Clinical Enzymology

 

  Clinical Enzymology  Enzymology  11-23/10/2011 1. L ecture 1: The Biochemical Role of En zymes / Introduction 2. L ecture 2: Kinetics of En zyme-Catalyzed Reactions/ Regulation of enzymes by substrate and product concentration. 3. L ecture 3: K Kinetics of Enzyme-Catalyzed Reactions/ Enzyme Enzyme Inhibitors kinetics of Enzyme-Catalyzed Reactions 4. L ecture 4: Plasma Enzymes in Diagnosis 5. L ecture 5: Clinically Important Enzymes and Diagnostic Applications 6. L ecture 6: Enzymes assays

Aim and objective of the above six lectures is to understand: 1. 2. 3. 4.

The biochem biochemical ical role of enzymes enzymes in the met metabolic abolic activity of the h human uman body. body. Kinetics of e enzymes nzymes inhibition inhibition and tthe he role of toxins an and d drugs The d disorder isorder of enzyme enzyme activity in d disease isease Diagnostic application of enzyme enzymes s

References: 1. "Biochemistry" by Lubert Stryer

(textbook) 

2. "Textbook of Biochemistry with Clinical Correlations" by T.M.Devlin (additional reading) 

3. "Lippincott's Illustrated Reviews in Biochemistry" by P.C.Champe, R.A.Harvey and D.R.Ferrier (additional reading) 

4. "Harper's Biochemistry" by R.K.Murray, D.K.Granner, P.A. Mayes and V.W.Rodwell. (additional reading) 

5. "C lin linic ical al Laborato Lab oratorry Sc ienc e Review" Review " By Ro Ro ber be rt R. R. Ha Ha rr 

(additional reading) 

 

 

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The Biochemical Role of Enzymes Introduction

11/10/ 2010 11/10/201 0 Prof. Dr. Hedef D. El-Yassin (Ph.D.,Pos (Ph.D .,Postt Doc torate torate))

Prof.Dr. H.D.El-Yassin 2011

 

 

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Enzyme-Catalyzed Reactions  A. Enzymes are catalysts that increase the rate or velocity,

v, of many physiologic

reactions.  a. In the absence of enzymes, most reactions in the body would would proceed so slowly slowly that life would be impossible.  impossible.  b. Enzymes can couple reactions that would not occur spontaneously to an energy-releasing reaction, such as ATP hydrolysis, that makes the overall reaction favorable.  favorable.  c. Another of the most important important properties properties of enzymes as catalysts is that they are not changed during the reactions they catalyze, which allows a single enzyme to catalyze a reaction many times.  times.  B. Enzymes specifically bind the reactants in order to catalyze biologic reactions. 

a. During the reaction, reaction, the the reactants reactants or substrates are acted on by the enzyme to yield the products. b. Each substrate substrate binds binds at its binding site on the enzyme, which may contain, be near to, or be the same as the active site harboring the amino acid side chains that participate directly in the reaction.  reaction.  c. Enzymes exhibit selectivity or specificity, a preference for catalyzing reactions with substrates having structures that interact properly with the catalytic residues of the active site.  site.  C. A deficiency in enzyme activity can cause disease. 

a. Inherited absence or mutations in enzymes involved in critical critic al metabolic pathways

eg, the urea cycle or glycogen metabolism

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are referred to as inborn

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errors of metabolism. If not detected soon after birth, these conditions can lead to serious metabolic derangements in infants and even death. death.   b. An enzyme deficiency can produce a deficien deficiency cy of the product of the reac r eaction tion it catalyzes, which may inhibit other reactions that depend on availability of that product.   product. c. Accumulation of the substrate or metabolic byproducts of the substrate due to an enzyme deficiency can have h ave profound physiologic consequences. consequences.   d. Most inborn errors of metabolism metabolism manifest after birth because the the exchange of metabolites between mother and fetus provides for fetal metabolic needs in utero. utero.   e. Therapeutic Therapeutic strategies strategies for enzyme deficiency deficiency diseases diseases include dietary modification modification and potential gene therapy or direct enzyme replacement (Table). Prof.Dr. H.D.El-Yassin 2011

 

 

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Table: Example of enzyme replacement replacement therapy for inherited disease

Disease

Enzyme deficiency

Normal Function of the Enzyme

Major Symptoms Or Findings on Examination

Physiologic Consequences and Prognosis

Weakness, fatigue, failure to thrive, lethargy

Glycogen accumulation in several organs, including heart and skeletal muscle Congestive heart failure  Accumulation of gluco Accumulation cerebroside in several organs, reduced lung and brain function, pain in upper trunk region, seizures, convul c onvulsions sions

Pompe disease

 Acid  -1,4-1,4- Hydrol Hydrolysis ysis of glucoglycogen sidase

Gaucher disease

GlucoCerebrosidase

Hydrolysis of the Hydrolysis glycollipid, glucocerebroside, a product of degradation of RBCs and WBCs

Easy bruising, fatigue, anemia, reduced platelet count

  -Galacto-Galactosidase A

Hydrol Hydrolysis ysis of the lipid, globotri aosylceramide

Severe paresthesi  Accumulation tion of  painful pfatigue, aresthesias as  Accumula globotriaosylcer-amide (numbness and in endothelial cells of tingling) of the feet the blood vessels, and arms, purplish purplish altered cellular structure skin lesions on of heart and glomeruli, abdomen and renal failure buttocks

Fabry disease

Prof.Dr. H.D.El-Yassin 2011

 

 

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Enzyme Classification 1. Enzymes can be made of either protein or RNA. 2. Most enzymes enzymes are proteins, which are grouped grouped according according to the six types of reactions they catalyze.. The top-level classification is

Class Name 1

Trivial Names and Trivial examples

Oxidoreductases Dehydrogenases Reductases

Type of reaction catalyzed  Addition or o r subtraction of electrons

Oxidases 2

3

Transferases

Hydrolases

KinasesPhosphotransferases  Aminotrans ferases  Aminotransferases Glycosidases Nucleases Peptidases

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Lyases

Decarboxylase Dehydratases Hydratases

Transfer of small groups: amino, acyl, phosphoryl, ph osphoryl, one-carbon, sugar  Add water wate r across bon bonds ds to cleave them

 Add the elements of water, ammonia, or carbon dioxide across a double bond (or the reverse reaction)

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Isomerases

Mutases Epimerases

Structural rearrangements

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Ligases

Synthases

Join molecules together

Synthetases

3. Several important physiologic catalysts are made of RNA, and these RNA-based

enzymes or r ibozymes are ibozymes  are of two general types: a. RNA molecules that undergo self-splicing self-splicing,, in which an internal portion of the RNA molecule is removed while the parts on either side of this intron intron   are reconnected.  Prof.Dr. H.D.El-Yassin 2011

 

 

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b. Other RNA molecules that do not undergo self-splicing can act on other molecules as substrates are true catalysts. catalysts. i. Ribonuclease P cleaves transfer RNA precursors to their mature

forms.  ii.

