INTRODUCTION TO ENZYMES

Enzymes are biological catalysts responsible for supporting almost all of the chemical
reactions that maintain animal homeostasis. Because of their role in maintaining life
processes, the assay and pharmacological regulation of enzymes have become key
elements in clinical diagnosis and therapeutics. The macromolecular components of
almost all enzymes are composed of protein, except for a class of RNA modifying
catalysts known as ribozymes. Ribozymes are molecules of ribonucleic acid that catalyze
reactions on the phosphodiester bond of other RNAs.

Enzymes are found in all tissues and fluids of the body. Intracellular enzymes catalyze
the reactions of metabolic pathways. Plasma membrane enzymes regulate catalysis within
cells in response to extracellular signals, and enzymes of the circulatory system are
responsible for regulating the clotting of blood. Almost every significant life process is
dependent on enzyme activity.

Common enzymes used for clinical diagnosis include:

• Acid phosphatase
• Alanine aminotransferase
• Alkaline phosphatase
• Amylase
• Angiotensin converting enzyme
• Aspartate aminotransferase
• Cholinesterase
• Creatinine kinase
• Gamma glutamyltransferase
• Lactate dehydrogenase
• Renin

There are a range of enzymes of which deficiency results in the inborn errors of
metabolism.

Enzymes in the Diagnosis of Pathology:

The measurement of the serum levels of numerous enzymes has been shown to be of
diagnostic significance. This is because the presence of these enzymes in the serum
indicates that tissue or cellular damage has occurred resulting in the release of
intracellular components into the blood. Hence, when a physician indicates that he/she is
going to assay for liver enzymes, the purpose is to ascertain the potential for liver cell
damage.

1. LDH:

The measurement of LDH is especially diagnostic for myocardial infarction because this
enzyme exists in 5 closely related, but slightly different forms (isozymes). The 5 types
and their normal distribution and levels in non-disease/injury are listed below.

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LDH 1 - Found in heart and red-blood cells and is 17% - 27% of the normal serum total.

LDH 2 - Found in heart and red-blood cells and is 27% - 37% of the normal serum total.

LDH 3 - Found in a variety of organs and is 18% - 25% of the normal serum total.

LDH 4 - Found in a variety of organs and is 3% - 8% of the normal serum total.

LDH 5 - Found in liver and skeletal muscle and is 0% - 5% of the normal serum total.

Following a myocardial infarct the serum levels of LDH rise within 24-48 hours reaching
a peak by 2-3 days and return to normal in 5-10 days. Especially diagnostic is a
comparison of the LDH-1/LDH-2 ratio. Normally, this ration is less than 1. A reversal of
this ration is referred to as a "flipped LDH". Following an acute myocardial infarct the
flipped LDH ratio will appear in 12-24 hours and is definitely present by 48 hours in over
80% of patients. Also important is the fact that persons suffering chest pain due to angina
only will not likely have altered LDH levels.

2. CPK:

CPK is found primarily in heart and skeletal muscle as well as the brain. Therefore,
measurement of serum CPK levels is a good diagnostic for injury to these tissues. The
levels of CPK will rise within 6 hours of injury and peak by around 18 hours. If the injury
is not persistent the level of CK returns to normal within 2-3 days. Like LDH, there are
tissue-specific isozymes of CPK and there designations are described below.

CPK3 (CPK-MM) is the predominant isozyme in muscle and is 100% of the normal
serum total.

CPK2 (CPK-MB) accounts for about 35% of the CPK activity in cardiac muscle, but less
than 5% in skeletal muscle and is 0% of the normal serum total.

CPK1 (CPK-BB) is the characteristic isozyme in brain and is in significant amounts in

smooth muscle and is 0% of the normal serum total.

Since most of the released CPK after a myocardial infarction is CPK-MB, an increased
ratio of CPK-MB to total CPK may help in diagnosis of an acute infarction, but an
increase of total CPK in itself may not. CPK-MB levels rise 3-6 hours after a myocardial
infarct and peak 12-24 hours later if no further damage occurs and returns to normal 12-
48 hours after the infarct.

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 3. Aldolase:

Aldolase is present most significantly in skeletal and heart muscle. Damage to skeletal
muscle produces high serum levels of aldolase, particularly in the case of progressive
muscular dystrophy. Aldolase may also be slightly increased in early stages of viral
hepatitis and advanced cancer of the prostate.

