1. Enzymes
Part 2
M. Zaharna Clin. Chem. 2009

2. Alanine Aminotransferase (ALT)
A transferase with enzymatic activity similar to AST
Converts alanine + α-ketoglutarate to pyruvate and glutamate
GPT or SGPT= serum glutamic pyruvic transaminase
The older terminology was serum glutamic-pyruvic transaminase (SGPT, or GPT)
M. Zaharna Clin. Chem. 2009

3. Alanine Aminotransferase
Pyridoxal phosphate is the coenzyme
Source is heart, liver, and skeletal muscle
Primarily associated with liver disease
It is considered the more liver-specific enzyme of the transferases.
Pyridoxal phosphate : It is the active form of vitamin B6
M. Zaharna Clin. Chem. 2009

4. Diagnostic Significance
Higher elevations are found in hepatocellular disorders than in extrahepatic or intrahepatic obstructive disorders.
In acute inflammatory conditions of the liver,
ALT elevations are frequently higher than those of AST
and tend to remain elevated longer as a result of the longer half-life of ALT in serum
Cardiac tissue contains a small amount of ALT activity, but the serum level usually remains normal in AMI
M. Zaharna Clin. Chem. 2009

5. Assay for enzyme activity
The typical assay procedure consists of a coupled enzymatic reaction using LDH in the second reaction
NADH is oxidized in the second reaction
The change in absorbance is at 340 nm is measured
ALT
Alanine + -Ketoglutarate Pyruvate + Glutamate
Pyruvate + NADH + H Lactate + NAD
LDH
Reference Range: 6-37 U/L (37°C)
M. Zaharna Clin. Chem. 2009

6. Alkaline Phosphatase (ALP)
Belongs to a group of enzymes that catalyze hydrolysis of phosphate monoesters at alkaline pH (9-10)
Removes inorganic phosphate from organic phosphate ester & generates alcohol
Requires Mg2+ as an activator
Phosphomonoesters (or phosphoric esters) are chemical compounds containing one ester bond and a phosphate group.
M. Zaharna Clin. Chem. 2009

7. Alkaline Phosphatase (ALP)
Present on all cell surfaces
Highest levels are found in:
intestine,
liver,
bone (osteoblasts),
spleen,
placenta,
and kidney
In Liver enzyme is located on both sinusoidal and bile canalicular membranes
M. Zaharna Clin. Chem. 2009

8. M. Zaharna Clin. Chem. 2009

9. Isoenzymes ALP exists as a number of isoenzymes
Major are those found in Liver, bone, placenta, and then intestinal fraction
Electrophoresis for isoenzyme analysis
Liver isoenzyme (fastest)
Bone isoenzyme
Placental isoenzyme
Intestinal isoenzyme (slowest)
Immunochemical methods now available
M. Zaharna Clin. Chem. 2009

10. Diagnostic Significance
Elevations of ALP are of most diagnostic significance in the evaluation of hepatobiliary and bone disorders.
Liver
Obstructive 3-10 x ULN
elevation is due to increased synthesis of the enzyme induced by cholestasis
Hepatocellular < 3 x ULN
Bone
Highest in Paget’s Disease
Osteomalacia, rickets, hyperparathyroidism,
Healing fractures
1.5 x normal during late pregnancy
Low in hypophosphatasia
Inherited inadequate bone calcification
Osteomalacia is the general term for the softening of the bones due to defective Bone demineralization is a chronic disorder that typically results in enlarged and deformed bones.
cholestasis is a condition where bile cannot flow from the liver to the duodenum.
M. Zaharna Clin. Chem. 2009

11. Assay for enzyme activity
Different methods due to non-specificity of the enzyme group
Bowers and McComb method based on absorption of p-nitrophenol at 405 nm
colorless
yellow
M. Zaharna Clin. Chem. 2009

12. Acid Phosphatase (ACP)
Belong to the same group of phosphatase enzymes as ALP
Catalyze hydrolysis of phosphate monoesters at acid (5.0) pH
High concentrations in the prostate gland,
Found also in bone, liver spleen, kidney, RBC and platelets
M. Zaharna Clin. Chem. 2009