The 23S ribosomal RNA is responsible for the peptidyl transferase activity transferase  activity of the bacterial ribosome. 

4. Isozymes Isozymes are  are protein-based enzymes that catalyze the same reaction but differ

in amino acid composition.  a. Because of their structural s tructural differences, differences, isozymes isozymes may often be

distinguished by separation in an electric field (electrophoresis) (electrophoresis) or  or by reactivity with selective antibodies. b. Several clinical clinical uses have been been made of isozym isozymes es selectively expressed expressed by different tissues. Example:

Diagnos Diag nosis is of Hea Heart rt Atta Attack ck and Musc Muscle le Da Dama magg e 1. The enzyme enzyme creating creating kinase (CK) is formed of two subunits that subunits that can either be of the brain (B) type or the muscle (M) type, and different combinations of these types lead to isozymes that predominate in the brain (BB), skeletal muscle (MM), and heart muscle (MB).  (MB).  2. Within 3-4 hours of a heart attack, attack, damaged myocardial cells release CK of the MB type, type, which can be detected in serum by a monoclonal antibody and is useful to confirm the diagnosis. 3. Skeletal muscle myopathy myopathy often  often leads to release of CK of the MM type. Rhabdomyolysis is one of the major side effects of treatment with the cholesterol-lowering drugs the statins statins.. o

  Inflammation of the muscle (myositis) (myositis) leads  leads to cell death.

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  The condition is characterized by muscle pain, weakness weakness,, elevated CK MM, MM, and myoglobinuria.

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Mechanisms of Enzyme Catalysis  A. Enzymes use a variety of strategies to catalyze reactions, and individual enzymes often use more than one strategy. B. Substrate binding binding by an enzyme enzyme helps catalyze the reaction reaction by bringing the reactants into proximity with the optimal orientation for reaction. C. Amino acid side chains within within active sites sites of many enzymes enzymes assist in catalysis by acting acting as acids or bases in reaction with the substrate. 1. In the mechanism of the pancreatic hydrolase hydrolase ribonuclease ribonuclease,, a specialized histidine within the active site acts as a general acid or proton donor  to  to begin cleavage of the phosphodiester linkage of the t he substrate RNA. 2. The digestive enzyme chymotrypsin  has a serine in its active site that acts as chymotrypsin has a general base or proton acceptor  during  during hydrolysis of peptide bonds in protein substrates (Figure).

Figure from (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000):

D. The binding binding of polysaccharide polysaccharide substrates substrates that have have six or more sugar groups groups to lysozyme, the enzyme in tears and saliva that cleaves such molecules, induces strain in the sugar nearest the t he active site making the nearby bond more susceptible to hydrolysis.

Figure from Garrett & Grisham, Biochemistry, 2nd ed., 1999) Lysozyme mechanism  mechanism 

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E. In covalent catalysis, catalysis, the enzyme becomes covalently coupled to the substrate as an intermediate intermediate in  in the reaction mechanism before release of the products. 1. The active site serine of chymotrypsin chymotrypsin attacks the protein protein substrate, substrate, which is is cleaved and a portion of it becomes temporarily connected through the serine by an acyl linkage to the enzyme. 2. The acyl-enzyme acyl-enzyme intermediate intermediate reacts further further by transfer of the polypeptide polypeptide segment to water, completing cleavage (or hydrolysis hydrolysis)) of the protein substrate. substrate. Examples: Snake Venom Enzymes: Hydrolases that Produce Toxic Effects  

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Snake venoms are composed of a toxic mixture of enzymes that enzymes that can kill or

immobilize immobiliz e prey.  prey.   

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Neurotoxic venoms Neurotoxic  venoms of cobras, mambas, mambas, and coral snakes inhibit the enzyme en zyme acetylcholinesterase..  acetylcholinesterase   This hydrolase hydrolase normally normally breaks breaks down the neurotransmitter neurotransmitter acetylcholine within

o

nerve synapses.   The resultant elevation of acetylcholine causes a transient period of

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contraction followed by prolonged depolarization in depolarization in the postsynaptic muscle cell, which induces relaxation and then paralysis of the victim.  

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Hemotoxic venoms Hemotoxic  venoms of rattlesnakes and cottonmouths contain as their principal toxin phosphodiesterase phosphodiesterase,, an enzyme that catalyzes hydrolysis ofphosphodiester bonds in A TP and other substrates. -

One consequence of this activity is altered metabolism metabolism of endothelial cells,

-

which leads to cardiac effects and rapid decrease in blood pressure. pressure. These venoms induce circulatory shock and shock and potentially death.

Enzymes as Therapeutic Agents  The catalytic efficiency and exquisite specificity of enzymes have been exploited for use as

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therapeutic agents in certain diseases.  Patients with cystic fibrosis use fibrosis use aerosol inhaler sprays of the DNA-hydrolyzing enzym enzyme e

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deoxyribonuclease to help reduce the viscosity of mucous secretions, which contain large deoxyribonuclease to amounts of DNA arising from destruction of WBCs as they fight lung infections.  Patients who have had a heart attack or  stroke are  stroke are frequently treated by intravenous

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administration administrati on of tissue plasminogen activator (tPA) or (tPA) or streptokinase streptokinase,, enzymes that break down d own fibrin clots that clog c log blood blood vessels. vessels. Prof.Dr. H.D.El-Yassin 2011

 

 

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 Allosteric Regulation of Enzymes  A. Key enzymes that catalyze rate-limiting steps of metabolic pathways or that are responsible for major cellular processes must be regulated to maintain homeostasis of individual cells and the organism overall. B. Allosteric regulation refers regulation refers to binding of a molecule to a site on the enzyme other than the active site and site and induces a subsequent change in shape of the enzyme causing an increase or decrease in its activity. C. Many allosteric enzymes have have multiple multiple subunits whose interaction accounts for their unusual kinetic properties. i. Enzymes that are are subject to allosteric regulation by either positi positive ve or negative negative effectors effectors   exhibit cooperativity cooperativity.. 2. In the presence of positive cooperativity, cooperativity, a plot of v versus [S] shows sigmoidal kinetics, ie, is S-shaped (Figure). a. This kinetic kinetic behavior signifies signifies that the enzyme's enzyme's affinity for the substrate increases as a function of substrate loading. b. This is analogous analogous to O2 binding by hemoglobin, hemoglobin, in which O2 loading to one subunit facilitates O2 binding to the next subunit, and so on.

