Enzyme Anabolism And Catabolism

RELEVANCE OF ENZYMES IN CHEMICAL PATHOLOGY

BY

WOYO, RAY CHIBUZOR

DE: 2011/2734

A SEMINAR PRESENTED TO THE DEPARTMENT OF MEDICAL LABORATORY SCIENCE (CHEMICAL PATHOLOGY OPTION), FACULTY OF SCIENCE, RIVERS STATE UNIVERSITY OF SCIENCE AND TECHNOLOGY, NKPOLU, PORT HARCOURT.

DR. DAVIES, T. G.

SUPERVISOR

DR. AMALA, S. E.

SEMINAR CO-ORDINATOR

MARCH, 2016

SUMMARY

Enzyme anabolism and catabolism is an important biological process that is essential for the survival of all specie. Their specific function is to speed up the rate at which chemical reactions process. Enzyme play critical role in the metabolic activities of all living organisms whether human animals plants or microorganisms and are widely applied in microbial technology and are widely applied in microbial technology and their diagnosis process. Abnormality of enzyme metabolism lead to a number of metabolic diseases. It is shown that many diseases associated with many component of enzyme metabolism system are now wisely applied in clinical examination as special marker for disease. An interesting discovery suggesting that new roles of enzymes as a potential link that associates to prevent metabolic disorders.The aim of this review is to discuss the relevance of enzyme in chemical pathology, general diseases of the enzyme, enzyme as diagnostic tools and enzyme as a reagents.

TABLE OF CONTENT

Pages

Title page i

Summary ii

Table of Content iii

CHAPTER 1

Introduction 1

CHAPTER II

2.1 Metabolism Diseases 4

2.2 Enzyme Defects Metabolic Disorders 5

2.3 Approaches to Treatment 6

2.4 Disorders of Mitochondrial Oxidative Metabolism 7

2.5 Disorders of Amino Acid Metabolism Phenlketonuria 8

2.6 Alkaptonuria 9

2.7 Disorders of Organic Acid metabolism 9

2.8 Disorders of Fatty Acid Metabolism hyperlipidemia and

Hypercholesterolemia 10

2.9 Disorders of Carbohydrate Metabolism 10

2.10 Glycogen Storage Disease 10

2.11 Galactosemia 11

2.12 Disorders or Purine and Pyrimidine 11

2.13 Lysosomal Storage Disorders 12

2.14 Gausher’s Disease 12

2.15 Tay-Sachs Disease 13

2.16 Enzymes as Markers for Disease 15

2.17 Relevance to Chemical pathology 19

2.18 Enzymes of Clinical Importance 21

2.19 Enzymes Used in Immunoassays 21

CHAPTER 3

3.1 Conclusion 24

3.2 Recommendations 24

References 25

CHAPTER 1

INTRODUCTION

Enzymes are biological catalyst that speed up the rate of chemical reaction without suffering a permanent harm. They are found within the living cell.

Most enzymes are present in cells at much higher concentrations than in plasma. Some occur predominantly in cells of certain tissues, where they may be located in different cellular compartments such as the cytoplasm or the mitochondria. 'Normal' plasma enzyme levels reflect the balance between the rate of synthesis and release into plasma during cell turnover, and the rate of clearance from the circulation.

Plasma contains several enzymes, some of which are functional in the plasma, while others are merely present in plasma due to leakage from tissues. Lipoprotein lipase, psuedocholinesterase and enzymes concerned in the coagulation of blood and the dissolution of the blood clot are enzymes that serve a function in the plasma. Though many other enzymes have no function in the plasma, they are still useful as diagnostic tools. Measurement of their levels in plasma offers valuable information about diseases involving the tissue of their origin.

It is easier to measure enzyme activity in body fluids, by monitoring changes in either substrate or product concentrations, than to measure enzyme protein concentration directly. (Drolet, et al., 2007).

Scientifically and functionally enzymes are biological catalyst, they seed up chemical reactions that takes place within the living cells, without themselves suffering any permanent harm or change. The reactant of enzyme catalysed reaction are termed substrate and each enzyme is quite specific in character, acting on a particular substrate to produce a particular product or products.

ENZYME CLASSIFICATION

Previously many enzymes were given trival names.th trival names consisted of the suffix – ase added to the substrate acted on (e.g. urease).

Presently the accepted nomenclature of enzyme is that recommended by enzyme commission (set up in 1955 by the InternationalUnion of Biochemistry in consultation with the international union of pure and applied chemistry.e to the formation of six (6) distinct classes of the enzyme namely

Oxidoreductase

Transferase

Hydrolase

Lyases

Isomerase and

Ligases or synthetases.

CHEMICAL PATHOLOGY

Chemical pathology also known as clinical chemistry, clinical biochemistry or medical biochemistry is the area of clinical pathology that is generally concerned with analysis of body fluids.

The discipline originated on the late 19th century with the use of simple chemical test for various component of blood and urine.

