Major Clinical Enzymes

phosphatases

•Alkaline Phosphate/ Alkaline Ortosphoric Monoester

Phosphohydrolase

• A nonspecific enzyme capable of reacting with many different substrates

• Functions: to liberate inorganic phosphate from an organic phosphate ester with the concomitant production of an alcohol.

• In healthy sera, alkaline phosphate (ALP) levels derive from liver and bone (osteoblasts)

• 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)

• In normal pregnancy, increased ALP activity can be detected between 16-20 weeks of pregnancy.

•The presence of intestinal ALP isoenzyme in serum depends on the blood group and secretor status of the individual – B or O blood group increases intestinal ALP after consumption of a fatty meal.•Major tissue sources: liver, bone, placenta, intestinal, renal•Reference values: 30-90 U/LDiagnostic Significance:•When total ALP levels are increased, it is the major liver fraction that is most frequently elevated- obstructed jaundice. ALP increase in obstructive jaundice due to greater rate of secretion.•For bone disorders, highest elevations occur in Paget’s disease (osteitis deformans).

Isoenzymes• Liver ALP- most anodal• Bone ALP• Placental ALP• Intestinal ALP- least

anodal

Carcino-placental ALP• Regan ALP- lung, breast,

ovarian, and gynecological cancers, bone ALP co-migrator; most heat stable ALP (65°C for 30 minutes); inhibited by phenylalanine reagent.

• Nagao ALP- adenocarcinoma of the pancreas and bile duct, pleural cancer, variant of Regan: inhibited by L-Leucine and phenylalanine.

• Notes to Remember:• Placental, intestinal, liver and bone- decreasing

order of ALP heat stability• Heat stability test is performed at 56 C for 10-15

minutes- placental ALP most eat stable; bone ALP most heat labile

• Placental and intestinal ALP are inhibited by Phenylalanine reagent; 3M urea inhibits bone ALP

• Levamisole reagent inhibits liver and bone ALP• Bowers and McComb (Szaz modification)- IFCC

recommended method • Hemolysis and diet (fat meals)-sources of analytical

errors; elevated serum ALP• Sensitive if stored at low temperature (4 C)- leads to

increase value• ALP is inhibited by phosphorus –addition of 2-amino-2-

methyl-1-propanol (AMP) buffer binds phosphorus under Bowers-McComb method.

Increased ALP• Osteitis deformans• Obstructive jaundice• Osteomalacia• Rickets• Osteoblastic bone tumors• Sprue• Hyperparathyroidism• Hepatitis and cirrhosis (slight

increased)• Bone cancer

•Acid Phosphatase/ Acid Orthophosphoric Monoester Phosphohydrolase

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

• useful in forensic clinical chemistry, in investigation of rape cases- vaginal washings are examined for seminal-fluid-acid phosphatase (ACP) activity, which can persist for up to 4 days.

• Diagnostic significance: detection of prostatic carcinoma

• Tissue Sources: prostate (major source), RBC, platelets and bone

• Reference values: 2.5-11.7 U/L (total

Aminotransferases

Aspartate aminotransferase from Escherichia coli bound

with cofactor pyridoxal 5-phosphate.[1]

A. Aspartate aminotransferase (AST)

an enzyme normally present in body serum and in certain body tissues, especially those of the heart and liver. This enzyme affects the intermolecular transfer of an amino group from aspartic acid to alpha-ketoglutaric acid, forming glutamic acid and oxaloacetic acid. The reaction is reversible. The enzyme is released into the serum because of tissue injury and thus may increase as a result of myocardial infarction and it is commonly measured clinically as a marker for liver health.

Enzymatic reaction:Aspartate (Asp) + α-ketoglutarate ↔ oxaloacetate + glutamate (Glu)

AST is similar to alanine transaminase (ALT) in that both enzymes are associated with liver parenchymal cells.

Diagnostic Significance: In the evaluation of myocardial infarction,

hepatocellular disorders and skeletal muscle involvement

In acute myocardial infarction, AST levels begin to rise 6-8 hours, peak at 24 hours and normalize within 5 days

It is released to a greater degree in chronic disorders of the liver with progressive damage.

• It has enzymatic activity similar to AST.

