ACP, ALP, AMYLASE, LIPASE, GGT Flashcards by Arriane Cyrel (MTI)

belongs to the same group of phosphatase enzymes as ALP and is a hydrolase that catalyze the same reactions

ACP

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Major difference between ACP and ALP is the pH where ACP activity takes place at a pH of

5.0 (acidic)

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ACP is buffered at pH
ALP is buffered at pH

6
10.2

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Belongs to a group of enzymes that catalyzes the hydrolysis of various phosphomonoesters in alkaline pH

ALP

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Considered a non-specific enzyme, able to react with many different substrate

ALP

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It liberates inorganic phosphate from an organic phosphate ester w/ the concomitant production of an alcohol

ALP

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is required as an activator of ALP

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GGT Chemical Name

Gamma-glutamyl transpeptidase

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Catalyzes the transfer of y-glutamyl residue from y-glutamyl peptides to amino acids, other peptides, or H2O

GGT

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Cell membrane-bound

GGT

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Largest amounts found in the kidney

GGT

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Small accumulations in the liver (hepatobiliary tree) and heart

GGT

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GGT Half-life:

7 - 10 days

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GGT In alcoholic liver disease:

28 days

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AMS Chemical Name:

α-1,4-glucan-4-glucanohydrolase

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Hydrolases are enzymes that catalyzes the breakdown of starch and glycogen

AMS

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Starch is made up of both

Amylose and Amylopectin

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-long unbranched chain of glucose linked by a,1-4 glycosidic bonds (glucose molecule)

Amylose

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-branched chain polisaccharide with a,1-6 linkage (polysaccharide)

Amylopectin

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a-AMS attacks only the a,1-4 glycosidic bonds to produce the degradation products:

o Glucose
o Maltose
o intermediate chain (Dextrin)

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MW: 45,000 daltons

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considered the smallest enzyme and can pass through glomerular filter

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top of blood vessels located between the bowman’s capsule within the kidney

Glomerulus

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microscopic filter”

Glomerulus

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Blood filtration in the kidney where fluids, ions, glucose, and waste products are being removed from the glomerular capillaries

Glomerulus

AMS Metalloenzyme:

Calcium

Requirement for the functional integrity of amylase

Calcium

AMS Optimum pH:

6.9 – 7.0 in serum

Normal serum contains both AMS

salivary and pancreatic AMS

Normal amylase / creatinine ratio =

1.0% – 4.0% (0.01 – 0.04)

A:C ratio (Acute Pancreatitis) =

>4.0% (up to 15%)

An elevated amylase-creatinine clearance ratio has been established as being highly specific for the diagnosis of

acute pancreatitis

Calculates the ratio of amylase to creatinine that if both filtered by the kidney

amylase-creatinine clearance ratio

LPS Chemical name:

Triacylglycerol Acylhydrolase

ACP SYSTEMATIC NAME

E.C. 3.1.3.2

ALP SYSTEMATIC NAME

E.C. 3.1.3.1

GGT SYSTEMATIC NAME

E.C. 2.3.2.1

AMYLASE SYSTEMATIC NAME

E.C. 3.2.1.1

LIPASE SYSTEMATIC NAME

E.C. 3.1.1.3

inhibited by tartrate

Prostatic ACP (band 1)

greatest activity in the prostate gland

Prostatic ACP (band 1)

fastest migrating ACP isoenzyme

Prostatic ACP (band 1)

2nd fastest migrating ACP isoenzyme

Bone - osteoclasts (band 5)

major form in plasma ACP

Platelets, RBCs & Monocytes (band 3)

Resistant to tartrate inhibition

Bone - osteoclasts (band 5)

differentiates prostatic fractions from, bone-osteoclast fractions in ACP

Inhibition

differentiates bone fractions from, liver fractions in ALP

Enzymes or lectins

Rate of mobility of ALP ISOENZYMES

3. Liver isoenzyme – migrates the fastest 4. Bone isoenzyme – 2nd 1. Placental isoenzyme – 3rd 2. Intestinal isoenzyme – 4th

Its activity starts in the salivary gland where it initiates the hydrolysis of starch while food is in the mouth and esophagus

S-type Isoamylase

Its action is terminated by the acidity of the stomach

S-type Isoamylase

Inhibited by monoclonal antibody

S-type Isoamylase

Inhibited by protein isolated from wheat

S-type Isoamylase

represent 2/3 of AMS activity of normal serum

S-type Isoamylase

Synthesized by the pancreatic acinar cells and secreted into the intestinal tract via the pancreatic duct system

P-type Isoamylase

Its action is favored by the mildly alkaline condition of the duodenum

P-type Isoamylase

Inhibited by monoclonal antibody

P-type Isoamylase

Inhibited by monoclonal antibody

P-type Isoamylase

M predominates in the normal urine

P-type Isoamylase

Migrates most quickly in terms of electrophoretic migration in AMS

S1 (fastest), S2, S3 P1 (follows S3), P2, P3

Other organs of S-type Isoamylase

Fallopian tube and lungs

Favoured by the mild alkaline conditions in the duodenum

P-type Isoamylase

The isoenzymes of salivary origin migrates most quickly (?) , P type are slower (?)