Subsequent to this other techniques were applied including the use and measurement of enzyme activity, spectrophetometry, electrophersis and immuno assay. All biochemical test come under chemical pathology, these are performed on any kind of body fluid, but mostly on serum or plasma, serum is the yellow watery part of blood that is left after blood has been allowed to clot and all blood cells have been removed. This is mostly done by centrifugation, which pack then denser blood cells and platelet to the bottom of the centrifuge tube. Leaving the liquid serum fraction resulting above the packed cells.

Plasma is in essence the same as serum, but it is obtained by centrifuging the blood without clotting (contains anticoagulant and fibrin). Although, the type of test required by the physician dictate what type of sample is used.

The large array of test can be further subcategorized into sub-specialties’ of:

General or routine chemistry test commonly ordered i.e. liver and kidney function test.

Special chemistry – Elaborate techniques such as electrophoresis and manual testing materials.

Clinical endocrinology – The study of hormone and diagnosis of endocrine disorders.

Toxicology – The study of drugs of abuse and other chemical

Therapeutic drug monitoring – Measurement of the therapeutic medication levels to optimize dosage.

Limaclysis – Chemical analysis of urine for a wide array of disease along with other fluids such as CSF and effusions.

Fecal analysis – Mostly for detection of gastrointestinal disorders.

CHAPTER 2

GENERAL DISEASE OF ENZYME

2.1 Metabolic Diseases

Metabolism is the sum of the chemical process and interconversions that take place in the cells and the fluid of the body. This includes the absorption of nutrient and minerals, the breakdown and buildup of large molecules and interconversion of small molecules and the production of energy from chemical reaction.

Virtually, every chemical steps of metabolism is catalysed by an enzyme. Disorders of this enzymes that results from abnormalities in these genes are known as inborn errors of metabolism. Inborn errors of metabolism were first discovered Sir Archibald Garrod, a British physician who noted in 1902 that the principles of Mendelian Inheritance applied to certain examples of human metabolic variation. He perceived the genetic basis for a particular metabolic conditions that leads to visible effects –alkaptonuma. Alkaptonurian = A recessive metabolic anomaly marked by ochronosis and presence of alkapton by the urine.

Alkapton = an acid formed as an intermediate product of the metabolism of tyrosine and phenglalanine.

Ochronosis – an accumulation of dark pigment in cartilage and other connective tissue usually a symptom of alkaptonuria or phend poisoning, since then, more advanced chemical methods have allowed the discovery of hundreds of enzyme defect that sex metabolic diseases.

Enzymes are protein that controls the rate of chemical reaction in the cells. In general enzyme control the rate of only one or a few reactions. Enzymes formation by binding to the molecules to be reacted (called substrate or precursors) and altering their chemical bonds, producing product. The binding occurs on the surface of the enzyme, usually in a packet groove called active site. The enzyme release the product after reaction, the active site has a specific three dimensional structure that is required for binding substrate. In addition, it may have other sites that bind regulatory molecules or cofactors. Some cofactor re vitamins, which perform some accessory function, critical for enzyme action.

Enzymes are often linked in multistep pathways, such that the product of one reaction becomes the substrate for another. In this way simple molecule can be changed step by step into a complex one,, or vice versa. In addition, the multiplestep provide additional level of regulation and intermediates can be shunted into other pathways to make other products. For instance, some intermediate in the breakdown of sugar can be sunted to make amino acids. When all the enzymes in a pathway are functional, intermediate rarely build up to high concentrations.

2.2 Enzyme Defeacts Metabolic Disorders

The causes of enzyme defects are genetic mutations that affect the structure or regulation of the enzyme protein or create problems with the transport, processing, or binding of cofactor. In general, the consequences of an enzyme deficiency are due to perturbation of cellular chemistry, because of either a reduction in the amount of an essential product, the buildup of toxic intermediates or the production of a toxic side-product shown most metabolic disorders are inherited as autosomal recessive condition. In this inheritance pattern two defective gene copies are needed (one from each parent) to develop the disease. The parents each of whom almost always has only one gene copy will not have the disease but are carriers. The chance that two carriers parents will have a child who inherits two defective gene copies is 25 percent for each birth. Metabolic disorders tend to be recessive, because they are due to inactrelating, or less-of-function, mutations. One working copy of the gene is usually enough to maintain sufficient levels of the enzyme and so with one copy present, no disease develop.

2.3 Approaches to Treatment

Treatment approaches for met

Major classes of metabolic disorders cells are constructed from four major types of molecules: carbohydrates, proteins, fats and nucleic acids. The metabolic pathways involving each are the basis for classification of many of the metabolic disorders. The mitochondria in cells are organelles that play a major role in most metabolic disorders. Carbohydrate are used primarily as fuel and can be built and broken down rapidly. The major storage form is glycogen. They are also added to proteins to make glycoproteins. Fatty acids are long chain molecules that are used to construct membranes. Fatty are derived from dietary fats. Excess fat is sued as fuel by mitochondria. Proteins are made of amino acids.