Figure: Relationship between V; and [S] for a reaction catalyzed by an allosteric enzyme, showing the effects of positive and negative effectors.

inhibition occurs when the end product of a metabolic pathway D.  Feedback inhibition occurs accumulates, binds to and inhibits a critical enzyme upstream in the pathway, either as a competitive inhibitor or an allosteric effector. 

Prof.Dr. H.D.El-Yassin 2011

 

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Pro Prof.Dr.H.D.El-Ya f.Dr.H.D.El-Ya ssin  

2011 13-16O C TO BE BER R 

K INET INETIC S O F ENZYME-C ATALYZED REAC TIO IONS NS 

I-REGULATION OF ENZYMES BY SUBSTRATE AND PRODUCT CONCENTRATION II-ENZYME INHIBITORS  Prof.Dr. H.D.El-Yassin 2011

 

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REGULATION OF ENZYMES BY SUBSTRATE AND PRODUCT CONCENTRATION The velocity of all enzymes is dependent on the con concentration centration of substrate. The hypothesis of enzyme kinetics assumes the rapid, reversible formation of a complex co mplex between an enzyme and its substrate (the substance upon which it acts to form a product). It also assumes that the rate of formation of the product, P, is proportional to the concentratio concentrationn of the complex. The velocity of such a reaction is greatest when  all the sites at which catalytic activity can take place on the th e enzyme molecules (active sites) are filled with substrate; i.e., when the substrate concentration is very high.

k1  E

+

S

k2  ES

E

+

P

k-1 E= enzyme, S= substrate, ES= enzyme-substrate complex, P= product k1, k-1 and k2 are rate constants Prof.Dr. H.D.El-Yassin 2011

 

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The Michaelis-Menton equation describes how reaction velocity varies with substrate concentration: Vmax[S] v 0 

= Km +[S]

Vmax=maximum velocity, v 0 = initial velocity, Km= (k-1 + k2)/k1 

To determine the maximum speed of an enzymatic reaction, the substrate concentration concentration is increased until a constant rate of product formation is achieved. This is the maximum m aximum velocity (Vmax) of the enzyme. In this state, all enzyme active sites are saturated with substrate. The Michaelis-Menten constant (Km)= B, is the substrate concentration required for an enzyme to reach one half its maximum velocity. Each enzyme has a characteristic Km  for a given substrate.

The key features of the plot are marked by points A, B and C. At high substrate concentrations concentrations the rate represented represented by point C the rate of the reaction is almost equal to Vmax, and the difference in rate at nearby concentrations of substrate is almost alm ost negligible. Prof.Dr. H.D.El-Yassin 2011

 

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If the Michaelis-Menten plot is extrapolated to infinitely high substrate concentrations, concentrations, the extrapolated rate is equal to Vmax. When the reaction rate becomes independent of substrate concentration, or nearly so, the rate is said to be zero order. (Note that the reaction is zero order only with respect to this substrate. If the reaction has two substrates, it it may or may not be zero order with respect to the second substrate). At lower substrate concentrations, concentrations, such as at points A and B, the lower reaction velocities indicate indicate that at any moment mom ent only a portion of the enzyme molecules are bound to the t he substrate. In fact, at the substrate concentration concentration denoted by point B, exactly half the enzyme molecules are in an ES complex at any instant and the rate r ate is exactly one half of Vmax.

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Inhibition of Enzyme Catalyzed Reactions  To avoid dealing with curvilinear plots of enzyme catalyzed reactions, reactions, biochemists Lineweaver and Burk introduced an analysis of enzyme kinetics based on the following rearrangementt of rearrangemen o f the MichaelisMenten equation:

A Lineweaver-Burk Plot  [1/v] = [Km (1)/ Vmax[S] + (1)/Vmax] 

Plots of 1/v versus 1/[S] yield y ield straight lines having a slope of K m/Vmax  and an intercept on the ordinate at 1/V max. The Lineweaver-Burk transformation of the Michaelis-Menton Michaelis-Menton equation is useful in the analysis of enzyme inhibition. Since most clinical drug therapy is based on inhibiting the activity of enzymes, analysi analysiss of enzyme reactions using the tools described above has been fundamental to the modern design of pharmaceuticals. Many Drugs Acts As Enzyme Inhibitors   Many drugs, including antibiotics and antiviral agents, operate by

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inhibiting critical enzyme-catalyzed reactions or serve as

alternative dead-e dead-end nd substrates of such reactions.   The antibiotic activity of penicillin is due to its ability to inhibit

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transpeptidases responsible for crosslink formation in construction

of bacterial cell walls, leading to lysis of the weakened cells.

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  Sulfanilamides (sulfa drugs) are antibiotics that serve as

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structural analogs of para-aminobenzoic acid ac id (PABA (PABA)), a substrate in the formation of folic acid by many m any bacteria. Substitution of the th e sulfanilamide compound in place of PABA in the reaction prevents formation of the critical coenzyme folic acid.   Inhibitors of the HIV protease pr otease are useful in antiviral therapy 

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strategies because this enzyme is absolutely required for processing of proteins needed for synthesis of the viral coat.   the use of methotrexate  in cancer chemotherapy to semi-

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selectively inhibit inhibit DNA synthesis of malignant cells, o

  the use of aspirin to inhibit the synthesis of prostaglandins prostaglandins

which are at least partly responsible for the aches and pains of arthritis. Examples of irreversible (suicidal) inhibitors Organophosphorous Pesticides: Suicide Inhibitors of Acetylcholinesterase   Organophosphates form stable phosphoesters with the active site

•

serine of acetylcholinesterase, the enzyme responsible for hydrolysis and inactivation of acetylcholine at cholinergic synapses.   Irreversible inhibition of the enzyme leads to accumulation of

•

acetylcholine at these synapses and consequent neurologic neurologic impairment. impairment.   Poisoning by pesticides that contain organophosphate compounds

•

produces a variety of symptoms, including nausea, blurred vision, fatigue, muscle weakness and, potentially, death caused by paralysis of respiratory muscles. mu scles. Prof.Dr. H.D.El-Yassin 2011