• It catalyses the transfer of an amino group from alanine to α-ketoglutarate with the formation of glutamate and pyruvate.

• The highest concentration is in the liver. More liver specific than AST.

• Other sources: kidney, pancreas, RBC, heart, skeletal muscle, and lungs.

B. Alanine Aminotransfer

ase

Diagnostic significance Significant in the evaluation of hepatic

disorder. Monitors the course of hepatitis

treatment and the effect of drug therapy. ALT measurement is a more sensitive

and specific screening test for postt ransfusion hepatitis or occupational toxic exposure.

ALT levels are also used to screen blood donors.

Aminotransferases require pyrixodal phosphate (vitamin B6) as coenzyme (prosthetic group).

Aminotransferases are present in human plasma, bile, CSF and saliva

In acute hepatitis, the De Ritis ratio (ALT:AST) is >1.0. Several viral or toxic hepatitis may produce elevations

of transaminases up to 20x the normal limits. The highest elevations of transaminases in chronic

hepatitis, hepatic cancer and infectious mononucleosis. Slighlty increased in hepatic cirrhosis, alcohol hepatitis

and obstructive jaundice. ALT is slightly increased in obstructive jaundice but

markedly increased in necrotic jaundice. Hemolysis should be avoided beause it increases AST

10x.

Notes to remember:

Increase Transferases

•Toxic hepatitis•Acute myocardial infacrtion- AST•Hepatic Cancer•Reye’s syndrome•Viral Hepatitis

•Woff-Parkinson White syndrome •Trichinosis

• Dermatomyositis- AST

Acute Pancreatitis

• Muscular dystrophy-AST

•Chronic alcoholism

An enzyme that hydrolyzes the ester linkages of fats to produce alcohol and fatty acid.

It catalyzes partial hydrolysis of dietary TAG in the intestine to the 2-monoglyceride intermediate, with the production of long chain fatty acid

Most specific pancreatic marker- secreted exclusively in the pancreas; not affected by renal disorders.

Concentrations are normal in conditions of salivary gland involvement.

Major tissue source; Pancreas Reference value; 0-1.0U/mL

Lipase (LPS)/ Tricyglycerol Acylhydrolase

Diagnostic Significance• Diagnosis of acute pancreatitis. • It is similar in this respect to AMS measurements but is

considered more specific for pancreatic disorders than AMS measurement.

• Both AMS and LPS levels rise quickly, but LPS elevations persist for approximately 5 days in acute pancreatitis, whereas AMS elevations persist for only 2-3 days.

• Elevated LPS levels also may be found in other intra-abdominal conditions but with less frequency than elevations of serum AMS.

• Elevations have been reported in cases of penetrating duodenal ulcers and perforated peptic ulcers, intestinal obstruction, and acute cholecystitis.

• In contrast to AMS levels, LPS levels are normal in conditions of salivary gland involvement.

• Therefore, LPS levels are useful in differentiating serum AMS elevation as a result of pancreatic versus salivary involvement.

CREATINE KINASEATP- CREATINE-N-PHOSPHOTRANSFERASE (CK)

CREATINE KINASE

• It catalyzes the transfer of phosphate group between creatine phosphate and adenosine di phosphate

• Involved in the storage of high-energy creatine PO4 in the muscles.

• CK-MM is the major isoenzyme (94-100%)• Bedridden patients may decrease CK

activity.• Intramuscular injections are known to

increase CK (<5x URL )• Ref. Value : Male – 15- 160 U/L• Female- 15-130 U/L

ISOENZYMES

3 TYPES

CK-BB (BRAIN TYPE)

CK-MB ( HYBRID TYPE)

CK-MM (MUSCLE TYPE)

Serum of adults which can be found in the neonatal sera, which contains CK-BB of brain origin due to its high molecular size.

Myocardium is the only tissue from which CK-MB enters the serum in significant quantities ( 20% )

CK-MM is both present in the cardiac and skeletal muscles.

DIAGNOSTIC SIGNIFICANCE

It is a very sensitive indicator of acute myocardial infection (AMI) and Duchenne disorder.

Total CK is markedly elevated after trauma to skeletal muscle from crush injury, convulsion, tetany, surgical incision or intramuscular injection.