Human must eat eight types of amino acid and then convert these into twelve other types of amino acids or make up the twenty amino acids found in our protein. Excess amino acid in the diet are used for fuel by mitochondria. Along the way, they generate organic acids.

Nucleic acids – DNA, RNA are the molecules that store and process genetic information. They must be built from smaller units, called nucleotides. The storage and interconversion o different types of nucleotides assures a steady supply.

2.4 Disorders of Mitochondrial Oxidative Metabolism

Most cellular energy is derived from mitochondrial electron transport chain, which reduces ocyten to water in a series of steps to derived the formatin of the high energy-compound ATP. The kerbs cycle create high energy intermediates that it feels to the electron transport chain, the energy of which ultimately is derived from a two carbon compound called acetate which is broken down successfully to carbon dioxide. Acetate is derived from several pathways of amino acid, carbohydrate and fat metabolism,

Thus, many pathways of metabolism feeds into the kreb cycle to drive oxidative metabolism in a web of processes requiring hundreds of enzyme. When there are defects in the krebs cycle or electron transport chain, one resultmay be ketoacidoses which is due to the allcumulation of lactic acid and ketone bodies. The lack of cellular energy may be manifest in many cellular processes and can affect several tissue and organ system, particularly those that are mostlydependant upon oxidative metabolism for energy. The brain and music are generally affected first, which can cause developmental delay, neurological crises including episodes of coma, stroke-like events and seizures and muscle weakness or cardiomyopathy. Kidney function, most often the tubular function required for retention of electrolytes may also be affected. Endocrine system may also be affected, resulting in conditions such as diabetes mellitus (caused by effects on the pancreas or by sensitivity to insulin in muscle and fat cells) or adrenal insufficiency (from effects on the adrenal glands).

Disorders of mitochondria oxidative metabolism are very variable in terms of age of onset, severity, specific symptoms and clinical course. Even the inheritance patients of mitochondria diseases are heterogeneous most are inherited from defects in the mitochondria DNA, which is passed on to the maternal line.

The mitochondria contains a circular chromosome of about 165000 bases. The codes for thirty component of the electron transport chain, as well as transfer RNA molecules and ribosomal RNAs required for their expression. Since there are multiple copies of the mitochondria DNA and there may be mixtures of normal and abnormal mitochondria DNA (a phenomenon known as heteroplasma). The precise proportion of mutated mitochondrial DNA vary in an unpredictable manner from individual to individual within a family and from tissue to tissue within an individual tissue overtime, adding to the unpredictability of mitochondrial disease and the difficulty in the diagnosis.

2.5 Disorders of Amino Acid Metabolism Phenlketonuria

Phenylketonuria (pku) is the most common disorder of amino acid metabolism and it is a paradigm for effective newborn screening.

Phenylaanine hydroxylase reaction which converts phenglanine to another amino acid tyrosine. A genetic defect in the phenylalanine hydroxylase enzyme is the basis of classical pku. Untreated PKU results in severe mental heteradiation, but pku can be detected by screening new born blood spots and the classical form can be very effectively treated by using medical formulas that are limited in their phenylanine content.

The hydroxylase enzyme requires a co-factor called biopterin which is also a cofactor for other enzymes, defects affecting the production of biopterin result in anester form, so called malignant pku. In this form the other biopterin – dependent hydroxylase are also affected resulting in deficient neurotransmitter synthesis and significant neurological symptoms.

2.6 Alkaptonuria

Alkaptonuria is a disorder of tyrosine breakdown the intermediate that accumulates called homogentisic acid, can polymerize to form pigment that bind to cartilage and causes progressive arthritis and bone disease and that also is excreted to darken the urine the effect that allowed Garrod to recognize the genetic inheritance of this inborn error of metabolism.

2.7 Disorders of Organic Acid Metabolism

Propionic Acidemia – PropionylcoA is formed mainly from the breakdown of four essential amino acids (isoleucine, valine, theronine, and methoionine). Defect of the enzyme propionyl-CoA carboxylase results in propionic academia, a life threating disease characterized by episodes of generalized metabolic dysfunction and ketoacidosis. The basis of treatment is a carefully applied diet containing limited amount of the amino acids that are precursors to propionyl-CoA.

Methylamine Acidemia– Methylmalonyl–coA is the product of propionyl-coA carboxylase. There are a variety of metabolic defects in the ferther metabolism of this compound, resulting in methylmalonicacedemia. The best known of these condition arises from defect in methylmalonyl-CoA mutase, the vitamin B12 dependent enzyme that converts methylmolony-CoA to succnglCoA, which enters the kreb cycle. There are other conditions resulting in methylmalonic academia that are due to defects in the enzyme system involved in vitamin B12 metabolism. In some cases, supplementation with large doses of vitamin B12 is effective but in most cases of methylmaonic academia, a special diet is required, similar to that used to treat propionce academia.

