MindMap Gallery Research on amylase
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Edited at 2023-09-05 17:33:34Case Study #4 Amylase & Lipase
1. Phophatases
an enzyme that accelerates the hydrolysis and synthesis of organic esters of phosphoric acid and the transfer of phosphate groups to other compounds
Two Clinically Important Phosphatases
1. Alkaline Phosphatase
Acid phosphatases (APs) are a family of enzymes that are widespread in nature, and can be found in many animal and plant species.
exists in different isoforms or isoenzymes depending on its tissue source which include
Intestinal Alkaline Phosphatase (ALP-I): Found in the intestines.
Placental Alkaline Phosphatase (ALP-PLAP): Found in the placenta during pregnancy.
Bone Alkaline Phosphatase (ALP-BAP): Predominantly found in bone tissue.
Liver/Biliary Alkaline Phosphatase (ALP-L/BALP) Found in the liver and biliary tract.
To differentiate between these ALP isoenzymes, various laboratory methods can be employed:
Electrophoresis
technique that separates molecules based on their electric charge and size. They will migrate to different positions based on their charge and molecular weight.
This separation can help identify the specific isoenzymes present in a sample.
Immunoassays
Antibodies specific to different ALP isoenzymes can be used to detect and quantify their presence in a sample. For example, monoclonal antibodies directed against ALP-I, ALP-PLAP, or ALP-BAP can be used in immunoassays to differentiate and measure the levels of these isoenzymes.
Inhibitor Studies
Certain inhibitors can selectively inhibit the activity of specific ALP isoenzymes. For instance, the inhibitor L-phenylalanine can inhibit ALP-I, while levamisole can inhibit ALP-BAP.
By using these inhibitors, one can selectively inhibit the activity of a specific isoenzyme and then measure the remaining ALP activity to determine the contribution of that isoenzyme.
Isoenzyme Staining
Different staining substrates may be specific to certain isoenzymes, leading to the production of distinct colored or precipitated products that can be visualized.
Methods of Measurement
Substrate used
B-glycerophosphate (introduced by Kay)
Bodansky (classic procedure)
measuring the rate of liberation of an inorganic PO4 as the enzyme hydrolyzed glycerol PO4 to glycerol. pH of 8.8 at 37°C
Shinowara, Jones, and Reinhart
Modified the method of Bodansky by using a more alkaline buffer which provides a pH of 10.8 for an enzyme reaction
Phenyl phosphate
King and Armstrong
Phenol is being measured to determine the enzyme activity
Folin-Ciocalteau- mixture of phosphomolybdic and phosphotungstic acid (reagent used)
Kind and King - Antipyrine serves as a chromogenic agent
Diazo reagent - phenol reduces reagent to a blue compound
p-nitrophenyl phosphate
Bessey, Lowry, and Brock: original
ester is colorless, but the final product is yellow at the pH of the reaction the rate of PO4 action is enhanced if certain amino alcohols are used as PO4 accepting buffer enzyme reaction can be followed by observing the formation of yellow color of the p-nitrophenol
Bowers and McComb
colorless substrate is hydrolyzed to a yellow p-nitrophenol an increase in absorbance at 405 nm is directly proportional to the ALP activity
phenolphthalein phosphate
Huggins and Talalay
phenolphthalein released is measured by its red color formed
Klein Babson Reid
Substrate is phenolphthaleinmonoPO4 uses Linear reaction kinetics
Sample Considerations
1. Serum or heparinized plasma, free of hemolysis
2. Complexing anticoagulants like citrate, oxalate, and EDTA must be avoided
3. Blood transfusion (containing citrate) causes a transient decrease in serum ALP same as #2
4. Freshly collected serum samples should be kept at RT and assayed ASAP preferable within 4 hrs after collection
5. Fasting serum is preferred because of rise in ALP in blood group B or O during blood absorption
6. Frozen specimens must be thawed and keep at RT for 18-24 hrs before measurement to achieve full enzyme reaction or incubate at 37°C *Urinary ALP is necessary to do dialysis to remove inhibitors
Clinical Significance
1. Hepatobiliary (obstructive) 3-10x ULN biliary tract obstruction (increase due to synthesis induced by cholestasis)
2. Hepatocellular - hepatitis and cirrhosis 3x ULN must correlate with other tests for interpretation
3. Bone disorders a. Paget’s (osteitis deformans) highest elevation of ALP b. Osteomalacia, rickets, hyperthyroidism, and osteogenic sarcoma
4. Increase levels in healing bone fractures and physiologic bone growth
5. Increase in pregnancy - hypertension, pre-eclampsia, eclampsia, and threatened abortion ● Normal Pregnancy: ○ 16-20 weeks - 1.5x ○ 3rd trimester - 2-3x ○ Return to normal 3-6 after delivery ● Decreased Levels: ○ Inherited condition of hypophosphatasia ○ Absence of bone isoenzyme ○ Inadequate bone calcification
2. Acid Phosphatase
they catalyze the hydrolysis of organic phosphate esters present in the extracellular space.
