Cofactors in enzyme activation
○ Regulation on the ATPase ion
pump
○ For the acid-base balance
○ Blood coagulation
○ Neuromuscular excitability
○ Production and use of the ATP
from glucose
● The method we use is protein-free
supernate
.
Materials and Instruments
● Spectrophotometer
● Centrifuge
● Pipette
● Cuvettes and test tubes
● Timer
Reagents
● Sodium color reagent
○ solution of uranyl acetate and
zinc acetate in aqueous acetic
acid-ethanol mixture.
● Precipitating agent
○ aqueous solution of TCA (
Trichloroacetic acid ) - to
remove proteins from our
sample. To make protein-free
supernate
● Sodium standard
○ sodium chloride in aqueous
TCA
○ value of the standard is 140
Specimen Collection and Preparation
● Serum
○ remove from clot promptly and
carefully to prevent hemolysis.
● Plasma
○ we can either use oxalate or the
heparin as our anticoagulant.
For the heparin we can use
Lithium heparin or ammonium
_____________________________________________________________________________________ 1
Introduction (LAB)- Sodium and Potassium Determination ( midterms )
2
nd
SEM, 2022
● Hyponatremia is defined as a serum
level of less than 135 mEq/L and is
considered severe when the serum
level is below 125 mEq/L.
Table 15-2 CAUSES OF HYPONATREMIA
INCREASED SODIUM LOSS
Hypoadrenalism ( hormone affecting
aldosterone)
Potassium deficiency ( as the kidney tubules
conserve the potassium the kidney will
excrete the sodium - electroneutrality )
Diuretic use
Ketonuria ( excretion of sodium loss )
Salt-losing nephropathy
Prolonged vomiting or diarrhea
Severe burns
INCREASED WATER RETENTION (
DILUTION )
Renal failure
Nephrotic syndrome
Hepatic cirrhosis ( edema )
Congestive heart failure
WATER IMBALANCE
Excess water intake ( polydipsia )
SIADH ( over-production of ADH hormone )
Pseudohyponatremia
Hypernatremia
● It is a serum sodium concentration
above the upper limit of the reference
interval.
Table 15-4 CAUSES OF HYPERNATREMIA
EXCESS WATER LOSS
Diabetes insipidus ( polyuria, hypotonic fluid
without sodium )
Renal tubular disorder
Prolonged diarrhea
Profuse sweating
Severe burns
DECREASED WATER INTAKE
Older persons
Infants
Mental impairment
INCREASED INTAKE OR RETENTION
Hyperaldosteronism
Sodium bicarbonate excess ( given by IV, to
correct acidosis )
Dialysis fluid excess
_____________________________________________________________________________________ 3
Introduction (LAB)- Sodium and Potassium Determination ( midterms )
2
nd
SEM, 2022
Potassium Determination
ADDED NOTES:
● Abbreviated as “K” or Kalium
● Major cation in the ICF within the cell.
● It is very important in our body in terms
of; neuromuscular excitability,
contraction of the heart, ICF volume,
regulation of hydrogen concentration.
● Potassium has an inverse relationship
with the sodium and hydrogen.
● Effects also in the balancing of pH of
the blood.
Principle
● Turbidimetric technique ( Hillmann and
Beyer )
○ Potassium ions in a protein-free
alkaline medium react with
sodium tetraphenyl boron —>
produce finely dispersed turbid
suspension of potassium
tetraphenylboron.
● Turbidity
○ Proportional to potassium
concentration
ADDED NOTES:
● Just like with sodium determination we
will be preparing a protein-free
supernate.
Materials and Instruments
● Spectrophotometer
● Centrifuge
● Pipette
● Cuvettes and test tubes
● Timer
Reagents
● Potassium Boron reagent
○ aqueous solution of sodium
tetraphenylboron acetic
acid-ethanol mixture.
● Sodium Hydroxide reagent
○ aqueous sodium hydroxide
● TCA Precipitating reagent
○ aqueous solution of TCA
● Potassium Standard
○ solution of potassium chloride in
aqueous TCA
ADDED NOTES:
● Potassium standard volume is 4.
Preparation of working reagent
● Mix 1 volume of Potassium Boron
reagent with 1 volume of Sodium
Hydroxide. Allow to stand for at least
15-30 minutes at room temperature
before use.
ADDED NOTES:
● 1:1 ratio of potassium boron reagent
with sodium hydroxide.
Specimen Collection and Preparation
● Serum
○ remove from clot promptly and
carefully to prevent hemolysis.