Disorders of Fatty Acid Metabolism Hyperlipidemia and hypercholesterolemia

Dietary facts are distributed through the body attached to proteins, in lipoprotein complexes. There are a number of disorders involving the regulation or utilization of lipoproteins, which result in hyperlipidemia and or hypercholesterolemia including the common conditions in adult that are associated with cardiovascular disease. Standard treatment approach include modifying the diet and administration drugs that inhibits fatty acid synthesis.

2.9 Disorders of Carbohydrate Metabolism

The most active pathways in carbohydrate metabolism are glucogenolysis (the breakdown of glycogen, a polymerized form of carbohydrate, which is store primarily in the liver and muscle), which produces glucose and distributes it through the bloodstream and glucoses, which releases energy produces private. Pyruvate is a three carbon molecule that can be converted acetate and enter the Kreb cycle or form several building block molecules. The reverse processes are referred to as glycogen synthesis and gluconeogenesis.

2.10 Glycogen Storage Disease

A number of defects may occur in glycogenolysis, given rise to the disorders known as glycogen storage disease. Glycogen storage disease may affect the liver (enlarging of or damaging it due to increased amount of glycogen) or muscle (weakening muscle or causing breakdown during times of exercise, due to inadequate glucose production). There may be additional problems including disturbed kidney tubular function (which causes loss of nutrients and minerals), and there is a risk of brain damage in cases that result in critically low blood sugar.

2.11 Galactosemia

Another common disorder of carbohydrate metabolism is galatosemia, which is due to the inability to form glucose that is found in milk. The classic form of galatisemia is due to a deficiency of the enzyme galatose-1-phospho uridyltransferase, and if untreated, it presents in infant with fatal liver failure. Galactose is important because new born screening (conducted by most developed countries on blood spots collected in the first days of life has been vary successful and simple alteration of the diet replacing milk with formula’s that contain glucose or glucose polymers) has permitted a generation of individual to survive with quite normal lives and in general normal intellect.

2.12 Disorders or purine and pyrimidine

Metabolism – Purine and pyrimidine are chemicals that form the nucleic acids (DNA and RNA). An important purin compound is adenosine triphophate (ATP) which is used to transfer chemical energy for processes such as biosynthesis and transport. There are several rare defects in the synthesis of pruines and pyrimidines. The most common symptom of purine overproduction is gout, which arises for several reasons, often not associated with an identifiable enzyme defect before rather due to an imbalance between pruine synthesis and disposal gout manifests when the ultimate product of purine degradation, uric acid, accumulate and crystallize in joints.

A very dramatic disorders of purine metabolism is Lesch-nyhan syndrome, which is due to a defect in the enzyme hypoxanthine phosphoribosyltransferase (HPRT), resulting in defective salvage of purines and accordingly, in an increase in the excretion of uric acid. For reasons that are still incompletely understood, a severe defect in HPRT also causes brain.Neurotransmitter dysfunction, resulting in a severe spastic form of movement disorders and also a stereotypical compulsion for self injurious behavior. Theconcentration of uric acid can be reduced by using the drugalluprenol, but there is no satisfactory treatment for the neurological symptoms associated with Lesh-rghan disease.

2.13 Lysosomal Storage disorders

Lysosome are intracellular compartment in which macromalecudes broken down in an acidic environment various class of lysosomal storage disorder arise when there are detects in specific enzymes and the mantestion of these disorders depend upon the class of macromolecule whose breakdown is affected.

2.14 Gaucher’s Disease

The most common lysosomal storage disorder is Gaucher disease, caused by a deficiency of enzyme cerebrosidase which is needed to breakdown cerebrosidase which is needed to breakdown cerebroside a component of the cell membrane in blood cells and neuron. Partial defect of cerebrosidase cause type 1 Gaucher’s disease, in which material accumulate in the lysosome of microphage cells in spleen, iliver and bone marrow where most of the cell turnover takes place. Significant accumulation usually occurs by childhood or early adulthood, resulting in dramatic enlargement of the spleen and liver. Later there may be painful and crippling effects on the bones. Type 1 Gauchers disease can be effectively treated with enzyme replacement, but the enzyme must be infrad intravenously approximately every two weeks for life. More severe defect or cerebrasidase cause type 2 Gauchers disease, which is rare appears in infancy and present with the same problems as in type 1 disease as well as death. Very rarely, defects of intermediate severity can give rise to type 3 Gaucher disease, which is a chronic neuropathic form.

2.15 Tay-Sachs Disease

Tay-Sach disease is due to a defect in the beta-hexosaminidaseA enzyme, which removes sugar from certain lipids called ganglosides, which build up in the lysosome. The disease causes neurological symptoms, and enlarged head and death in early childhood.