 

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In addition, many poisons, such as cyanide, carbon monoxide and polychlorinated biphenols (PCBs), produce their life- threatening effects by means of enzyme inhibition. inhibition. Enzyme inhibitors fall into two broad classes: 1.  Those causing ir r ev evers ers ibl ible e inactivati inactivation on ooff enzymes: Inhibitors of the first class usually u sually cause an inactivating, covalent modification of enzyme structure . Cyanide is a classic example e xample of an irreversible irrever sible enzyme inhibitor inh ibitor:: by covalently binding mitochondria m itochondriall cytochrome oxidase, it inhibits all the reactions associated with electron transport. The kinetic effect of irrever irreversible sible inhibitors is to decrease the concentration of active enzyme, thus decreasing the maximum possible concentration of ES complex. Since the limiting enzyme reaction rate is often k 2[ES], it is clear that under these circumstances the reduction of enzyme concentration will lead to t o decreased reaction rates. Irreversi Irreversible ble inhibitors are usually considered considered to be poisons and are generally unsuitable for therapeutic purposes purposes.. 2.  Those whose inhibitory effects can be reversed . Reversible inhibitors can be divided into two main m ain categories; competitive inhibitors and noncompetitive inhibitors, with other three

categories, , rarely encountered, partially completive inhibitors uncompetitive inhibitors and mixed inhibitors. 

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Inhibitor Type 

Competitive Inhibitor

Binding Site on Enzyme  

Specifically at the catalytic site, where it competes with substrate for binding in a dynamic equilibrium- like process. Inhibition is reversible by substrate.

Noncompetitive Inhibitor

Partially competitive Inhibitor

Binds E or ES complex other than at the catalytic site. Substrate binding unaltered, but ESI complex cannot form products. Inhibition cannot be reversed by substrate.

similar to that of non-competitive, except that the EIS-complex has catalytic activity, which may be lower or even higher (partially competitive activation) than that of the enzyme-substrate (ES)

Kinetic effect 

Vmax is unchanged; Km, as defined by [S] required for 1/2 maximal activity, is increased.

Km appears unaltered; Vmax is decreased proportionately to inhibitor concentration.

lower Vmax, but an unaffected Km value

complex. 

Binds only to ES complexes at locations other than the catalytic Uncompetitive Inhibitor

Mixed inhibition

site. Substrate binding modifies enzyme structure, making inhibitor- binding site available. Inhibition cannot be reversed by substrate.

Inhibitor bind to both the enzyme and the ES complex. It has the properties of both competitive and uncompetitive inhibition.  inhibition. 

Apparent Vmax  decreased; Km, as defined by [S] required for 1/2 maximal activity, is decreased.

Both a decrease in Vmax  and an increase in the Km value are seen in mixed inhibition.  inhibition. 

Prof.Dr. H.D.El-Yassin 2011

 

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The feature of all the t he reversibl reversible e inhibitors is that when the inhibitor concentration drops, enzyme activity is regenerated. Usually these inhibitors bind to enzymes by non-covalent forces and the inhibitor maintains a reversible reversible equilibrium with the enzyme. The equilibrium equilibrium constant for the dissociation of enzyme enzyme inhibitor complexes is known as KI: KI = [E][I]/[E--I--complex] 

The effects of K I are best observed in Lineweaver-Burk Lineweaver-Burk plots. Probably the best known reversible reversible inhibitors are competitive inhibitors, which always bind at the catalytic or active a ctive site of the enzyme. Most drugs that alter enzyme activity are of this type. Competitive inhibitors are especially attractive as clinical modulators of enzyme activity act ivity because they offer two routes for the reversal of enzyme inhibition: 1.  First, as with all kinds of reversible reversible inhibitors, a decreasing concentration of the inhibitor reverses reverses the equilibri equilibrium um regenerating active free enzyme. 2.  Second, since substrate and competitive inhibitors both bind at the same site they compete com pete with one another for b binding inding.. Raising the concentration of substrate (S), while holding the concentration of inhibitor constant, provides the second route for reversal of competitive inhibition. The greater the proportion of substrate, the greater the proportion of enzyme present in competent ES complexes. Prof.Dr. H.D.El-Yassin 2011

 

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High concentrations of substrate can displace virtually all competitive inhibitor inhibitor bound to active sites. Thus, it is apparent that Vmax should be unchanged by competitive competitive inhibitors. This characteristic of competitive com petitive inhibitors inhibitors is reflected reflected in the identical vertical-axis intercepts of Lineweaver-Burk Lineweaver-Burk plots, with and without inhibitor. panel B.

Lineweaver-Burk Plots of Inhibited Enzymes

Analogously, panel C illustrates that noncompetiti no ncompetitive ve inhibitors appear to have no effect on the intercept at the x-axis implying that noncompetitive inhibitors inhibitors have no effect on the K m of the enzymes they inhibit. Prof.Dr. H.D.El-Yassin 2011

 

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Since noncompetitive inhibitors do not interfere in the equilibra equilibration tion of enzyme, substrate and ES complexes, the K m's of Michaeli Michaelis-Menten s-Menten type enzymes are not expected to be affected by noncompetitive inhibitors, as demonstrated by x-axis intercepts in panel C. However, because complexes that contain inhibitor (ESI) are incapable of progressing to reaction products, the effect of a noncompetitive n oncompetitive inhibitor is to reduce the concentration concentration of ES complexes that can advance to product. Since Vmax = k2[Etotal], and the concentration of competent Etotal is diminished by the amount of ESI formed, noncompetitive inhibitors are expected to decrease V max, as illustrated by the y-axis intercepts in panel C. A corresponding analysis of uncompetitive inhibition leads to the expectation that these inhibitors should change the apparent values of Km as well as Vmax. Changing both constants leads to double reciprocal plots, in which intercepts on the x and y axes are proportionately changed; this leads to the production of parallel lines in inhibited and uninhibited reactions.

Prof.Dr. H.D.El-Yassin 2011

 

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Clinical Problems

A Polish man and his friend who is of Japanese descent descent are sharing conversation over drinks at a party. After the Polish man finishes his second bottle of beer, he notices that his friend, despite having drunk only half his drink, appears flushed in the th e face. His friend then complains of dizziness and headache and asks to be driven home. 1.  The marked difference in tolerance to alcohol illustrated by these men is most likely due to a ge gene ne encoding which of the following following enzymes? A.  Alcohol dehydrogenase B.  Acetate dehydrogenase C.  Alcohol reductase D. Aldehyde dehydrogenas

E.  Aldehyde aminotransferase aminotransferase

Comment: The answer is D. Many Asians lack a low-K m, form of acetaldehyde dehydrogenase, which is responsible for detoxifying acetaldehyde generated by oxidation o xidation of ethanol in the liver. Acetaldehyde accumulation in the blood of such individuals leads to the facial flushing and neurologic effects exhibited by the man of Japanese descent.