13. Diagnostic Significance
ACP used as an aid in the detection of prostatic carcinoma particularly metastatic carcinoma of the prostate
Prostatic specific antigen (PSA) now better
Total ACP determination relatively insensitive technique
Chemical inhibition methods to differentiate the prostatic portion
Serum incubated with and without addition of tartarate (inhibits P. ACP)
Tartarate inhibits P. ACP
M. Zaharna Clin. Chem. 2009

14. Diagnostic Significance
Total ACP – ACP after tartrate inhibition = prostatic ACP
Other methods
Immunological test
Other diagnostics
Vaginal washings for rape cases (persist for up to 4 days)
Serum levels in bone disease (osteoclasts)
Idiopathic thrombocytopenic purpura – platelet destruction
ITP= Idiopathic thrombocytopenic purpura
M. Zaharna Clin. Chem. 2009

15. Assay for enzyme activity
Same as ALP but at acidic pH
P-Nitrophenolphosphate p-nitrophenol + phosphate ion
Products are colorless at acidic pH
Addition of alkali stops the reaction and transforms the products into chromogens, can be measured spectrophotometery
ACP
pH 5
M. Zaharna Clin. Chem. 2009

16. γ-Glutamyltransferase (GGT)
Transfers γ-glutamyl residue from γ-glutamyl peptides (usually glutathione) to amino acids, H2O and other small peptides
Glutathione + Amino Acid
glutamyl-peptide + L-cysteinylglycine
High concentrations in liver tissue
Also in pancreas and kidney
Glutathione, an antioxidant, protects cells from toxins such as free radicals , GGT is involved in the transfer of amino acids across the cellular membrane
GGT
M. Zaharna Clin. Chem. 2009

17. Diagnostic Significance
Increased plasma GGT is associated with
Hepatobiliary disease
Highest seen in biliary obstruction
Alcoholic cirrhosis
Used with ALP to differentiate between liver and bone diseases
M. Zaharna Clin. Chem. 2009

18. Assay for enzyme activity
γ- glutamyl residue of γ glutamyl-p-nitroanilide is transferred to glycylglycine, releasing p-nitroaniline
L-γ-glutamyl-p-nitroanilide + glycylglycine
p-nitroaniline + y-glutamyl glycylglycine
The rate of liberation of p-nitroaniline is directly related to the GGT activity in the sample and is quantitated by measuring the increase in absorbance at 405 nm.
GGT
Reference Range: male, 6-45 U/L (37°C); female, 5-30 U/L (37°C)
M. Zaharna Clin. Chem. 2009

19. Amylase (AMS) Hydrolyzes starch and glycogen
Starch = amylose & amylopectin
Amylose = long, unbranched α,1-4 linkages
Amylopectin = branched chain α,1-6 linkages
Glycogen similar to amylopectin but more branches
α-Amylase attacks only α-1-4 glycosidic bonds
Generates glucose, maltose, and dextrins (branch points)
Requires Ca+ and Cl- for activation
Cellulose is not attacked by α-Amylase due to α-1-6 bonds
Amylose is a linear polymer of glucose
Amylopectin is a polysaccharide and highly branched polymer of glucose found in plants
M. Zaharna Clin. Chem. 2009

20. Acinar cells of pancreas and salivary glands major tissue sources
AMS is smallest enzyme – ,000 daltons
Readily filtered into urine
Digestion of starches begins in the mouth
Inactivated by stomach acid Pancreatic AMS takes over in intestine
Acinar cell can refer to:exocrine cells of the pancreas, which release hydrolytic enzymes into the small intestine (duodenum
M. Zaharna Clin. Chem. 2009

21. Isoenzymes Two isoenzymes are found in normal serum;
P isoamylase … derived from pancreas
S isoamylase … derived from salivary glands
They are identified using electrophoresis chromatography or isoelectric focusing.
M. Zaharna Clin. Chem. 2009

