SUMMARY OF ENZYMES AND THEIR CLINICAL SIGNIFICANCE ACCORDING TO DEAN

RODRIGUEZ:

1. Alkaline Phosphate/Alkaline Orthophosphoric Monoester Phosphohydrolase

 Bone isoenzyme increases due to osteoblastic activity and is normally elevated in children during periods of
growth and in adults older than age 50 years (geriatric).
 The presence of intestinal ALP isoenzyme in serum depends on the blood group (secretor gene and H
substance) of the individual. B or O blood group increases intestinal ALP after consumption of a fatty meal.
 For bone disorders, highest elevations occur in Paget's disease (osteitis deformans). Bone ALP isoform,
B1x, was detected in the serum of dialysis patients.

Carcinoplacental ALP:

1. Regan ALP-is found in lung, breast, ovarian and gynecological cancers; bone ALP co-migrator; most heat stable
ALP (65°C for 30 minutes); inhibited by phenylalanine reagent

2. Nagao ALP-found in adenocarcinoma of the pancreas and bile duct, pleural cancer; variant of Regan ALP,
inhibited by L-leucine and phenylalanine.

Methods:

1. Electrophoresis

Liver and bone ALPs are the most anodal isoenzymes; intestinal ALP is the least anodal Use of neuraminidase and
wheat germ lectin improves separation of bone and liver ALPS.

Liver-Bone-Placental-Intestinal

2. Heat Fractionation/Stability Test

 It is performed at 56°C for 10-15 minutes.

 Placental ALP is the most heat stable; bone ALP is the most heat labile. Decreasing order of ALP heat
stability: placental, intestinal, liver and bone

3. Chemical Inhibition Test

 This method uses different concentrations of phenylalanine, synthetic urea and levamisole solutions.

 Placental and intestinal ALPs are inhibited by phenylalanine reagent and 3M urea inhibits bone ALP.
Levamisole reagent inhibits liver and bone ALP.

4. Bowers and Mc Comb (Szasz modification) Is considered as the most specific method.

Notes to Remember:

 Decreased ALP is seen in zinc deficiency.

Increased ALP

1. Osteitis deformans

2. Obstructive jaundice

3. Osteomalacia
4. Rickets

5. Osteoblastic bone tumors-osteosarcoma

6. Sprue

7. Hyperparathyroidism

8. Hepatitis and cirrhosis (slight increased)

2. Acid Phosphatase/ Acid Orthophosphoric Monoester Phosphohydrolase

 It catalyzes the same reaction made by ALP, except that it is active at pH 5.0.

 ACP activity >50 IU/L indicates the presence of seminal fluid in the sample.

 Tissue sources: prostate (major source)

Diagnostic Significance:

 For detection of prostatic adenocarcinoma.

 It also useful in forensic clinical chemistry, in the investigation of rape cases

Notes to Remember
 Thymolphthalein monophosphate is the specific substrate; substrate of choice for quantitative endpoint
reaction.
 Serum sample must be free from hemolysis.
 Tartrate-resistant acid phosphatase (TRAP) is present in certain chronic leukemias and some lymphomas,
most notably in hairy cell leukemia.

Increased ACP (with Metastatic Bone Involvement)

1. Prostatic carcinoma

2. Breast, lung and thyroid carcinoma

3. Gaucher's disease

4. Niemann Pick Disease

TRANSFERASES/TRANSAMINASES

A. Aspartate Aminotransferase (AST)

 It has 2 isoenzyme fractions, cytoplasm and mitochondrial ASTs-the cytoplasmic isoenzyme is the
predominant form in serum.

 Major tissue source: cardiac tissue, liver and skeletal muscle

Diagnostic Significance:

 In the evaluation of myocardial infarction, hepatocellular disorders and skeletal muscle involvement.

 In acute myocardial infarction (AMI), AST levels begin to rise 6-8 hours, peak at 24 hours and normalize
within 5 days
Method:

Karmen Method pH 7.5; 340nm

 It uses malate dehydrogenase (MD) and monitors the change in absorbance at 340 nm. AST

B. Alanine Aminotransferase (ALT)

 It has enzymatic activity similar to AST.

 The highest concentration is in the liver; more liver-specific than AST.

 Major tissue source: liver

Diagnostic Significance:

 It is significant in the evaluation of hepatic disorders markedly increased concentration in acute inflammatory
conditions than AST.
 ALT measurement is a more sensitive and specific screening test for posttransfusion hepatitis or
occupational toxic exposure compared to AST.