4. Lipase:

Lipase is an enzyme secreted by the pancreas into the duodenum. Damage to the
pancreas, as in acute pancreatitis results in lipase in the blood from the secretory cells.

5. Transaminases (GOT and GPT):

Glutamic-Oxaloacetic Transaminase (GOT) also known as Aspartate Transaminase

(AST) occurs in large concentrations in the heart and liver with moderate amounts in
skeletal muscle, kidneys, and pancreas. GOT levels can be used to diagnose myocardial
infarction within 10-48 hours. Other conditions with elevated GOT include arrhythmias
and severe angina of the heart, and liver damage.

Glutamic-Pyruvic Transaminase (GPT) also known AlaineTransaminase (ALT) is

found in significant quantities in liver, kidney, and skeletal muscle, in decreasing order.
When liver cells are damaged, GOT and GPT levels rise especially early in the disease.
In hepatitis, transaminase levels rise several days before jaundice begins. The enzyme
levels are especially useful in assessing subtle and early changes in biliary obstruction
and active cirrhosis.

6. Gamma-glutamyl Transpeptidase (GGT):

GGT catalyzes the transfer of the glutamyl groups among different polypeptides and
amino acids. Clinically significant GGT found in the blood comes from cells that line the
biliary tract. GGT levels rise dramatically with obstructive diseases of the biliary tract
and liver cancers. GGT is especially useful in assessing liver function associated with
alcohol-induced liver disease.

7. Amylase:

Amylase is an enzyme that breaks starch down into sugar. Amylase is present in human
saliva, where it begins the chemical process of digestion. Foods that contain much starch
but little sugar, such as rice and potato, taste slightly sweet as they are chewed because
amylase turns some of their starch into sugar in the mouth. The pancreas also makes
amylase (alpha amylase) to break down dietary starch into di- and trisaccharides which
are converted by other enzymes to glucose to supply the body with energy. Plants and
some bacteria also produce amylase.

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Classification:

α-Amylase:

The α-amylases are calcium metalloenzymes, completely unable to function in the

absence of calcium. By acting at random locations along the starch chain, α-amylase
breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from
amylose, or maltose, glucose and "limit dextrin" from amylopectin. In human physiology,
both the salivary and pancreatic amylases are α-Amylases.

β-Amylase:

Another form of amylase, β-amylase is also synthesized by bacteria, fungi, and plants.
Working from the non-reducing end, β-amylase catalyzes the hydrolysis of the second α-
1, 4 glycosidic bond, cleaving off two glucose units (maltose) at a time. Animal tissues
do not contain β-amylase, although it may be present in microorganisms contained within
the digestive tract.

γ-Amylase:

In addition to cleaving the last α (1-4) glycosidic linkages at the nonreducing end of
amylose and amylopectin, yielding glucose, γ-amylase will cleave α (1-6) glycosidic
linkage. Unlike the other forms of amylase, γ-amylase is most efficient in acidic
environments and has an optimum pH of 3.

Diagnostic use:

Amylase may be measured for purposes of medical diagnosis. A normal concentration is

in the range 21-101 U/L. A higher than normal concentration may reflect one of several
medical conditions, including acute inflammation of the pancreas, macroamylasemia,
perforated peptic ulcer, and mumps. Amylase may be measured in other body fluids,
including urine and peritoneal fluid.

8. Alkaline phosphatase:

It is a hydrolase enzyme responsible for removing phosphate groups from many types of
molecules, including nucleotides, proteins, and alkaloids. The process of removing the
phosphate group is called dephosphorylation. As the name suggests, alkaline
phosphatases are most effective in an alkaline environment.

Diagnostic use:

The normal range is 39-120. High ALP levels can show that the bile ducts are blocked.
Level is significantly higher in children and pregnant women.

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Lowered levels of ALP are less common than elevated levels. The following conditions
can cause abnormal levels of ALP:

Elevated levels (hyperphosphatasemia):

If it is unclear why alkaline phosphatase is elevated, isoenzyme studies using

electrophoresis can confirm the source of the ALP. Heat stability also distinguishes bone
and liver isoenzymes ("bone burns, liver lasts").

• Liver (Liver ALP):

o Cholestasis, cholecystitis, cholangitis, cirrhosis, hepatitis, fatty liver, liver
tumor, liver metastases, drug intoxication
o Drugs: e.g. verapamil, carbamazepine, phenytoin, erythromycin,
allopurinol, ranitidine.