Classification
Orthophosphoric monoester phosphohydrolase, E.C no. 3.1.3.2
Where is ACP normally found in the body?
Prostate
Leukocytes
Bone, liver, spleen, kidney, RBCs and platelets
isoenzymes of ACP in the human body
Tartrate-resistant acid phosphatase (TRAP)
Prostatic Acid Phosphatase (PAP)
Function
Dephosphorylation of organic phosphate esters at acidic pH.
Clinical Significance
Detection of Prostate Carcinoma
Other prostate conditions such as prostate hyperplasia, and prostate surgery.
Forensic rape Investigations
Elevations in Bone diseases
Elevations during platelet disorders
ACP Measurement Method
Bodansky and Shinowara
B-glycerophosphate
Lengthy and non-specific
King-Armstrong, modified by Gutman & Gutman
Phenylphosphate
Non-specific
Hudson
P-nitrophenylphosphate
In this assay, the hydrolysis is forward Rapid, nonspecific method
Babson and Reed
a-naphthylphosphate
introduced the substrate specific for prostatic CA.
Roy et al, modified by Ewen and Spitzer
Thymolphthalein monophosphate
more linear and more specific for prostatic forms of the enzyme
Reitz and Guilbault
4-methylumbellieferone phosphate
Uses fluorescence, the method has some improved sensitivity
2. Amylase and Lipase
3. Mr Q <3
What is the inhibitor used to determine prostatic ACP and how is it computed?
Acid Phosphatase-Tartrate Resistant. -This test is a chemical inhibition test used to measure Prostatic ACP. -authored by Hillman’s Principle: based on Bobson and Reed. -Na tartrate is the one that inhibits the sensitivity of the isoenzyme. -being measured here is the non prostatic ACP -Prostatic ACP conc = total ACP - non prostatic ACP.
What is the purpose of adding alkali after hydrolysis?
Neutralization: Hydrolysis reactions often involve the use of strong acids to break down substances or molecules. After the hydrolysis is complete, the reaction mixture may be highly acidic. Adding an alkali, such as sodium hydroxide (NaOH) or potassium hydroxide (KOH), can neutralize the excess acid. Adjusting pH: Hydrolysis reactions may occur under acidic conditions, which are necessary for specific chemical transformations. Reaction Control: The alkali can react with any remaining acid and reduce the acidity, which can slow down or stop the reaction. Product Isolation: After hydrolysis, the reaction mixture may contain a mixture of products, which are acidic or unstable under acidic conditions. By adding an alkali, pH is adjusted to conditions that favor the stability and isolation of the desired product. Precipitation: In some chemical reactions, adding an alkali can lead to the precipitation of specific compounds or products. The specific purpose of adding alkali after hydrolysis will depend on the nature of the reaction, the properties of the substances involved, and the goals of the overall process.