Mucopolysacharidosis – Mocopolysacharidoses are lysosomal storage disorders affecting the breakdown of mucopolasaccharides, which are carbohydrate –protein mascromolecules found on several cell type Harler syndrome (a-iduronidase deficiency) and Hunter syndrome (iduronatesultalase deficiency) are two disorders that affects the breakdown of the mucopolysacchraidesdermationsulphate and heparin sulphate which are component of connective tissue throughout the body. The usual clinical manifestations of these syndrome are enlargement of laser and spleen, skeletal deformitesm coarse facial features, staff joints and methl retardation. Most cases are severe and progress to death within five to fifteen year, but there are exceptions. By 2002, there were several experimental approaches with enzyme replacement for mucoplysaccheridoses.

ENZYME AS A DIAGNOSTIC TOOLS

The Relevance of Enzyme as Diagnostic Tools

Diagnostic Enzyme of Importance

AST – Aspartate aminotrasnferase

ALT – Alanine aminotransferase

ACP – Acid Phospahatase

ALP – Alkaline phospahase

GG – Gamma glutamytransferase

LDH – Lactate dehydrogenase

CK/CPK – Creatine kinase/Creatinephospoh kinase

2.16 Enzymes As Markers For Disease

Some enzymes are found only in specific tissues or in a limited number of such tissues. For example, lactase dehydrogenase (LDH) has 2 different forms, called isozymes, in heart and skeletal muscle. Two forms differ slightly in amino acid composition and Can be separated on the basis of charge as a result. Since LDH is a tetramer of four subunits, it too can exist in 5 different forms depending on the source of the subunits. An increase of any form of LDH in the blood indicates some kind of tissue damage. Aheart attack can usually be diagnosed with certainty if there is an increase of LDH from heart. Also, there are different forms of Creatine Kinase (CK), an enzyme that occurs in the brain, heart and skeletal muscle. Appearance of the brain type can indicate a stroke or a brain tumour, whereas the heart type indicates a heart attack. After a heart attack, CK shows up more rapidly in the blood than LDH. Monitoring the presence of both enzymes extends the possibility of diagnosis, which is useful, since a very mild heart attack might be difficult to diagnose. An elevated level of the isozyme from heart in blood is a definite indication of damage to the heart tissue (Drolet et al., 2007).

Another useful enzyme assayed is acetyl cholinesterase (AChE), which is important in controlling certain nerve impulses. Many pesticides affect this enzyme, so farm workers are often tested to be sure that they have not received inappropriate exposure to these important agricultural toxins. There are several enzymes that are typically used in the clinical laboratory to diagnose diseases. There are highly specific markers for enzymes active in the pancrease, red blood cells, liver, heart, brain, prostate gland and many of the endocrine glands. Since these enzymes are relatively easy to assay using automated techniques, they are part of the standard blood test veterinary and medical doctors are likely to need in the diagnosis and treatment/management of diseases.

Some enzymes are found only in specific tissue or in a limited number. In such tissue for example lactase dehydrogenase (LDH) has two different forms called isozymes. In heart and selectal muscle. Two forms differ slightly in amino acid composition and can be separated on the basis of change as a result since LDH is a tetramer of four subunits, it too can exist in 5 different form depending on the source of subunits.

An increase in any form of LDH in the blood indicates some kind of tissue damage. A heart attack can usually be diagnosed with certainty if there is an increase of LDH from heart, also, there are different form of creatine, kinase (ck), an enzyme that occurs in the brain, heart and skeletal muscle. Appearance of the brain type can indicate strike or brain tumour whereas the heart type indicates a heart attack.

After a heart attack, ck shows up more rapidly in the blood than LDH monitoring the presence of both enzyme extends the possibility of diagnosis, which is useful, since a very mild heart attack might be difficult to diagnose. An elevated level of isozyme from heart in blood is a definite indication of damage to the heart tissue (Dreletet al., 2007). Another useful enzyme assay is acetylecholanesterase (Ache) which is important in controlling certain nerve impulses. Many pesticides affect this enzymes so form workers are often tested to be sure that they have not reserved inappropriate exposure to these important agricultural toxins.

There are several enzymes that are typically used n clinical laboratory diagnosis. There are highly specific morters for enzyme activity in the pancrease red blood cells, liver, heart, brain, prostate gland and many of the endocrine gland.

The measurement of serum level f numerous enzyme has shown to be of diagnostes significance. This is because the presence of these enzyme in the serum indicate that tissue damage has occurred resulting in the release of intracellular component into the blood (Michettiet al., 1998). Hence, when a physician indicate that he or she is going to assay for liver enzymes, the purpose is to ascertain for the potential of liver cell damage commonly assayed enzymes are the amino transferase. Alanine transaminase, ALT (sometimes still refereed to as serum glutamate pigment chlino-transferase, SGOPT) and aspertate aminotransferase, AST (also referred to as serum glutmerase oxalate aminotransferase SGDT) lactase dehydrogenase LDH; creatine kinase ck, (also called creatinephosphotinese, CPK), gamma-glutamyltranspestide GGT. Other enzyme are assayed under a verity of different clinical situation many enzymes are indicated in clinical diagnosis of various disease in human (Nordsenet al., 1986).