Injury in both cardiac and skeletal muscles accounts for the majority of CK-MM elevations

Demonstration of elevated levels of CK-MB, >6% of the total CK, is considered the most specific indicator of myocardial damage.

Duchenne muscular dystrophy (DMD) is a recessive X-linked form of muscular dystrophy, affecting around 1 in 3,600 boys, which results in muscle degeneration and eventual death.[1] The disorder is caused by a mutation in the dystrophin gene, located on the human X chromosome, which codes for the protein dystrophin, an important structural component within muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane.

HISTOPATHOLOGY

DIAGNOSTIC SIGNIFICANCE

Following AMI, the CK-MB levels begins to rise within 4-8 hours, peak at 12-24 hours and normalize within 48-72 hours.

CK is not elevated in angina.

PATIENTS WITH DUCHENNE MUSCULAR DYSTROPHY

INCREASED CREATINE KINASE

Duchenne’s muscular dystrophy Myocardial Infraction Hypothyroidism Pulmonary infraction Reye’s syndrome Strenous exercise and intramuscular

injection Cerebral vascular accident (occasional) Rocky mountain spotted fever- CK-MB Carbon Monoxide poisoning

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

Isoenzymes:Aldolase A-Skeletal MusclesAldolase B-WBC, liver, kidneyAldolase C-Brain Tissue

Other clinically significant enzymes

1)31 Nucleotidase (51N)

● a phosponic monoester hydrolase: predominantly secreted from the liver.

● a marker for hepatobiliary disease and infiltratiy lesions of the liver.

● methods used: Dixon and Purgon, Campell, Belfield & Goldberg.

● reference values: 0-1.6 units.

2. Gamma glutamyl transamine peptidase/ transferase (GGT)

● it catalyzes the transfer of glutamyl groups between peptides or amino acids through linkage at a gammy carboxyl group.

● located in the canaliculi of the hepatic cells and particularly in the epithelial cells lining the biliary ductules; also in the kidney, prostate and pancreas.

● useful in differentiating the source of an elevated ALP levels.

● elevated in all hepatobiliary disorders- biliary tract obstructions.

● sensitive indicator of alcoholism (occult alcoholism) – most sensitive marker of acute alcoholic hepatitis.

● useful in monitoring the effects of abstention from alcohol.

● it affects the cell membrane and microsomal fractions - elevated among individuals undergoing warfarin, Phenobarbital and phenytoin therapies.

● also increased in pancreatitis and prostatic disorders.

● substrate: y-glutamyl-p-nitroanilide

● methods used: Szass, Rosalki & Tarrow, Orlowski

● reference values: 5-30 U/L (F)/ 6-45 U/L (M)

3.Cholinesterase (CHS)/Pseudocholisterase

Marker for insecticide/pesticide poisoning (organophosphate poisoning) –low CHS.

Methods used: ellman technique and potentiometric

Reference values: 0.5-1.3 pH units (plasma)

Index of parachymal function; secreted by the liver.

It is used to monitor the effect of muscle relaxants (succinylcholine) after surgery.

Also known as peptidyldipeptidase A or Kininase II. It converts angiotensin I to angiotensin II within the

lungs. Possible indicator of neuronal dysfunction

(Alzheimer’s disease- CSF) Increased: sarcoidosis, acute and chronic bronchitis

and leprosy Main source: macrophages and epitheloid cells

4. Angiotensin- Converting Enzyme (ACE)

5. Ceruplasmin

6. Ornithine Carbamoyl Transferase (OCT)

Copper-carrying protein and also enzyme.

A marker for Wilson’s disease (hepato lenticular diseases).

For hepatobiliary diseases

Reference value: 8-20 mU/mL

It functions to maintain NADPH in the reduced form in the erythrocytes.

It is an newborn screening marker.

It is found in the adrenal cortex, spleen ,RBC and lymp nodes.

Deficiency of this enzyme can lead to drug-induced hemolytic anemia (primaquine,antimalarial drug).

Increased levels: myocardial infarction, megaloblastic anemia.

Specimen: red cell hemolysate, serum

Reference values: 10-15 U/g hemoglobin or 1200-2000 mU/ml packed RBC

7. Glucose 6- Phosphate dehydrogenase

(G-6 PD)