Case Study #4 Amylase & Lipase
1. Phophatases
an enzyme that accelerates the hydrolysis and synthesis of organic esters of phosphoric acid and the transfer of phosphate groups to other compounds
Two Clinically Important Phosphatases
1. Alkaline Phosphatase
2. Acid Phosphatase
they catalyze the hydrolysis of organic phosphate esters present in the extracellular space.
Classification
Function
Clinical Significance
ACP Measurement Method
2. Amylase and Lipase
Amylase
It catalyzes the breakdown of starch and glycogen, which are complex carbohydrates, into simpler sugars such as maltose and maltodextrin through a process called hydrolysis.
The catalytic action of alpha amylase involves the cleavage of the alpha-1,4-glycosidic bonds present in the starch molecule
The aspartate amino acid in the upper portion of the amylase enzyme reacts with the positively charged carbon, causing a conformational change in the enzyme.
This interaction leads to the breakage of the alpha-1,4-glycosidic bond, resulting in the detachment of the glucose molecules from the enzyme
different methodologies in Amylase determination
Amyloclastic/ Iodometric method
Measures the disappearance of starch substrate. The decrease in color is proportional to the AMY concentration
Saccharogenic/ Reductiometric method
-Measures the appearance of the product -Measure the reducing sugars (glucose and maltose) produced as a result of enzymatic activity
Chromogenic Method
Measures the increasing color from production of product coupled with a chromogenic dye
Continuous Monitoring / Coupled Enzyme/Kinetic Method
Coupling of several enzyme systems to monitor amylase activity
Enzymatic Colorimetric Method
Wallenfels et al introduced p-nitrophenyl glycoside as a defined substrate for alpha amylase determination 1st Principle 2nd Principle
Lipase
An enzyme that hydrolyzes the ester linkages of fats to produce alcohols and fatty acids
catalytic action of lipase
Substrate Recognition: Lipase recognizes triglycerides as its substrate.
Active Site Binding: Lipase has an active site, which is a specific region on the enzyme where the substrate (triglyceride) binds.
Hydrolysis: Once the triglyceride is bound to the active site of lipase, the enzyme catalyzes a hydrolysis reaction. In this reaction, a water molecule is used to break the ester bonds that hold the fatty acids to the glycerol backbone of the triglyceride.
Formation of Fatty Acids and Glycerol: As a result of the hydrolysis reaction, the ester bonds between the fatty acids and glycerol are broken, yielding three individual fatty acid molecules and one glycerol molecule.
Solubility: Fatty acids and glycerol, being smaller and more polar than triglycerides, are now water-soluble.
Nutrient Absorption: The liberated fatty acids and glycerol are absorbed through the walls of the small intestine into the bloodstream.
different methodologies of Lipase Determination
Titrimetric Method
Tietz- Fierick Method
Oleic acid is titrated with 0.05 N NaOH with 1% thymolphthalein → definite/ persistent Blue end color
Tietz -Templeton Method
Cherry and Crandall Method
Difference between tietz- Fierick & Cherry and Crandall: Buffer & incubation period
Henry Method
Disadvantages of the Titrimetric Method -Preparation and use of unstable oil emulsion -Long incubation time -Difficulty in detecting endpoint
Turbidimetric method
Triglycerides form emulsions that scatter light, hydrolysis of these triglycerides to soluble monoesters and glycerol react to a decrease in light scattering measured spectrophotometrically
Disadvantages: -The method is not easy to perform technically, nor has it been as reproducible as serum AMS. -The rate of enzyme activity is not linear throughout the incubation period. -It is seldom used as it takes too long to be employed as an emergency procedure/set. -Lack of standardization for the enzyme assay
Coupled Enzyme Assay
Diagnostic Significance of AMY and LPS determination
AMY
ACUTE PANCREATITIS
○ Serum amylase levels begin to rise 2-12 hours after onset of attack ○ Peak at 24 hours and return to normal levels within 3-5 days ○ Values generally range from 250-10000 Somogyi units/ dL (2.55 X ULN)