The enzymes facilitate or enhance rapid diagnosis of these disease. These enzymes could be classified into many classes, they are: Alkaline phosphate, these enzymes were the earlier enzymes to be recognize to have clinical significance when in the 19200, it was discovered that they increase in bone and liver disease since there may have been the subject of more publication than any other enzyme (Rome et al., 2003). Alkaline phosphates are a group of iso forms which hydrolyse many type phosphate esters, whose natural substrate or substrate are unknown. The term alkaline refers to the optimal alkaline pH of this class of phosphatase investors in both human and animal, the major source of ALPS are the liver, bone, kidney and placenta. In human it is involved in bone and hepatoballwry disease.

Creatinine kinase isoenzyme are the most organ specific serum enzymes in clinical use, they catalyse the reverablephosporglation of creatine by ATP to form creatinephosperate, the major storage form of high energy phosphate required by muscle. Cereatine kinase are found in many part of the body like the heart, brain, skeletal muscle and smotie muscle, but they have their highest specific activity in the skeletal muscle (Aksemosaet al., 2000). In human creatine kinase is associated with myocardiacinfaction and muscle diseases. Increase in creatine kinase in cerebiospinal fluid has been associated with a number of disorder creatinine kinase are such sensitive, inducetors of muscle damage that generally only large increase in serum activity are of clinical significance alanine aminotransferase; it was formerly known as glutamic pynaeatetransmainase (GTP). It catalyses

2.17 Relevance to Chemical Pathology

Clinical application of enzyme assay

The diagnose usefulness of measuring plasma enzyme activities lies in the fact that changed in their activities provide very sensitive indicators of tissue damage or cell proliferation. These changes help on detect and in some cases, locate the tissue damage but also to monitor treatment the progress of disease. However, these usefulness is diminished by the lack specificity i.e. the difficulty of identifying an increase enzyme activity with tissue which has been damaged.

This is because most if not all enzyme are not confined to specific tissue or organs, neither most of them are widely distributed to different tissue, and their plasma activities may be raised in diseases involving different tissues or one. In practice this problem of lack of specificity is partially surmounted by measuring several parameters which may include several enzymes as the relative concentrations of enzymes in different tissue very considerably for instance although both alanine and aspirate transminese (ALT, and AST) are equally abundant in liver tissue, AST is present in much greater concentration than ALT in a ratio of 20:1 in heart muscle.

Simultaneous measurement of both enzymes can thus provide a clearer indication of the probable site of tissue damage.

The diagnostic specificity can also be enhanced by performing isoenzyme analysis. Isoenzymesare enzymes which have similar catalytic activities but differs in their physical and chemical properties. Physical properties(Electrophoretic mobility,kinetic properties).Chemical properties(sequence of amino and composition). is an analyses of an enzyme of different molecular forms which can catalyse the reaction characteristics of that enzyme with organ specific in origin, e.g. the lactate dehydrogenase (LD) isoenzymes LD is found mainly in the whilst LD occurs mainly in cardiac muscle. Awareness of the time cause of elevation of various enzyme after certain pathological event may be applied judiciously to this additional useful information e.g. the interpretation of the cardiac enzyme profile in post myocardial infection the basis of this is that each individual enzyme has a fairly constant halfe life which is characteristic of that enzyme.

A knowledge of the halfe lives of the different enzyme affected by the acute illness will be of help in assessing the time of its onset. Non-pathological factors affecting result of plasma enzyme assays. There are number of factors other than disease process that can affect the result of plasma enzyme assay.

Perturbation – The state of feeling anelous about something that has happen change in quantity, behavior, movement and also change in temperature. Therefore changes in the body plasma enzyme may seen in conditions which are not as a result of disease, e.g. creatine kinase (ck) increase in serum after exercise, and also in pregnancy the ALP isoenzyme will increase due to placenta contributions. The formation of complex with other protein such as immunoglobulins e.g. LD (cheasatedehydrogenese) and ck complex with IgG, so this may delay there clearance from the circulation and raise their plasma activities in the absence of liver r renal disease.

Most enzyme assays in current usage measures the enzyme activities rather than there concentration. Analytical variations will affect the results of such assay, introducing bias and in precisions such as the choice of substrate cofactor and product concentrations the type and strength of buffer, the assay pH and reaction temperature as well as the spurious (false but seerning to be genure) resece of inhibitor, activators and interparents in the sample or reagent are the main causes of analytical variability.

The pharmacological effects of drug taken by the patient may also affect the result e.g. many drugs including the anticonulsantphenytom and Phenobarbital cause element plasma GGT activities via microsomal induction.

Plasma enzyme results must be interpreted bearing in mind these and other relevant points, before attributing a change in plasma enzyme activity to a specitic disease process.