Method:
 Aminotransferases require pyridoxal phosphate (vitamin B as coenzyme (prosthetic group).
 Hemolysis should be avoided because it increases AST 10x.
 Heparin may inhibit the activity of AST (but not all methods).

Increased Transferases

1. Toxic hepatitis

2. Acute Myocardial Infarction - AST

3. Wolff-Parkinson White Syndrome

4. Trichinosis-AST

5. Chronic alcoholism

6. Dermatomyositis - AST

8. Reye's syndrome

9. Viral hepatitis hepatitis AST

10. Muscular Dystrophy-AST

11. Acute pancreatitis AST

Notes to Remember
 The highest elevations of transferase is seen in acute hepatitis.

 Severe viral or toxic hepatitis may produce elevations of transferase up to 20x the normal limits.

 In acute hepatitis, the De Ritis ratio (ALT:AST) is > 1.0.

  Moderate elevation of transferase in chronic hepatitis, hepatic cancer and infectious mononucleosis.

 Slightly increased in hepatic cirrhosis, alcoholic hepatitis and obstructive jaundice.

 ALT is slightly increased in obstructive jaundice but markedly increased in necrotic jaundice.

III. AMYLASE/ ALPHA-1-4 GLUCAN-4-GLUCOHYDROLASE (AMS)

 It catalyzes the breakdown of starch and glycogen an important enzyme in the physiologic digestion of
starch.

 It is the smallest enzyme in size (with a MW of 50,000 to 55,000 daltons)

 It is the earliest pancreatic marker.
 Isoenzymes: S-type (ptyalin) and P-type (amylopsin) both present in normal healthy sera
Methods:
Substrate for all the methods: Starch

1. Saccharogenic

 It is the classic reference method expressed in Somogyi units.

 It measures the amount of reducing sugars produced by the hydrolysis of starch by the usual glucose
methods.

2. Amyloclastic

 It measures amylase activity by following the decreases in substrate concentration (degradation of starch).

3.Chromogenic

 It measures amylase activity by the increase in color intensity of the soluble dye-substrate solution produced
in the reaction.

4. Coupled-enzyme

• It measures amylase activity by a continuous-monitoring technique.

Increased Serum Amylase

1. Acute pancreatitis

2. Ectopic pregnancy

3. Peptic ulcers

4. Alcoholism

5. Mumps-Parotitis

IV. LIPASE (LPS)/TRIACYLGLYCEROL ACYLHYDROLASE

 Plasma concentrations are normal in conditions of salivary gland involvement.

 Major tissue source: Pancreas

Methods:
1. Cherry Crandal (reference method)

Principle: Hydrolysis of olive oil after incubation for 24 hours at 37°C and titration of fatty acids using NaOH.

Substrate: 50% olive oil / Triplein (pure TAC)

End product: Fatty acid

2. Tietz and Flereck
3. Peroxidase coupling most commonly used method; does not use 50% olive oil.

V. LACTATE DEHYDROGENASE (LD)

 Is a hydrogen-transfer enzyme that uses the coenzyme nicotinamide dinucleotide (NAD+), It is a tetrameric
molecule containing four subunits of two possible forms (H and M).
Diagnostic Significance:

 Highest serum levels are seen in pernicious anemia and hemolytic disorders.
 In AMI, LD levels begin to rise within 12-24 hours, peak levels within 48-72 hours and remains elevated for
10-14 days.
 Hepatic carcinoma and toxic hepatitis will have 10-fold increased.
 Viral hepatitis and cirrhosis would give LD slightly increased values (2-3x URL).
 LD-1> LD-2 also known as the "flipped pattern" is seen in myocardial infarction and hemolytic anemia,
 LD-5 is moderately increased in acute viral hepatitis and cirrhosis and markedly increased in hepatic
carcinoma and toxic hepatitis.
 LD-6 represents the alcohol dehydrogenase enzyme; 6th band in electrophoresis; elevated in drug
hepatoxicity and obstructive jaundice; it is responsible for the metabolic conversion of methanol and
ethylene glycol to toxic compounds; present in patients with arteriosclerotic failure

Methods
1. Wacker Method (forward/direct reaction) reaction is at pH 8.8 Is the most commonly used method
2. Wrobleuski La Due (reverse/indirect reaction)-reaction is at pH 7.2

 Decreased values of LD are observed when samples are frozen (LD-S is cold-labile), therefore samples
should be processed within 24 hours after collection and stored at 25°C.

VI. CREATINE KINASE/ATP-CREATINE-N-PHOSPHOTRANSFERASE (CK)

 It is a dimeric molecule with small molecular size, composed of a pair of two different monomers called M
and B.