MACROAMYLASEMIA
Not a disease but an acquired benign condition
amylase molecule combines with immunoglobulins to form a complex that is too large to be filtered across the glomerulus
Other Conditions
1.Mumps and parotitis- salivary gland lesions; Hallmark symptoms of Mumps is the swelling of the salivary glands
2. Perforated peptic ulcer, intestinal obstruction, mesenteric infarction and acute appendicitis-intra abdominal diseases - Usually less than 500 Somogyi units/ dL
3. Renal insufficiency/failure
4. Diabetic Ketoacidosis ○ body produces high levels of blood acids called ketones ○ body cannot produce enough insulin
LPS
ACUTE PANCREATITIS
● Higher levels of serum lipase may be indicative of Acute pancreatitis ● Mainly caused by gallstones and alcohol abuse ● In acute pancreatitis, both AMS and LPS will increase ● Elevated levels may also be found in other intra-abdominal conditions but with less frequency than elevations of serum AMS ● Elevations have been reported in cases of: ○ Penetrating ulcers ○ Intestinal obstruction ○ Acute cholecystitis ● Amylase is a poor test for the diagnosis of pancreatitis
3. Mr Q <3
Mr. Q recently passed the Medical Technology board exams and decided to take on a medical technologist job in his quiet, suburban hometown since he had to look after his father who just suffered from a mild stroke. In his first day of work, he was assigned in the Clinical Chemistry department and since the laboratory was undermanned, he had to do all the clinical chemistry assays for that day. Anyway, there were senior med techs assigned in other departments as well if ever there was a need to consult. He first performed an Amylase CC FS assay. For the procedure, he mixed 20 ml of reagent 1 and 4 ml of reagent 2 to make a mono-reagent. The chief med tech suddenly came in and asked him if he could carry out a stat procedure for an emergency patient, so he forgot to store the mono-reagent in the refrigerator. He proceeded to perform the amylase assay 2 hours after. Category of Estimation: Kinetic, spectrophotometric at 405 nm. The kinetic measurement was concluded after 4 minutes. The result was below the normal range. Mr. Q wasn’t confident of his performance since it was his first day, so he went to consult a senior colleague and showed him the results. He then proceeded to discuss what he did during the assay.
Did Mr. Q perform the test correctly?
NO
Wrong volume mixture for mono reagent
Wrong total reaction time
Mono reagent not stored correctly
2-hour time delay
What is the inhibitor used to determine prostatic ACP and how is it computed?
Acid Phosphatase-Tartrate Resistant. -This test is a chemical inhibition test used to measure Prostatic ACP. -authored by Hillman’s Principle: based on Bobson and Reed. -Na tartrate is the one that inhibits the sensitivity of the isoenzyme. -being measured here is the non prostatic ACP -Prostatic ACP conc = total ACP - non prostatic ACP.
What is the purpose of adding alkali after hydrolysis?
Neutralization: Hydrolysis reactions often involve the use of strong acids to break down substances or molecules. After the hydrolysis is complete, the reaction mixture may be highly acidic. Adding an alkali, such as sodium hydroxide (NaOH) or potassium hydroxide (KOH), can neutralize the excess acid. Adjusting pH: Hydrolysis reactions may occur under acidic conditions, which are necessary for specific chemical transformations. Reaction Control: The alkali can react with any remaining acid and reduce the acidity, which can slow down or stop the reaction. Product Isolation: After hydrolysis, the reaction mixture may contain a mixture of products, which are acidic or unstable under acidic conditions. By adding an alkali, pH is adjusted to conditions that favor the stability and isolation of the desired product. Precipitation: In some chemical reactions, adding an alkali can lead to the precipitation of specific compounds or products. The specific purpose of adding alkali after hydrolysis will depend on the nature of the reaction, the properties of the substances involved, and the goals of the overall process.