2.18 Enzymes of Clinical Importance

Alkaline phosphatase (ALP) ALP is not a single enzyme, rather it is the generic name for group of enzymes that catalyse hydrolysis of phosphoric acid mono-esters at alkaline pH of (9.0 – 10.5) it is found in most tissue having one or more iso-enzyme or isoforms. The clinicalimportantsisoenzymes are those of the bone (osteoblast) the liver cell living the biliary canaliculi) the intestine (epithelia cells) the kidney (renal tabular cells) and the placenta.

ENZYME AS DIAGNOSTIC REAGENT

2.19 Enzymes Used In Immunoassays

Enzymes may also be used as an alternative to radioisotopes as markers in immunoassays have been used for the determination of a variety of proteins and hormones. The role of enzymes in mmunoassay used to replace radioisotopes as markers, since they are not hazardous to health and can be detected by techniques which are more generally available. Any enzyme with a sensitive and convenient assay procedure can be used for this purpose. Two common examples of enzyme immunoassay (EIA) procedures are enzyme-linked immunosorbent assay (ELISA) and Enzyme-Multiplied Immunoassay Test (EMIT). ELISA is a highly sensitive assay that can be used to detect either antigen or antibody. Applications of ELISA include diagnostics for noninfectious diseases involving hormones, drugs, serum components, oncofetal proteins, or autoimmune diseases, as well as diagnostics for infectious diseases caused by bacterial, viral, mycotic or parasitic organisms. The enzymes frequently used in ELISA are Horseradish peroxidase, alkaline phosphatase and βgalactosidase. In EMIT, the activity of malate dehydrogenase is assayed by standard enzyme methodology for the detection of thyroxine by Enzyme-labelled immunoassay.

Reaction of glucose oxidase reagent with glucose

Chapter 3

3.1 Conclusion

Most enzymes are present in the cell at a much higher concentration than in plasma. Some occur predominantly in cells of certain tissue where they may be located in different cellular compartment such as cytoplasm or the mitochondria. Normal plasma enzyme levels reflect the balance between the rate of synthesis and release into plasma during cell turn over, and the rate of clearance from tissue.

Plasma contains several enzyme some of which are functional in the plasma and other merely present due to leakage from tissues. The measurement of there levels in plasma offers valuable information about diseases involving the tissue of their origin.

3.2 Recommendations

Since enzymes are affected by certain physicological factors such as pH, temperature, substrate concentration and inhibitors, therefore, it is recommended that enzymes assay should be done with the following parameters being put into consideration.

Because of enzyme specificity it is recommended that enzymatic assay is employed in clinical chemistry to give out a consistent result.

There are wide use of enzyme such as industrial use, medicinal use, and in research. It is also recommended that a good knowledge of enzyme and their behaviour should be known by pathologist to come up with a universal acceptable result.

REFERENCES

Abir, F. S., Alva, L., Kaminski, L., Donald A. and E. Walter, (2005). The role of arachidonic acid regulatory enzymes in colorectal disease. Dis. Colon Rectum, 48, 257-303.

Aksenova, M., Butterfield, D.A. and Markesbery, W.R. (2000). Oxidative modification of creatine kinase BB in Alzheimer`s disease brain. Journal of Neurochemistry, 74, 2520-2527.

Bittinger, M.A., Nguyen, L. P. and Bradfield, C.A. (2003). Aspartate aminotransferase generates proagonists of the aryl hydrocarbon receptor. Molecular Pharmacology, 64. 550-556.

Chatterjea, M. N. and Shinde, R. (2002). Textbook of Medical Biochemistry, (5th Edn.), Jaypee Brothers, Medical Publishers Pvt. Ltd., New Delhi.

Coombs, J., (1992). Dictionary of Biotechnology (2nd Edn.), Stockton Press, New York, USA., pp: 555.

Daini, O.A., (2000). Fundamentals of Genetic Engineering. 1st Edn., Samrol Ventures and Printing Co., Ijebu Igbo, Ogun State, Nigeria, pp: 78-82.

Devlin, T.M., (1986). Textbook of Biochemistry with Clinical Correlations, (2nd Edn.), John Wiley and Sons, Inc., New York, USA., pp: 165.

Drolet, R., M. Simard, J. Plante, P. Laberge, A. and Tremblay, Y. (2007). Human type 2 17 betahydroxysteroid dehydrogenase mRNA and protein distribution in placental villi at mid and term pregnancy. Reproduction Biology Endocrinology, 5, 30-30.

El-Kabbani, O., C., Darmanian. R. and Chung, R.P.T. (2004). Sorbitol dehydrogenase: Structure, function and ligand design. Current Medical. Chemistry, 11, 465-476.

Ellis, J.M., (2005). Cholinesterase inhibitors in the treatment of dementia. Journal of American Osteopathy Association., 105, 145-158.

El-Yassin, H. D. (2012). Plasma in enzymes in diagnosis. Circulation, 2(3), 121 – 125.