● Plasma
○ we can use lithium heparin or
lithium oxalate.
● Sample stability
○ Potassium levels remain stable
for at least 14 days at 20-
degrees celsius or in the room
tempt.
● Interfering substances
○ contaminated glassware is the
greatest source of error. All
glassware should be washed
with 10-20% nitric acid in
distilled water.
Preparation of Protein-Free Supernate
1. Label test tube as sample and add 0.
_____________________________________________________________________________________ 4
Introduction (LAB)- Sodium and Potassium Determination ( midterms )
2
nd
SEM, 2022
CELLULAR SHIFT
Alkalosis
Insulin Overdose ( cellular uptake )
DECREASED INTAKE
Hyperkalemia
Table 15-8 CAUSES OF HYPERKALEMIA
DECREASED RENAL EXCRETION
Acute or chronic renal failure ( GFR,
<20mL/min )
Hypoaldosteronism
Addison’s disease
Diuretics
CELLULAR SHIFT
Acidosis
Muscle/cellular injury ( affects the cell )
Chemotherapy
Leukemia
Hemolysis
INCREASED INTAKE
Oral or IV potassium replacement therapy
ARTIFACTUAL
Sample hemolysis
Thrombocytosis
Prolonged tourniquet use or excessive fist
clenching
_____________________________________________________________________________________ 6
Introduction (LAB)- Chloride and Magnesium Determination ( midterms )
2
nd
SEM, 2022
Chloride Determination
ADDED NOTES:
● Properties of chloride
○ Major extracellular anion
○ Together with sodium, chloride found
outside the cell ( ECF )
○ The movement of chloride is
secondary to the movement of sodium
○ Chloride also affect pH of our body.
Direct relationship to bicarbonate.
● Functions of chloride
○ Maintenance of the osmolality
○ Blood volume together the sodium
○ Maintenance for electroneutrality
Analytical Methods
● Ion-selective electrode ( ISE ) commonly use
method for the measurement of the
electrolytes
● Mercurimetric Titration ( Schales - Schales
method )
○ For chloride determination
● Colorimetric method uses mercuric
thiocyanate and ferric nitrate to form a reddish
- colored complex.
● Coulometric-Amperometric Titration ( Cotlove
Chloridometer )
Principle
● Classical method
○ Combination of chloride with either
silver or mercury —> undissociated
chloride compound
● Most popular method
○ Titrimetric ( Schales and Schales ) —>
standard solution of mercuric nitrate
and diphenylcarbazone as indicator. (
titration method )
● Colorimetric method
○ ( Zall et. al, Skeggs and Hochtrasser )
● Chloride reacts directly with mercuric
thiocyanate to release thiocyanate ions. The
latter immediately combines with ferric ion to
form ferric thiocyanate. The absorbance of this
stable colored compound is measured at 500
nm and compared to that of a chloride
standard similarly treated.
● Hg + 2 (SCN) + Cl —> HgCl2 + 2 (SCN)
● Fe + 3 (SCN) —> Fe (SCN) Reddish
ADDED NOTES:
● For chloride determination not only serum and
plasma are used, we can also use urine and
spinal fluid.
Materials and Instrument
● Spectrophotometer
● Centrifuge
● Pipette
● Cuvettes and Test tubes
● Timer
Reagents
● Direct Chloride Color Reagent
○ Solution of mercuric thiocyanate, ferric
nitrate, mercuric nitrate in methanol
and water. Also contains nitric acid
and surfactants.
● Direct Chloride standard ( 100 meq/L )
○ Aqueous solution of Sodium chloride
● Reagent storage and stability
○ Reagent and standard are both stable
at room temperature until expiration
date.
ADDED NOTES:
● Specimen ( chloride ) stable for at least 1
week when it is placed at refrigerator or to the
room temperature.
Specimen collection and preparation
● Blood should be drawn with a minimum of
venous stasis and the serum or plasma
separated from cells as soon as possible to
prevent diffusion of chloride into the red cells.
● Chloride in serum, plasma, urine and spinal
fluid has been reported stable for at least 1
week at refrigerator or room temperature.
● Interfering substances
○ Hillogen ( chlorine )
○ Bromide will be calculated as chloride,
as will iodide, thiocyanate and sulfhyril
ions.
● Chloride residue to glasswares —> 10-20%
nitric acid —> wash thoroughly with deionized
water or distilled water.