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An alcoholic has consumed antifreeze as as a substitute s ubstitute for ethanol. Ethyleneglycol, an ingredient ingredient in antifre ant ifreeze, eze, is also a substrate for the enzyme alcohol dehydrogenase (ADH), which normally converts ethanol to acetaldehyde. Ethylene glycol, however, is converted by ADH to a highly toxic product. Ethanol is administered as a treatment in this case of poisoning. 2.  Why is ethanol an effective treat treatment ment for ethylene glycol poisoning? A. ADH exhibits a much higher affinity (K m for ethanol than for ethylene glycol

B.  Ethanol is an allosteric allosteric effector of ADH C.  Ethanol combines with the toxic product formed by the reaction of ADH with ethylene glycol and renders r enders it harmless D.  Acetaldehyde is of therapeutic value. E.  Ethanol induces another enzyme that is capable of metabolizing ethylene glycol

Comment: The answer is: 1-A: Aldehyde dehydrogenase (ADH), which exhibits a broad substrate specificity for alcohols, has a much higher affinity for ethanol [i.e., a lower Km] than for ethylene glycol. Saturating ADH with ethanol by administration of therapeutic levels prevents it from converting ethylene glycol to the toxic aldehyde, and allows ethylene glycol to eventually be excreted unmetabolized.

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Ethylene Glycol is a competitive com petitive inhibitor inhibitor of alcohol dehydrogenase (ADH) activity. Ethylene glycol is converted by ADH, to oxalic o xalic acid, calcium oxalate crystals in kidney. •

  In Non-Asians, ethanol is administered as antidote to ethylene

glycol ingestion •

  In Asians, ethanol administration is not efficacious due to low,

non-inducible ADH activity.

Interesting story in Japanese press several years ago Drunk Japanese businessman was spared from death due to ethylene glycol poisoning siphoning antifreeze antifreeze from a friend's car habitual alcohol user, had higher steady-state ADH levels.

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3.  If one compares Lineweaver-Burk Lineweaver-Burk plots for the reactions of ADH with ethanol and ethylene e thylene glycol, which of the following would be observed? A.  They exhibit identical slopes. B. They exhibit identical y-intercepts.

C.  They exhibit identical x-intercepts. D.  Only the plot for the reaction of ethanol is linear E.  Only the plot for the reaction of ethylene glycol is linear

Comment: The answer is 2-B: If ADH obeys Michaelis-Menten kinetics, then the Lineweaver-Burk plot, which is a linear transform of the Michaelis-Menten equation, will be linear for both substrates. ADH exhibits different Km values for ethanol and ethylene glycol. Therefore, the x-intercept (1/ K m and the slope (Km/Vmax) are different. If only the affinity for the alternative substrate is different, then the Vmax will be the same as will the y-intercept (1/Vmax).

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4.  Blood was taken from this patient and analyzed for the serum levels of certain enzymes. Which one of the t he following enzymes will most likely be present at elevated lev levels? els? (A) Amylase (B)

Creatine kinase

(C)

Alanine aminotransferase aminotransfera se

(D)

Acid phosphatase

(E)

Lactate dehydrogenase

Comment: he answer is 3-C :Chronic alcoholics are likely to exhibit signs of liver damage. Alanine aminotransferase is present in the cytosol of liver cells, and its release into the serum is diagnostic of hepatocellular damage. Lactate dehydrogenase and creatine kinase isozymes are analyzed to diagnose heart attacks. Amylase levels are elevated in patients with acute pancreatitis, and elevated acid phosphatase levels may be diagnostic of prostate cancer.

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5. 

A noncompetitive enzyme inhibitor A.  Decreases Vmax and increases Km. B. Decreases Vmax max and has no effect on K m.

C.  Has no effect on Vmax or Km. D.  Has no effect on Vm:K and increases Km. E.  Has no effect on Vmxi and decreases Km. 

Comment: The answer is B. A noncompetitive inhibitor binds to the enzyme at a site other than the substrate binding site, so it has little measurable effect on the enzyme's affinity for substrate, as represented by the K m. However, the inhibitor has the effect of decreasing the availability of active enzyme capable of catalyzing the reaction, which manifests itself as a decrease in Vmax max.

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6. 

A 47-year-old man is evaluated for a 12-hour history of nausea, vomiting and, a nd, more recent, difficulty difficulty breathing. breathing. His past medical history is unremarkable, unremarkable, and he takes no medications. However, he is a farmer who has had similar episodes in the past after working with agricultural chemicals in his fields. Just yesterday he reports applying diazinon, an organophosphate insecticide, to his sugar beet field. After consultation with the poison center, you conclude that this patient's condition is most likely due to inhibi inhibition tion of which of the following enzymes? A.  Acetate dehydrogenase B.  Alanine aminotransferase C.  Streptokinase D.  Acetylcholinesterase  E.  Creatine kinase

Comment: The answer is D. Organophosphates react with the active site serine residue of hydrolases such as acetylcholinesterase and form a stable phosphoester modification of that serine that inactivates the enzyme toward substrate. Inhibition of acetylcholinesterase causes overstimulation of the end organs regulated by those nerves. The symptoms manifested by this patient reflect such neurologic effects resulting from the inhalation or skin absorption of the pesticide diazinon. Prof.Dr. H.D.El-Yassin 2011

 

  28

7.  Accidental ingestion of ethylene glycol, an ingredient of

automotive antifreeze, is fairly common among childre ch ildrenn because of the liquid's pleasant color and sweet taste. Ethylene glycol itself is not very toxic, but it is metabolized by alcohol dehydrogenase to the toxic compounds com pounds glycolic acid, glyoxylic acid, and oxalic acid, which can produce acidosis and lead to renal failure and death. Treatment for suspected ethylene glycol poisoning is hemodialysis to remove the toxic metabolites and administration administration of a substance that reduces the metabolism of ethylene glycol by displacing it from the enzyme. Which of the following compounds would be best suited for this therapy? A.  Acetic acid B. Ethanol