Griffith, K.L., Shah, I. M., and Wolf, R.E. (2004). Proteolytic degradation of Escherichia coli transcription activators SoxS and MarA as the mechanism for reversing the induction of the superoxide (SoxRS) and multiple antibiotic resistance (Mar) regulons. Molecular Microbiology, 51(2), 1801-1816.

Gupta, K.B., Ghalaut, R., Gupta, S,. Tandon A. and Prakash, P. (2001). Estimation of serum and pleural fluid amylase and iso-enzyme in cases of malignant pleural effusion. Indian Journal of Tuberculosis, 48, 87-87.

Hoskeri, H.J., Krishna, P. and C. Amruthavalli, 2010. Effects of extracts from lichen Ramalina pacifica against clinically infectious bacteria. Researcher, 2: 81-85.

Jiang, X., L., Jing, V. and Lan, M. (2007). Metabolic enzymes link morphine withdrawal with metabolic disorder. Cell Research., 17, 741-743.

Kaneko, J.J., 1989. Clinical Biochemistry of Domestic Animals. 4th Edn., Academic Press, New York, California, USA., pp: 898.

Kikuchi, H. S. ,Hirose, S. Toki, A., Kazuhito E.and F. Takaiwa, 1999. Molecular characterization of a gene for alanine aminotransferase from rice (Oryza sativa). Plant Molcular Biology, 39, 149-159.

Lone, M. A., Wahid, S. M., Saleem, P., Koul, G.H., Nabi, D.and Shahnawaz, A. (2003). Alkaline phosphatase in pleural effusions. Indian Journal of Chest Disease and Allied Science, 45, 161-163.

Machetti, M., M. Feasi and N. Mordini, M.T. Van Lint and A. Bacigalupo et al., 1998. Comparison of an enzyme immunoassay and a latex agglutination system for the diagnosis of invasive aspergillosis in bone marrow transplant recipients. Bone Marrow Transplantation, 21, 917-921.

Murray, R.K., D.K. Granner, P.A. Mayes and V.W. Rodwell, 2000. Harper`s Biochemistry. 25th Edn., McGraw-Hill Health Professions Division, McGraw-Hill Co., USA., pp: 74-76.

Nduka, N., 1999. Clinical Biochemistry for Students of Pathology. 1st Edn., Longman Nigeria Plc, Ikeja, Lagos, pp: 125-126.

Nielsen, K.H., L. Kelly, D. Gall, S. Balsevicius, J. Bosse, P. Nicoletti and W. Kelly, 1996. Comparison of enzyme immunoassays for the diagnosis of bovine brucellosis. Prev. Vet. Med., 26: 17-32.

Palmer, T., 2001. Enzymes: Biochemistry, Biotechnology and Clinical Chemistry. 1st Edn., Horwood Publishing Ltd., Chichester, West Sussex, England, pp: 345-350.

Porcelli, A. M., A. Ghelli, C. Ceccarelli, M. Lang and G. Cenacchi et al., 2010. The genetic and metabolic signature of oncocytic transformation implicates HIF1{alpha} destabilization. Human Mol. Genet., 19: 1019-1032.

Qijun, W., Y. Zhang, C. Yang, H. Xiong and Y. Lin et al., 2010. Acetylation of metabolic enzymes coordinates carbon source utilization and metabolicflux. Science, 327: 1004-1007.

Raja, A., P. Prabakaran and P. Gajalakshmi, 2010. Isolation and screening of antibiotic producing psychrophilic actinomycetes and its nature from rothang hill soil against viridans Streptococcus sp. Res. J. Microbiol., 5: 44-49.

Reetz, M.T., R. Wenkel and D. Avnir, 2000. Entrapment of lipases in hydrophobicsol-gel materials: Efficient heterogeneous biocatalysts in aqueous medium. Synthesis, 6: 781-783.
Shinoda, T. and K. Itoyama, 2003. Juvenile hormone acid methyltransferase: A key regulatory enzyme for insect metamorphosis. PNAS, 100: 11986-11991

Soares, C.M.F., H.F. de Castro, M.H.A. Santana and G.M. Zanin, 2001. Selection of stabilizing additive for lipase immobilization on controlled pore silica by factorial design. Applied Biochem. Biotechnol., 91: 703-718.

Stenesh, J., 1989. Dictionary of Biochemistry and Molecular Biology. 2nd Edn., John Wiley and Sons, Inc., Hoboken, New Jeresey.

Van Roon, J., R. Beeftink, K. Schroen and H. Tramper, 2002. Assessment of intraparticle biocatalytic distribution as a tool inrational formulation. Curr. Opin. Biotechnol., 13: 398-405.

Vellard, M., 2003. The enzyme as drug: Application of enzymes as pharmaceuticals. Curr. Opin. Biotechnol., 14: 444-450.

White, J.S. and D.C. White, 1997. Source Book of Enzymes. CRC Press, Boca Raton.