(S1,S2,S3) (P1,P2,P3)

The most commonly observed fractions of AMS

P2, S1 and S2

In acute pancreatitis and renal failure: predominates

Abnormal amylase (usually the S-type) in combination with Immunoglobulins (IgA or IgG) or other high MW proteins

Macroamylases

Not found in urine and concentrated in serum/plasma

Macroamylases

No clinical significance

Macroamylases

ELEVATIONS Prostatic isoenzyme

1. Prostatic Cancerm 2. Prostatic hyperplasia & prostatic infarction 3. Ureteral Obstruction, carcinoid tumors of rectum & prostatic massage 4. Medico-legal

: ACP is inferior to PSA

Prostatic Cancer

DECREASE Bone isoenzyme

• Active osteoclast-mediated bone resorption • Gaucher's cells • Hairy cell leukemia

best marker for prostatic cancer; leading cause of death from cancer in the Ph

Prostate specific acntigen

benign condition where the prostatic gland enlargement; not cancerous

Prostatic hyperplasia

common among men aging > 60

Ureteral Obstruction

prostatic gland enlargement blocks urine flow

Ureteral Obstruction

a tumor presses the division that separates the prostate gland and the rectum

Carcinoid tumors of rectum

performed to extract a prostatic secretions

Prostatic massage

performed by stoking the prostate several times

Prostatic massage

performed in combination w/ the digital rectal exam

Prostatic massage

o px is asked to bend forward o doctor inserts finger to the rectum until prostate gland is reached to check for enlargement

digital rectal exam

usage of bone rigidity, causing loss of important enzymes

Active osteoclast-mediated bone resorption

infiltration of the bone marrow and other tissues by Gaucher cells, which are rich in ACP activity

Gaucher's cells

slow growing cancer of the bones

Hairy cell leukemia

affects the bone marrow making too much B lymphocytes

Hairy cell leukemia

ALP ELEVATIONS

• Obstructive hepatic disorder (Liver isoenzyme) • Paget’s disease (Osteitis deformans) • increased osteoblastic activity (Bone isoenzyme) • DM, renal failure and cirrhosis (Intestinal isoenzyme)

ALP DECREASE

Chronic Kidney Disease patients

Low ALP and low Bone Turnover rate

ALP

- bone ALP isoform in the serum of dialysis patients

B1x

 Blockage of bile ducts and pancreatic ducts

Obstructive hepatic disorder (obstructive jaundice)

 Can cause affected bones to become fragile

Paget’s disease

ALP Isoenzymes in cancer

Carcinoplacental Alkaline Phosphatases

Regan isoenzyme Nagao isoenzyme Kasahara isoenzyme

patients w/ a particular type of lung CA

Regan isoenzyme

Caused by ectopic production of by cancerous tissues

Regan isoenzyme

Low incidence but used in monitoring therapy

Regan isoenzyme

Adenocarcinoma of the pancreas and the bile ducts

Nagao isoenzyme

Variant of regan isoenzyme

Nagao isoenzyme

– hepatoma

Kasahara isoenzyme

GGT ELEVATIONS

• Liver damage • Smoking • Ethanol abuse • Medication (anticonvulsant)

GGT DECREASE

• Pregnancy • Oral contraceptives

GGT - major source

Liver damage

- moderate = 10%

Smoking Ethanol abuse

- heavy = 20%

• Medication (anticonvulsant)

o Carbamazepine o Phenytoin o Barbiturates o Valproic acid

1st trimester (Cholestasis)

Pregnancy:

dec. 20%

Oral contraceptives:

originates primarily from the hepatobillary system

GGT

may leak in the blood stream in cases of liver or bile duct damage

GGT

Bile is important for digestion

GGT

is the first liver enzyme to increase in the blood when any of the liver bile ducts becomes constricted

GGT

is one of the most sensitive liver enzyme test for binding bile duct issues

GGT

is tested alongside ALP

GGT

• 2-3X ULN

Chronic alcoholism (heavy drinkers)

• Affects the microsomal structure of the liver cell

Medication (anticonvulsant)