• Bone disease (Bone ALP):

o Paget's disease, osteosarcoma, bone metastases of prostatic cancer (High /

very high ALP values)
o Other bone metastases
o Fractured bone
o Multiple myeloma (only when associated with fractures)
• Skeletal involvement of other primary diseases:
o Osteomalacia, rickets, vitamin D deficiency, (Moderate rise)
o Malignant tumors (ALP originating from tumor)
o Renal disease (secondary hyperparathyroidism)
o Primary hypothyroidism
• Polycythemia vera
• Myelofibrosis
• Leukemoid reaction to infection
• Women using hormonal contraception
• Pregnancy
• Biliary obstruction
• Transient hyperphosphatasaemia of infancy: benign, often associated with
infection

Lowered levels (hypophosphatasemia):

• Hypophosphatasia, an autosomal recessive disease

• Postmenopausal women receiving estrogen therapy because of osteoporosis
• Men with recent heart surgery, malnutrition, magnesium deficiency,
hypothyroidism or severe anemia
• Children with achondroplasia and cretinism
• Children after a severe enteritis
• Pernicious anemia
• Aplastic anemia
• Chronic myelogenous leukemia

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 9. GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme in the pentose phosphate

pathway. G6PD converts glucose-6-phosphate into 6-phosphoglucono-δ-lactone and is
the rate-limiting enzyme of this metabolic pathway that supplies reducing energy to cells
by maintaining the level of the co-enzyme nicotinamide adenine dinucleotide phosphate
(NADPH). The NADPH in turn maintains the supply of reduced glutathione in the cells
that is used to mop up free radicals that cause oxidative damage. The G6PD / NADPH
pathway is the only source of reduced glutathione in red blood cells (erythrocytes). The
role of red cells as oxygen carriers puts them at substantial risk of damage from oxidizing
free radicals except for the protective effect of G6PD/NAPDH/glutathione.

People with G6PD deficiency are therefore at risk of hemolytic anemia in states of
oxidative stress. Oxidative stress can result from severe infection and from chemical
exposure to medication and certain foods.

10.CERULOPLASMIN:

Ceruloplasmin (or caeruloplasmin) is officially known as ferroxidase or

iron(II):oxygen oxidoreductase. It is the major copper-carrying protein in the blood,
and in addition plays a role in iron metabolism. Like any other plasma protein, levels
drop in patients with hepatic disease due to reduced synthesizing capabilities.

Decreased levels:

Lower-than-normal ceruloplasmin levels may indicate:

• Menkes disease (Menke's kinky hair syndrome) (very rare)

• Wilson's disease (a rare copper storage disease)
• Overdose of Vitamin C
• Copper deficiency
• Aceruloplasminemia

Elevated levels:

Greater-than-normal ceruloplasmin levels may indicate:

• Pregnancy
• Lymphoma
• Acute and chronic inflammation (it is an acute-phase reactant)
• Rheumatoid arthritis

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 11.ACID PHOSPHATASE

Acid phosphatase is a phosphatase, a type of enzyme, used to free attached phosphate

groups from other molecules during digestion. It is basically a phosphomonoesterase. It is
stored in lysosomes and functions when these fuse with endosomes, which are acidified
while they function; therefore, it has an acid pH optimum.

Different forms of acid phosphatase are found in different organs, and their serum levels
are used as a diagnostic for disease in the corresponding organs. For example, elevated
prostatic acid phosphatase levels may indicate the presence of prostate cancer.

12. PROSTATE SPECIFIC ANTIGEN (PSA)

Prostate specific antigen (PSA) is a protein produced by the cells of the prostate gland.
PSA is present in small quantities in the serum of normal men, and is often elevated in
the presence of prostate cancer and in other prostate disorders. A blood test to measure
PSA is the most effective test currently available for the early detection of prostate
cancer. Rising levels of PSA over time are associated with both localized and metastatic
prostate cancer.

Diagnostic use:

PSA is normally present in the blood at very low levels. The reference range of 0-4.0
ng/mL —the upper limit of normal is much less than 4 ng/mL. Increased levels of PSA
may suggest the presence of prostate cancer. However, prostate cancer can also be
present in the complete absence of an elevated PSA level, in which case the test result
would be a false negative. Obesity has been reported to reduce serum PSA levels.
Delayed early detection may partially explain worse outcomes in obese men with early
prostate cancer.

PSA levels can be also increased by prostate infection, irritation, benign prostatic
hyperplasia (BPH), and recent ejaculation, producing a false positive result.