22. Diagnostic Significance
In Acute pancreatitis
Amylase begin to rise 2-12 hours after onset of attack
Peak 24 hours, return to normal 3-5 days
2.5 x ULN
Also elevated in salivary gland disorders and Intraabdominal disease (e.g. Perforated peptic ulcer)
M. Zaharna Clin. Chem. 2009

23. Assay for enzyme activity
Measurement – 4 different methods
Amyloclastic – disappearance of starch substrate
Starch substrate with Iodine attached
As hydrolyzed Iodine is released = decrease in dark blue color
Saccharogenic – appearance of product
Classic Somogyi method
Starch substrate is hydrolyzed to CHO molecules that have reducing ability
Amount of reducing sugars present = amount of AMS present
M. Zaharna Clin. Chem. 2009

24. Assay for enzyme activity
Somogyi units
A measure of the level of activity of amylase in blood serum.
One Somogyi Unit is defined as the number of milligrams of glucose released in 30 minutes at 37°C under specific assay conditions.
M. Zaharna Clin. Chem. 2009

25. Assay for enzyme activity
Chromogenic
Chromogenic methods use a starch substrate to which a chromogenic dye has been attached, forming an insoluble dye-substrate complex.
As AMS hydrolyzes the starch substrate, smaller dye-substrate fragments are produced, and these are water soluble.
The increase in color intensity of the soluble dye-substrate solution is proportional to AMS activity.
Continuous – coupling enzymes to monitor AMS activity (NADH produced)
M. Zaharna Clin. Chem. 2009

26. Lipase (LPS)
Lipase is an enzyme that hydrolyzes the ester linkage of fats to produce alcohols and fatty acids
LPS catalyses the partial hydrolysis of dietary triglycerides in the intestine
Pancreatic LPS specific for fatty acid residues at position 1 and 3 of triglyceride
Accelerated by presence of bile salts & colipase
M. Zaharna Clin. Chem. 2009

27. Lipase (LPS) Primarily found in pancreas
Some from stomach and small intestine
M. Zaharna Clin. Chem. 2009

28. Diagnostic Significance
Used in diagnosis of acute pancreatitis
Similar pattern to AMS but lasts longer
May be elevated in other abdominal conditions
Normal in salivary gland conditions (AMS↑)
M. Zaharna Clin. Chem. 2009

29. Assay for enzyme activity
Cherry-Crandall method – classic
Triolein as substrate
Titrate amount of fatty acids generated
Triglyceride + 2 H2O monoglyceride + 2 fatty acids
LPS pH
M. Zaharna Clin. Chem. 2009

30. Assay for enzyme activity
Turbidimetric methods
Fats in solution = cloudy
LPS hydrolysis causes clearing of solution
Colorimetric
Coupled reactions with peroxidase or glycerol kinase
M. Zaharna Clin. Chem. 2009

31. Glucose-6-Phosphate Dehydrogenase (G6PD)
Oxidoreductase catalyzes oxidation of G6P to 6-phosphogluconate or 6-phosphogluconolactone
First step in pentose-phosphate shunt of glucose metabolism
M. Zaharna Clin. Chem. 2009

32. Little activity is found in normal serum Primarily focus of RBC Keeps
Adrenal cortex, spleen, thymus, lymph nodes, lactating mammary gland, and RBC
Little activity is found in normal serum
Primarily focus of RBC Keeps
NADPH in reduced form
Helps to generate glutathione
Which in turn protect Hb from oxidation
Cells hemolyze without it
M. Zaharna Clin. Chem. 2009

33. Diagnostic Significance
Deficiency of G6PD inherited sex-linked trait
Drug-induced hemolytic anemia (primaquine)
Increased levels reported in AMI and megaloblastic anemia
primaquine is a medication used in the treatment of malaria
M. Zaharna Clin. Chem. 2009

34. Assay for enzyme activity
G6PD
G-6-P + NADP phosphogluconate + NADPH + H+
A red cell hemolysate is used to assay for deficiency of the enzyme
Serum is used for evaluation of enzyme elevations
M. Zaharna Clin. Chem. 2009