• An acute type of liver problem

Cholestasis

• Slower than normal flow of bile

Cholestasis

• Itching, yellowing of the skin and eyes

Cholestasis

GGT levels typically rise after (?), which may continue for several weeks

heavy alcohol intake

2 to 6 weeks of abstinence – levels may gradually increase and come down to normal values

GGT

Evaluation of liver injury: primary use

GGT

The higher the level, the greater the liver damage

GGT

Test for alcoholic abuse

GGT

Most widely used marker for alcoholic abuse

GGT

LPS ELEVATIONS

• perforated peptic ulcer • duodenal ulcer • intestinal obstruction • mesenteric vascular obstruction

o pancreas o can be caused by carcinoma

intestinal obstruction

NOT elevated in mumps

LPS

Useful in differentiating serum amylase elevations as a result of pancreatic vs. salivary involvement

LPS

In vitro reaction

GGT

GGT catalyzes the transfer of (?) to the peptide group to another peptide or H2O

gamma-glutamyl group

GGT transfer (?) to (?) to form (?)

gamma-glutamyl glycyl-glycine Gamma-glytamyl-glycyl-glycine

GGT Liberation of

3-carboxy-4-nitroaniline

- most widely used substrate in GGT analysis

gamma-glutamyl-p-nitroanilide

In the presence of GGT, (?) will be transferred and attach to (?) while liberating (?) forming the products, (?)

gamma-glutamyl glycyl-glycine p-nitroanilide Gamma-glytamyl-glycyl-glycine and nitroaniline

Stable for: o 1 month at 4C o 1 year at -20C

GGT

-a chromogenic product w/ strong absorbance at 405-420nm

Nitroaniline

Activity is stable at least 1 month at 4dc and 1 year at -20 degree Celsius

GGT

GGT is preffered but EDTA plasma has also been used

Non hemolyzed sample

– produce turbidity in the reaction mixture

Heparin

– depress GGT activity by 10 to 15 %

Citrate, oxalate and fluoride

GGT Normal Value:

6-45 U/L men, 3-30 U/L women

Responsible for starch digestion

AMYLASE

Starts in the mouth with the hydrolytic action of the

salivary amylase

happens in a short duration only

Digestion

Upon swallowing, (?) is inactivated by the acidity of the stomach

amylase

perform the majority and the rest of digestion

Pancreatic amylase

Splits complex CHO made up of a-D glucose units

AMYLASE

AMS TISSUE SOURCES

Pancrease Acinar cells Salivary gland

– greatest source ANS

Pancrease

AMS ACTIVATORS Most effective

Bromide Chloride

Hydrolyzes glycerol esters of long chain fatty acids in the presence of H2O

LPS Primary products:

alcohols and fatty acids

Acts only on emulsified substrate

LPS

o To force two immiscible liquids to combine into suspension o Ex. Oil in water – will not dissolve in each other to form a uniform soln

LPS

LPS TISSUE SOURCES

1. Pancreas 2. GIT 3. Leukocytes 4. Adipose cells 5. Colostrum

LPS (1° source)

Pancreas

Lipase conc is about (?) greater than any organs or tissues

9,000x

– first form of milk produced by the mammary glands

Colostrum

Responsible for triglyceride metabolism

LPS

 Bile salts  Colipase

LPS

A protein secreted by pancreas that fxs a a cofactor for pancreatic lipase, which will form a stoichiometric complex with lipase

Colipase

Binds to the bile salt over triglycerol interface, thus allowing the enzymes to anchor itself to the water lipid interface to allow hydrolysis by lipase

Colipase

Uses p-NPP substrate

Bowers and McComb Method

• sample is serum • plasma is rarely used

Bowers and McComb Method

• Chelators falsely lower activity

Bowers and McComb Method

• Enzyme activity increases slightly on storage: due to loss of inhibitors

Bowers and McComb Method

• Relatively stable at 4°C up to a week

Bowers and McComb Method

• Most specific method recommended by IFCC

Bowers and McComb Method

• Kinetic or continuous monitoring method method (pH 10.2) which is based on the molar absorptivity of para-nitrophenol

Bowers and McComb Method

• (?) (colorless cmpd) is hydrolyzed (?) (yellow) and subsequent liberation of (?)

p-nitrophenylphosphate p-nitrophenol phosphate ions

• increase in absorbance is directly proportional to ALP activity

Bowers and McComb Method

Bowers and McComb Method SUBSTRATE END-PRODUCTS

PNPP p-nitrophenol

- reduces activity of intestinal & placental isoenzymes

Phenylalanine

- inhibits bone & liver isoenzymes

Levamisole

measurement before and after heating

Heat fractionation

identification of enzyme based on stability

Heat fractionation

ALP isoenzyme – most stable – most labile

o Placental isoenzyme o Bone isoenzyme

Heat fractionation isoenzyme

o Placental isoenzyme o Liver isoenzyme o Bone isoenzyme

Gutman and gutman SUBSTRATE END-PRODUCTS

Phenylphosphate Inorganic PO4

Shinowara SUBSTRATE END-PRODUCTS

PNPP (pnitrophenol phosphate) p-nitrophenol

Babson, Read and Philips SUBSTRATE END-PRODUCTS

Alpha-naphthyl PO4 Alpha-naphthol

Roy and Hillman SUBSTRATE END-PRODUCTS

Thymolphthalein monophosphate Free thymolphthalein

Liberation of phosphate ions.