_____________________________________________________________________________________ 1
Introduction (LAB)- Chloride and Magnesium Determination ( midterms )
2
nd
SEM, 2022
Magnesium Determination
ADDED NOTES:
● Second most abundant intracellular ion
● Distributed in bones, muscles as well soft
tissue also in the serum and other rbc
● Majority in the bone just like the calcium. 53%
in the bone, 46% muscles and soft tissue while
the remaining 1% measure in the blood.
● Different types of magnesium
○ Bound to albumin
○ Free ionized tip
○ Complex with other ions
● Importance of magnesium
○ Co-factors for more than 300 enzymes
○ Glycolysis
○ Transcellular ion transport
○ Neuromuscular transmission
○ Synthesis of carbohydrates, proteins,
lipids and nucleic acids.
Analytical Methods
● Total magnesium ( bound albumin, ionized,
complex with other ions )
○ Atomic Absorption Spectrophotometry
( AAS ) is the reference method but it
is not routinely done in the clinical
laboratory.
○ Photometric methods on automated
analyzers. These method employ
metallochromic indicators or dyes
such as calmagite, formazan dye,
magon and titan yellow dye.
● Ionized ( free ) magnesium - active form
● Ion-selective electrode for magnesium
○ Intracellular magnesium
● Fluorescence measurement using furapta (
magnesium binder )
● Nuclear magnetic resonance spectroscopy
● Ion selective microelectrode
● Electroprobe microanalysis
Principle
● Magnesium and Xylidyl Blue - 1 —> combine
under alkaline conditions —> form water
soluble red-purple chelate —> 520 nm.
● GEDTA ( Glycoletherdiamine - N,N,N’.N’ -
tetraacetic acid ) - prevent interference from
calcium.
ADDED NOTES:
● Calcium number 1 interference
Materials and Instrument
● Spectrophotometer
● Centrifuge
● Pipette
● Cuvettes and Test tubes
● Timer
Reagents
Magnesium Reagent
● Tris buffer
○ 0 mmol/L
● Potassium carbonate
○ 70 mmol/L
● GEDTA
○ 40 mmol/L
● Xylidyl blue
○ 0 mmol/L
● Sodium Azide ( preservative )
○ 0%
● Activators
Magnesium standard ( 2 meq/L )
● Aqueous solution of Magnesium chloride with
sodium azides as preservative.
Specimen collection and preparation
● Sample stability
○ Serum and plasma for magnesium are
reportedly stable for 7 days at 2-8C
○ 1 month at -20C
○ Treated urine is reportedly stable for 7
days at 2-8C
● Interfering substances
○ EDTA
■ unsatisfactory anticoagulant
for this procedure.
■ can bind to magnesium
○ Do not use serum that has visible
hemolysis → erythrocytes contain
highest level of magnesium.
○ The test is not influenced by bilirubin
concentrations up to 20 mg/dL, nor by
lipemic serum.
○ Contaminated glassware is the
greatest source of error.
■ It is recommended that
disposable tubes and pipette
tips be used in this procedure.
ADDED NOTES:
● Urine is treated with concentrated hydrochloric
acid to adjust the pH and reach to 3-4pH
Test Procedure
Step 1
● Pipette into labeled tubes or cuvettes the
following volumes (mL). Mix each tubes
promptly after addition of color reagent.
_____________________________________________________________________________________ 3
Introduction (LAB)- Chloride and Magnesium Determination ( midterms )
2
nd
SEM, 2022
Reagent
Blank (
RB )
Standard (
S )
Sample ( U
)
Color
reagent
1 1 1.
Magnesium
standard
- 0 -
Specimen - - 0.
Step 2
● Incubate all tubes at 37C for 3 mins or at room
temperature for 10 mins.
Step 3
● Read absorbance of S and U vs RB at 520 nm
within 3 hours.
ADDED NOTES:
● Reagent use to zero the absorbance
Calculations
● Serum/plasma mEq/L = Au / As x 2.
Reference values
● Normal ranges
○ Serum / Plasma
■ 1 - 2 meq/L
○ 24 hour urine
■ 6 - 10 meq/L
ADDED NOTES:
● When expressing to mmol/L conversion factor
is 0.
Hypermagnesemia
● It is a condition with high level of serum
magnesium.
● Increased magnesium level in the blood is rare
and usually iatrogenic.
● Elderly and patients with bowel disorder and
renal insufficiency are the most at risk.