C.  Aspirin D.  Acetaldehyde E.  Glucose Comment: The answer is B. The therapeutic rationale for ethylene glycol poisoning is to compete for the attention of alcohol dehydrogenase by providing a preferred substrate, ethanol, so that the enzyme is unavailable to catalyze oxidation of ethylene glycol to toxic metabolites. Ethanol will displace ethylene glycol by mass action for a limited time, during which hemodialysis is used to remove ethylene glycol and its toxic to xic metabolites from the patient's bloodstream. Prof.Dr. H.D.El-Yassin 2011

 

  29

8.  Glucose taken up by liver cells is rapidly phosphorylated to glucose 6-phosphate with ATP serving as the phosphate donor in the initial step of metabolism and assimilation of the sugar. Two enzymes, which may be considered isozymes, are capable of catalyzing this reaction in the liver cell. Hexokinase has a low Km of -0.05 mM for glucose, whereas glucokinase exhibits sigmoidal kinetics with an approximate Km of ~5 mM. After a large meal, the glucose concentrationn in the hepatic portal vein may approximate 5 mM. concentratio After such a large meal, which of the following scenarios describes the relative activity levels for these two enzymes? Hexokinase Glucokinase A.  Not active Not active B.  v ≅ ½Vmax Not active C.  v ≅ Vmax  Not active v ≅  ½V max   D. v ≅V max max E.  v ≅Vmax  v≅Vmax  Comment: The answer is D. This problem provides a practical illustration of the use of the Michaelis-Menten equation. The high concentration of glucose in the hepatic portal vein after a meal would promote a high rate of glucose uptake u ptake into liver cells, necessitating rapid phosphorylation of the sugar. The glucose concentration far exceeds the K m  of hexokinase, ie, [S] > K m , meaning that the enzyme will be nearly saturated with substrate and v ≅Vmax. However, the [S] = K m  for glucokinase, which will be active in catalyzing the phosphorylation reaction and v ≅  ½V max  

Prof.Dr. H.D.El-Yassin 2011

 

qwertyuiopas g j zxcv nmqwert   30  

yuiopasdfghjklzxcvbnmqwertyuiop asdfghjklzxcvbnmqwertyuiopasdfg hjklzxcvbnmqwertyuiopasdfghjklzx Plasma Enzymes in Diagnosis [Prof. Dr. H.D.El-Yassin]

cvbnmqwertyuiopasdfghjklzxcvbn 18 October [2011] 

mqwertyuiopasdfghjklzxcvbnmqwe rtyuiopasdfghjklzxcvbnmqwertyuio pasdfghjklzxcvbnmqwertyuiopasdf ghjklzxcvbnmqwertyuiopasdfghjklz xcvbnmqwertyuiopasdfghjklzxcvbn mqwertyuiopasdfghjklzxcvbnmqwe rtyuiopasdfghjklzxcvbnmqwertyuio pasdfghjklzxcvbnmqwertyuiopasdf ghjklzxcvbnmqwertyuiopasdfghjklz Prof.Dr. H.D.El-Yassin 2011

 

  31

Plassma Enzy Pla Enzyme mess in D i agnosis nosis Most enzymes are present in cells at much higher concentrations than in plasma. Some occur  predominantly  predom inantly in cells of certain tissues, where they may be located in different cellular 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,  plasm a, they are still usef useful ul as diagnostic diagnostic tools. Measure Measuremen mentt of thei theirr leve levels ls in plasma plasma offers 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 concentr concentration ation directly.

The enzyme activity in plasma may be:   Increased due to proliferation of cells, an increase in the rate of cell turnover or dam damage age

•

or in enzyme synthesis (induction), or to reduced clearance from plasma   Lower than normal, very occasionally oc casionally due to reduced synthesis or congenital deficiency.

•

Changes in plasma enzyme activities may sometimes help to detect and localize tissue cell damage or proliferation, or to monitor treatment and progress of disease

Prof.Dr. H.D.El-Yassin 2011

 

  32  Assessment of Cell Damage Damage and Proliferati o on n Plasma enzyme levels depend on: 1. The rate of release from damaged cells which, in turn, depends on the rate which damage is occurring: o ccurring: 2. The extent of cell damage. In the absence of cell damage d amage the rate release depends depends on: 1. The rate of cell proliferation: 2. The degree of induction of e enzyme nzyme synthesis synthesis These factors are balanced by: !  The rate of enzyme clearance cle arance from circulation. circulation.

 Acute cell damage, for example in viral hepatitis hepatitis,, may may cause very high high plasma plasma enzyme activities that fall as the condition resolves. By contrast, the liver may be much more extensively involved in advanced cirrhosis but the rate of cell damage is often low and consequently plasma enzyme activities may be only slightly raised or be within the reference range. In very severe liver disease plasma enzyme activities may even fall terminally, when the number of hepatocytes is grossly reduced.

It is not known how most enzymes are removed from, or their action inhibited in the circu circulation. lation. Relatively small peptides, such as  --amylase, amylase, can be cleared by the kidneys: most enzymes are large proteins and are probably catabolized by plasma proteases before being taken up by the reticuloendothelial system. In health each enzyme has a fairly constant and characteristic biological half-life; knowledge of this half-life may be of help in assessing the time since the onset of an acute illness. After a myocardial infarction, for example, plasma levels of creatine kinase and aspartate transaminase fall to normal before those of lactate dehydrogenas, which has a longer half-life. The half-life may be lengthened if there is circulatory impairment. Renal glomerular impairment i mpairment may delay the rate of fall of those plasma enzymes cleared through the kidneys. For example plasma amylase activity m may ay be high due to renal glomerular impairment, rather pancreatic damage

Prof.Dr. H.D.El-Yassin 2011

 

  33

L ocalization ocalization of Damag Damag e Most of the enzymes commonly measured to assess tissue ti ssue damage are present in nearly all cells, although their relative concentrations in certain tissues may differ. Measurement of the  plasma activity of an enzyme enzyme known to be in high concentration within cells of a particular tissue may indicate an abnormality of those cells, but the results will rarely enable a specific diagnosis to be be ma made. de. For example example if there is circulatory failure after a cardiac arrest very high high plasma levels level s of enzymes originatin originating g fr from om many many tissues tissues m may ay occur occur because of o f hypoxic hypo xic damage to cells cell s and reduced rates of clearance: clearance: the raised plasma levels of 'cardiac' enzymes do not necessarily mean that a myocardial infarct caused the arrest The diagnostic precision precision of plasma enzyme enzyme analysis may be iimproved mproved by 1. Estimation of more than one enzyme. .Ma .Many ny enzymes are widely distributed, but their relative concentrations may vary in different tissues. For instance, although both alanine and aspartate transaminases are abundant in the liver, the concentration of aspartate transaminase is much greater than that of alanine transaminase in heart muscle 2. Isoenzyme determ determination. ination. Some enzymes exist in more than one form: these isoenzymes may be separated by their different physical or chemical properties. If they originate in different tissues such identification will give more information than the measurement of plasma total enzyme activity: for example, creatine kinase may be derived from skeletal or cardiac muscle, but one of its isoenz i soenzymes ymes is found  predominantly  predominant ly in the myocar myocardium dium 3. Serial enzym enzyme e estimations. The rate rate of change of plasma enzyme enzyme activity is related to to a balance between the rate of entry and the rate of removal removal from the circulation. A  persistently raised plasma plasma enzyme enzyme activity is suggestive of a chronic disorder or occasionally of impaired clearance. 