ACP

• Reaction is measured before addition of Tartrate (Total ACP)

Chemical Inhibition

• Inhibitor: Tartrate

Chemical Inhibition

• After addition: Residual activity

Chemical Inhibition

= Prostatic ACP

• Total ACP – Residual activity

– preferred substrate for quantitative endpoint reactions (Modified by Roy)

• Thymolphthalein monophosphate

• reaction is catalysed in a citrate buffer at pH 6.0

Prostatic ACP

– continuous monitoring methods (Hillman method)

• α-naphthyl phosphate

is the most commonly employed substrate for ACP

• α-naphthyl phosphate

The primary reason for an elevated amylase is Acute pancreatitis

AMYLASE LIPASE

AMS Rise upon onset of symptoms

2 – 12 H 5 – 8 hours

LPS Rise upon onset of symptoms

4 - 8 H 4 – 8 hours

AMS Peak activity (fourfold to sixfold)

48 Hours 12 - 72 hours

LPS Peak activity

4 - 8 H 4 – 8 hours

AMS Returns to normal

24 Hours 24 Hours

LPS Returns to normal

8 3rd – 5th day 3rd – 4th day

The degree of elevation of AMS is helpful in differentiating diagnosis of

acute pancreatitis

AMS Other conditions

2. Mumps 3. Perforated peptic ulcer 4. Appendicitis 5. Ruptured ectopic pregnancy 6. Dissecting aortic aneurysm 7. Biliary tract disease

– caused by a virus; young adults/children experience fever, muscle pain, headache, poor appetite, tiredness followed by painful swelling of one or both carotid salivary glands

Mumps

AMS Most popular method

Continuous Monitoring Method

Improved the reaction stoichiometry (quantitative study of reactants and products in a chem rx)

Continuous Monitoring Method

more controlled and consistent hydrolysis conditions

Continuous Monitoring Method

makes use of coupling of several enzyme systems to monitor amylase activity

Continuous Monitoring Method

AMS Substrates: Small oligosaccharides

 Maltopentose  Maltopentrose  4-NP-glcoside

AMS Historical methods

Uses starch substrate hydrolyzed to carbohydrate molecule w/ reducing properties

Saccharogenic

The amount of reducing sugars are then measured where the concentration is directly proportional to AMS activity

Saccharogenic

Classic reference method for amylase activity

Saccharogenic

Units: Somogyi

Saccharogenic

Hydrolysis of starch by amylase causes a decrease in color intensity

Amyloclastic (Iodometric method)

Starch substrate-attached to iodine, AMS hydrolyze starch and iodine is released-decrease in the intensity of the initial dark blue complex-the decrease is proportional to AMS conc.

Amyloclastic (Iodometric method)

Measures the disappearance of starch substrate

Amyloclastic (Iodometric method)

Use a starch substrate to which a chromogenic dye has been attached, forming an insoluble dye–substrate complex.

Chromogenic (Klein, Foreman, Searcy)

Opposite to amyloclastic

Chromogenic (Klein, Foreman, Searcy)

The increase in color intensity of soluble dye-substrate solution is proportional to AMS activity, measure increase in color from production of product couples with a chromogenic dye

Chromogenic (Klein, Foreman, Searcy)

Measures the change in turbidity of starch solution over a short reaction period

Turbidimetry and Nephelometry (Peralta & Reinhart)

Grouped depending of what substrate is used in a particular method

LPS

LPS Specimen

serum (stable at RT), pleural fluid, ascitic fluid

LPS Inhibitors

heavy metals, quinine & some esterase inhibitors

increases concentration of LPS

Bacterium

Some important lipase-producing bacterial genera include

Bacillus, Pseudomonas and Burkholderia

LPS Not inhibited by

fluoride or arsenilate

Most important dietary lipids. Digestion depends on the interplay among pancreatic lipase, colipase, and bile acids/salts.

For Long-Chain Triglycerol Substrate

– hydrolyses these triglycerides into fatty acid molecules and mono-acyl glycerols

Pancreatic lipase

fatty acid is titrated w/ alkali solution

Titrimetric Methods

titration is carried out by potentiometry