TABLE 15-13 CAUSES OF HYPERMAGNESEMIA
Decreased excretion
Acute or chronic renal failure
Hypothyroidism
Hypopituitarism ( decreased GH )
Increased intake
Antacids
Enemas
Cathartics
Therapeutic - eclampsia, cardiac arrhythmia
Miscellaneous
Dehydration
Bone carcinoma
Bone metastases
Hypomagnesemia
● It is a condition with low level of serum
magnesium
● The most common causes of
hypomagnesemia are:
○ Loss of magnesium in the GI tract as
in chronic diarrhea and malabsorption
steatorrhea
○ Diabetes mellitus secondary to
glycosuria and osmotic diuresis
○ Alcohol
○ Stress
TABLE 15-11 CAUSES OF HYPOMAGNESEMIA
REDUCED INTAKE
Poor diet / starvation
Prolonged magnesium-deficient IV therapy
Chronic alcoholism
DECREASED ABSORPTION
Malabsorption syndrome
Surgical resection of small intestine
Nasogastric suction
Pancreatitis
Diarrhea
Laxative abuse
Neonatal
_____________________________________________________________________________________ 4
Introduction (LAB)- Calcium determination ( midterms )
2
nd
SEM, 2022
Calcium determination
ADDED NOTES:
● Calcium is one of the major cation in the body
Pre-analytical Phase
● More than 99% of body calcium exists in
bones and teeth. The remaining 1% is present
in blood and soft tissues and serves as a
cofactor in blood coagulation, metabolism and
neuromuscular physiology. Serum calcium is
present in three different forms:
1. Nearly 45% is bound by serum
proteins,
2. About 5% is complexed in a
nonionized form and
3. The remaining 50% serum calcium is
in an ionic (free) form. It is
physiologically active ionic fraction that
is important in terms of biological
functions.
● Many factors influence serum calcium levels:
○ Hypercalcemia - increased serum
calcium
■ Is observed in
hyperparathyroidism,
hypervitaminosis, sarcoidosis,
myeloma, and certain
cancers of the bone
○ Hypocalcemia - decreased serum
calcium
■ Is encountered in
hypoparathyroidism, rickets,
nephrosis, nephritis,
streatorrhea, and pancreatitis.
Any decrease in serum
proteins frequently results in a
decrease of the total serum
calcium level.
Method
Quantitative Enzymatic-Colorimetric
Determination of Calcium in Serum or
Urine.
● Calcium reacts with cresolphtalein
complexone in 8-hydroxyquinoline to form a
colored complex (purple color) that absorbs at
570 nm (550 – 580). The intensity of the color
is proportional to the calcium concentration. Color
interfiers and a stabilizer are present to minimize
interference by other metallic ions
Alkaline
Calcium + O-Cresolphthalein Complexone —---------->
Calcium-Cresolphthalein Complexone Complex (
purple color )
ADDED NOTES:
● The darker purple color produces the more
calcium present in the sample.
Materials
● Spectrophotometer
● Centrifuge
● Accurate pipetting devices
● Cuvettes and test tubes
● Interval timer
Reagents
● When reconstituted as directed, the reagent
for calcium contains the following:
1. Calcium Color Reagent (A)
○ O-Cresolphtalein Complexone,
8-Hydroxyquinone
2. Calcium Buffer
○ Diethylamide, Potassium Cyanide,
Nonreactive ingredients and stabilizers
in both reagent A and B.
3. Calcium Standard
○ Calcium Carbonate in dilute
hydrochloric acid. (10mg/dl).
Reagent Preparation
1. Combine equal volumes of Calcium Color
Reagent (A) and Calcium Buffer (B), mix and
let stand for 10 minutes at room temperature
before use. Ratio is 1:
2. Reagents should be combined in clean
vessels. Water and glassware containing
calcium will react with the reagent. All
glassware should be rinsed with diluted
hydrochloric acid before use.
_____________________________________________________________________________________ 1
Introduction (LAB)- Calcium determination ( midterms )
2
nd
SEM, 2022
Specimen collection and Preparation
1. Fasting nonhemolyzed serum is specimen of
choice.
2. Anticoagulants other than Heparin should not
be used.
3. Remove serum from clot as soon as possible
since red blood cells can absorb calcium
4. Older serum specimens containing visible
precipitate should not be used
5. Serum calcium is stable for twenty-four (24)
hours at room temperature, one (1) week
refrigerated (2-8 ̊ C) and up to five (5) months
frozen, and protected from evaporation.
ADDED NOTES:
● We can use plasma with heparin.
Manual Procedure
1. Prepare working reagent.
2. Label tubes Blank, Standard, Controls,
Patients, etc.