The distribution of enzymes within cells may differ. Alanine transaminase and lactate dehydrogenase are predominantly located in cytoplasm and glutamate dehydrogenase in mitochondria, whereas aspartate transaminase occurs in both these cellular compartments. Different Differe nt dis disease ease pr process ocesses es in the same same ti tissu ssue e may affect the cell in different ways, causing alteration in the relative plasma enzyme activities

Prof.Dr. H.D.El-Yassin 2011

 

  34

Non-s pecific C aus es of R ais ed Plas Plas ma E nzyme nzyme Activit Activities   Before attributing a change in plasma enzyme activity to a specific disease process it is important to exclude the presence of factitious or nonspecific causes. Slight rises in plasma aspartate transaminase activities are common, non-specific findings in many illnesses. Moderate exercise, or a large intramuscular injection, may lead to a rise in  plasma creatine kinase activity; isoenzyme determination may identity skeletal muscle as the tissue of origin Some drugs, may induce synthesis of the microsomal enzyme, gamma-glutamyltransferase, and so increase its plasma activity in the absence of disease. Plasma enzyme activities may be raised if the rate of clearance from: the circulation is reduced. In the absence disease this may occur if for example the plasma enzyme forms.!  macromolecules  macromolec ules (aggregates), (aggregat es), such as in ma macroam croamylas ylasaem aemia ia . !   complexes with immunoglobulins. as occasionally occur with lactate dehydrogenase, alkaline

 phosphatas  phosp hatase e creatine creatine kinas kinase. e.

Facttors Affect Fac Affecting ing R esults esults of P las ma Enzyme A s s ays   1. Analytical factor factors s affecting results. The total concentration of all plasma enzyme  proteins is less than 1 g/L. Results of enzyme enzyme assays are not usually expressed as concentrations, but as activities. Changes in concentration may give rise r ise to proportional changes in catalytic activity, but the results of such measurements depend on many analytical factors. These include the concentrations of the substrate and product, the pH and temperature at which the reaction is carried out. The type of buffer, b uffer, and the  presence of activators activators or inhibitors. Because Because the definition of 'international 'international un units' its' does not take these factory into account, results from different laboratories, apparently expressed in the same units, may not be directly comparable. Therefore, plasma enzyme activities must be interpreted in relation to the reference ranges from the issuing laboratory.

Prof.Dr. H.D.El-Yassin 2011

 

  35 2. Physiological factors affecting enzyme activities include for example: a. Age: plasma aspartate transaminase activity is m moderat oderately ely higher during the neonatal period than in adults: plasma alkaline phosphatase activity of bony origin is higher in children than in adults and peaks during the pubertal bone growth spurt before falling to adult levels. b. Sex: Sex: plasma " -glutamyltransferase -glutamyltransferase activity is higher in men than-in-women. c. physiological conditions: Plasma alkaline phosphatase activity rises during the last trimester of pregnancy because of the presence of the placental isoenzyme: isoenzyme: several enzymes, such as the transaminases transaminases and creatine kinase rise moderately in plasma during and immediately after labour or strenuous exercise. Plasma enzyme act activities ivities must b be e int interpreted erpreted in relation to the sex sex and age-ma age-matched tched refe reference rence ranges of the issuing laboratory.

Prof.Dr. H.D.El-Yassin 2011

 

  36

G ene nettic ba bass is for e enzym nzyme e s ynt ynthe hess is : Since all enzymes are proteins, their synthesis follows the general pattern of protein synthesis and is regulated by genes. For every enzyme, there is said to be one gene (one gene, one enzyme hypothesis); where an enzyme is a complex protein containing more than one protein subunit more than one gene may be concerned in it its s synthesis. Genetic mutation mutation results in abnormal DNA code and synthesis of an abnormal enzyme protein. Since the abnormal enzyme cannot serve the normal function, a metabolic abnormality occurs and this is transmitted to the progeny. Such transmittable abnormalities of metabolism due to abnormal enzyme molecules are known as:

'Molecular diseases' or 'Inborn Errors of Metabolism' . Phenylketonuria, alkaptonuria,  pentosuria  pento suria,, glycogen glycogen stor storage age disease, disease, g galacto alactosemia semia a and nd cystinuria cystinuria are but but a few of several several known known molecular diseases.

1. Phenyl Phenylketonur ketonuria: ia: is an autosomal autosomal recessive disorder disorder ca caused used by an abnormality abnormality of the  phenylal  pheny lalan anin ine e hyd hydro roxyl xylas ase.. e.. Becau Because se ph phen enyl yl alan alanine ine can no nott be co conver nverte ted d to tyr tyros osine ine it accumulates accumula tes in plasma and is secreted in the urine u rine with its metabolites, metabolites, such as phenylpyrovic phenylpyrovic acid; the disease acquired its name from the detection of the latter "phenylketone" in the urine.(see diagram below) Phenylalanine

phenylpyruvic acid (phenylketone) etc.