 It is found in small amounts throughout the body, but is found in high concentrations only in muscle and
brain, although CK from brain virtually never crosses the blood-brain barrier to reach plasma.
 CK-MB <6% of total CK
 CK-1 is the most anodal and fabile isoenzyme; CK-3 is the least anodal. CK-BB is the dominant isoenzyme
of CK found in brain, intestine, and smooth muscle.
 Intramuscular injections are known to increase CK (<5x URL).

Diagnostic Significance:

 It is a very sensitive indicator of acute myocardial infarction (AMI) and Duchenne disorder. Highest elevation
of total CK is seen in Duchenne's muscular dystrophy (50x URL).

 CK-MB is found mainly in myocardial tissue-it is used as a serodiagnostic test for AMI.
Methods:
1. Tanzer-Gilbarg Assay (forward/direct method)
2. Oliver-Rosalki (reverse/indirect method) - most commonly used method; faster reaction; pH 6.8; 340nm

Increased Creatine kinase

1. Duchenne's muscular dystrophy - 50x

2. Myocardial Infarction
3. Hypothyroidism
4. Pulmonary infarction
5. Reye's syndrome
6. Strenous exercise and intramuscular injections
7. Cerebral vascular accident (occasional)
8. Rocky Mountain Spotted Fever-CK-MB
9. Carbon monoxide poisoning

VII. ALDOLASE/FRUCTOSE 1,6-DIPHOSPHATE ALDOLASE

Is a glycolytic enzyme that splits fructose-1,6-diphosphate into two triose phosphate molecules in the metabolism of
glucose.

Increased: skeletal muscle disease, leukemia, hemolytic anemia and hepatic cancer

Isoenzymes:
Aldolase A=Skeletal muscles
Aldolase B=WBC, liver, kidney
Aldolase C=Brain Tissue

OTHER CLINICALLY SIGNIFICANT ENZYMES:

1. 5' Nucleotidase (5'N)-2º markey for obstructive jaundice

 Is a phosphoric monoester hydrolase; predominantly secreted from the liver.

 is a marker for hepatobiliary disease and infiltrative lesions of the liver.

Method: Dixon & Purdon, Campbell, Belfield & Goldberg

2. Gamma glutamyl transamine peptidase/transferase (GGT)

Substrate: g-glutamyl-p-nitroanilide
Method: Szass, Rosalki & Tarrow

Diagnostic Significance:

 It is useful in differentiating the source of an increased ALP level.

 It is elevated in all hepatobiliary disorders, biliary tract obstructions.

 It is a sensitive indicator of alcoholism (occult alcoholism) must sensitive marker of acute alcoholic hepatitis.

 It is useful in monitoring the effects of abstention from alcohol.

3. Pseudocholinesterase (PCHE)
  It is secreted by the liver - it reflects synthetic function rather than hepatocyte injury. It catalyzes the removal
of benzyl group from cocaine-it acts as a "antixenobiotic enzyme."

 It is a marker for insecticide/pesticide poisoning (organophosphate poisoning) - low serum PCHE.

 Is used to monitor the effect of muscle relaxants (succinylcholine) after surgery.

 Tissue source: liver, myocardium and pancreas

 Decreased: acute hepatitis, cirrhosis, carcinoma metastatic to liver and malnutrition

 Method: Ellman technique and potentiometric

SUMMARY OF ENZYMATIC METHODS

ENZYME METHODS SUBSTRATE
ALP BODANSKY-SHINOWARA-JONES- BETA-glyceroPO4
REINHART
BESSY-LOWRY-BROCK NITROPHENOL PO4
BOWERS AND MCCOMB NITROPHENOL PO4
KLEIN, BABSON AND READ PHENOLPHTHALEIN PO4
ACP BABSON-READ-PHILIPS
ROY/ROY-HILLMAN THYMOLPHTHALEIN
AST KARMEN
ALT REITMAN AND FRANKEL
AMYLASE SACCHAROGENIC STARCH
AMYLOCLASTIC STARCH
CHROMOGENIC STARCH
COUPLED-ENZYME STARCH
LIPASE CHERRY-CRANDAL 50% OLIVE OIL
LD WACKER METHOD (FORWARD) LACTATE
WROBLEUSKI LA DUE (REVERSE) PYRUVATE
CK TANZER-GILBARG (FORWARD)
OLIVER-ROSALKI (REVERSE)
5-NT DIXON AND PURDON
CAMPBELL
GGT SZASZ/SZASS GAMMA-GLUTAMYL-P-
NITROANILIDE
PSEUDOCHOLINESTERASE ELLMAN
METHOD IN BOLD=MOST PREFERRED