3. Transfer 1 mL of working reagent into each
tube.
4. Add 0 mL (10μl) of sample to respective
tubes and mix.
5. Let stand for atleast sixty (60) seconds at
room temperature.
6. Zero spectrophotometer with blank at 550 nm.
(Wavelength ranges:550-580)
7. Read and record absorbance of all tubes.
Final color is stable for twenty (20) minutes.
Reagent
blank ( RB
)
Standard (
S )
Unknown (
U )
Standard 0 mL
Specimen 0 mL
Working
reagent
1 mL 1 mL 1 mL
ADDED NOTES:
● Stanbio
● 3mL of working reagent
Calculations
Calcium (mg/dl)= Abs. of Unknown x Conc. Of std. =
Abs. of Standard
Reference value
● Serum - 8.5-10 mg/dl
● Children under 12, usually have high values
which decrease with aging
_____________________________________________________________________________________ 2
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Cchm-lab-midterms - Lecture notes
Course: Clinical Chemistry 2 (MDT 3122L)
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CLINICAL CHEMISTRY 2
Introduction (LAB)- Amylase and Lipase Determination ( midterms )
2nd SEM 2022
Amylase Determination
● Reagent Manufacturer:
○ STANBIO LABORATORY
Principles
● Amylase catalyzes the hydrolysis of
starch into simpler molecules with
maltose as the end product. Amylase
is formed in the pancreas and is found
in increased amounts in the serum and
urine in varying degrees of pancreatic
disturbance. Normally small amounts
of amylase are found in the blood.
Increased amounts of pancreatic
amylase is found in the blood during
the early stage of acute pancreatitis.
● The greatest concentration of amylase
is in the pancreas where the enzymes
is synthesized and secreted into the
intestinal tract for digestion of starch.
Amylase is also secreted by the
salivary glands and is present in
saliva, where it initiates hydrolysis of
starch while the food still in the mouth
and esophagus.
● Amylase is increased in acute
pancreatitis, carcinoma of head of
pancreas, duodenal ulcer, perforation
of gastric ulcer, hyperthyroidism,
mumps, acute injury of spleen and
obstruction. It is decreased in cirrhosis
and carcinoma of the liver, liver
abscess, hepatitis, acute alcoholism
and toxemia of pregnancy.
● The enzymatic procedure presented is
based on modifications of Wallenfels,
using as substrate
p-Nitrophenyl-D-maltohepatoside (
PNG7 ) with the terminal glucose
blocked to reduce spontaneous
degradation of the substrate by
glucosidase and glucoamylase. The
test is performed in a kinetic mode with
a very short lag time and offers much
greater stability than previous amylase
methodologies.
● Amylase hydrolyzes
p-Nitrophenyl-D-maltohepatoside (
PNG7 ) to
p-Nitrophenyl-D-maltostriose ( PNPG3
) and maltotetraose. Glucoamylase
hydrolyzes PNPG3 to
p-Nitrophenylglycoside ( PNPG1 ) and
glucose. Then is hydrolyzed by
glucosidase to glucose and
p-Nitrophenol, which produce a yellow
color, the rate of increase in
absorbance is measured at 405 nm is
proportional to the amylase activity in
the sample.
ADDED NOTES:
● Amylase and Lipase are pancreatic
markers.
● Amylase also called alpha
glucan-glucanohydrolase
● Amylase is under the hydrolase group
● Amylase is considered as the smallest
enzyme, it is filtered by the kidneys
and appears into the urine.
● Two major isoenzymes: S-type (
ptyalin ) and P-type ( amyloid )
● Amylase earliest pancreatic marker:
rise between 2-12 hours, peak 24
hours, normalize 2-5 days
● Multiple measurement, kinetic method
● Amylase aids in the digestion of
carbohydrates ( strach and glycogen )
● Enzymes glucosidase and
glucoamylase came from microbial
source
● Amylase tissue source are pancreas
and salivary gland
Materials
● Spectrophotometer
● Accurate pipetting devices
● Cuvets
● Interval timer
● Test tubes
Reagents
● Amylase reagent ( powder )
○ Composition when reconstituted
_____________________________________________________________________________________ 1
ARAULLO, ASGARE, BAIS, BALATBAT, BANAWA, BRIONES, DE CASTRO, DE LEON, DELOS TRINOS, DURAN, GALANG, MENDOZA, MUZADA, OLBES, ORDONA, OSDON, PUNZALAN, RASING, SALVO, RODRIQUEZ, TOLENTINO, VENTURA
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