1

Adrenaline

3

Melanin

3

Dihydroxyphenylalanine DOPA

Alkapton

4

Thyroid hormones

TYROSINE

Homogentistic acid

2

TCA cycle Diagram showing the metabolism of tyrosine and some inborn errors of the aromatic amino acid pathways. Substances highlighted may be present in abnormal amounts in certain inborn errors of metabolism. 1 phenylalanine hydroxylase phenylketonuria 2 homogentisic acid oxidase alkaptonuria 3 tyrosinase albinism 4 thyroid enzymes thyroid dyshormonogenesis

Prof.Dr. H.D.El-Yassin 2011

 

  37 2. Alkapt Alkaptonuria: onuria: is an autosomal recessive recessive d disorder isorder due due to the d deficiency eficiency of homogentisic homogentisic acid oxidase. Homogentisic acid accumulates in tissue and blood and is passed in the urine. Oxidation and polymerization of homogentisic acid produces the pigment alkapton in much the same way as polymerization of DOPA produce melanin. Deposition of alkapton in cartilages, with consequent darkening is called ochronosis and result in visible darkening of the cartilages of the ears.. Conversion of homogentisic acid to alkapton is accelerated in alkaline conditions and most obvious abnormality in alkaptonuria is darkening of the urine as it becomes more alkaline on standing. The condition is compatible with normal life span despite the tendency for  patie  pa tient nt to dev develo elop p arthr arthriti itis s in later later life. life. .(r .(refe eferr to previ previou ous s diag diagra ram) m)

3. glycoge glycogen n storage disease: disease: a deficie deficiency ncy of one of the enzymes involved involved in the glycoge glycogenesis nesis or glycogenolysis glycoge nolysis results in the accumulation of normal normal or abnormal glycogen with hepatomegaly: hepatomegaly: in von Gierk's disease, the least rare glycogen storage disorder, there is a deficiency of glucose-6-phospate. Fasting hypoglycemia occurs because the enzyme is essential for the conversion of glucose-6-phosphate to glucose. 4. galactosaemia an autosomal recessive disorder due to a deficiency of galactose-1-phosphate uridyltransferase , may cause cirrhosis of the liver if untreated.

5. cystinuria: is an autosomal autosomal recessive inherited abnormality of tubular reabsorption, reabsorption, with excessive urinary excretion of the dibasic amino acids: cystine, arginine and lysine.

Prof.Dr. H.D.El-Yassin 2011

 

  38  

Clinically Important Enzymes and  Diagnostic Applications

Prof. Dr. D r. H.D.El-Yassin H.D.El-Yassin  20 Oc O c tob e r 2011

Prof.Dr. H.D.El-Yassin 2011

 

  39

C linic a lly Imp I mport orta a nt Enz nzyy me mess and Diag no nost stic ic A pplic pplic at ation ionss Di s tribution and applic applic ation ation of clinical cli nically ly i mporta mportant nt enzymes

Enzyme

P rinciple S ources of

C linic al app applicat lications ions

E nzyme in blood blood Li ver

Hepatic Hepatic pa parenchymal renchymal diseases dis eases

 A lkal lk alii ne

Li ver, bone, intestinal

B one dis dis eases eases , hepatob hepatobiliry iliry dis eas eas es

 phos phetase phetas e

mucos a, placenta placenta

 A lanine aminotransferase

 A mylase mylas e

 S alivary g lands, lands ,

P an ancrea creatic tic diseases

 pancreae  panc reae  A s partate

Li ver, s keletal keletal muscle,

Hepatic Hepat ic pa parenchymal renchymal dis dis ease, mus cle

aminotrasferase

heart erythrocy tes tes

disease

Cholinesterase

Liver

Organophosphorus insecticide  poiss oning  poi oni ng , hepatic hepati c parenc hymal disease

C reatine reatine ki nase

S keletal keletal muscle, heart heart

Muscle Mus cle dis eases (M.I.)

 -g -g lutam lutamyl yl

Liver

Hepatobiliary Hepat obiliary diseas es es,, marker marker of alcohol abus abus e

transferase Lactate

Heart, liver, sk eleta eletall

Hemolys Hemol ys is , hepatic hepatic pa parenchymal renchymal

dehydrogenase

muscle, erythroctes,

dis eas eas es, tumor tumor marker

 platelets  platel ets,, lymph nodes lipase lipa se  5'-nuc  5'-n ucleotidas leotidas e Try psin

P ancreas ncreas

P an ancrea creatic tic diseases

L iv er

Hepatbili He patbiliary ary dis di s ease s

pancrea pa ncreass

P an ancrea creatic tic diseases Prof.Dr. H.D.El-Yassin 2011

 

  40

Pancreatic enzymes:  A cute cu te pancr eatitis  is an inflammatory inflammatory process in which whi ch pancreat pancreatic ic enzymes enzymes are activated and cause autodigestion of the gland. It is a result of anatomical changes that arise from two events. 1. The first is the autodigestion of the acinar c cells ells by inappropriate inappropriate activation of the  pancreatic enzymes enzymes (especially trypsinoge trypsinogen) n) within the cell. 2. The second is the cellular injury response response that is mediated by proinflammatory proinflammatory cytokines. The mechanisms by which the digestive enzymes become activated within the acinar cell are unclear. However, such inappropriate activation of pancreatic enzymes leads to destruction of the acinar cell and surrounding fat deposits, and it weakens the elastic fibers of the blood vessels, resulting in leakage. There are some enzymes that are synthesized and stored as the

-amylase, carboxyl ester lipase, active enzymes in the zymogen granules. These include ! -amylase lipase, colipase, RNase, and DNase. - Amylase: (EC3.2.1.1; 1,4-  -Amylase: 1,4-  ! -D-glucan - D-glucan glucanohydrolase; AML)   is an enzyme of

1.

! 

-glycosidic linkages in the hydrolyase class that catalyzes the hydrolysis of 1,4-   -glycosidic  polysac  poly sacch chari arides. des. AMYs AMYs are are calc calcium ium metaloenz etaloenzyme ymes, s, with with the the calciu calcium m absolut absolutely ely required for functional integrity.   AMYs  AMY s normally normally occ occurri urring ng in hum human an plas plasma ma are sma small ll molecu molecules les wi with th molecu molecular lar we weigh ights ts varying from 54 to 62 kDa. The enzyme is thus small enough to pass the glumeruli of the kidneys and AMY is the only plasma enzyme physiologically found in urine. The AMY activity  pr  presen esentt in normal normal serum serum and urine iis s of pan pancre creat atic ic (P-AMY) (P-AMY) and sa saliv livary ary gla gland nd (S-A (S-AMY) MY) origin.

Clinicall S ig nificance Clinica nificance Normal values of amylase: 28-100 U/L =0.48-1.7 µkat/L CAUSES OF RAISED PLASMA AMYLASE ACTIVITY   •

 

Marked increase (five to 10 times the upper reference limit):   acute pancreatitis:

o

  severe glomerular impairment:

o

o

•

   perforated peptic ulcer especially if there is perforation into the lesser sac.

  Moderate increase (up to five times the upper reference limit): other acute abdominal disorders:  perforated peptic ulcer: acute cholecysti c holecystitis: tis:

Prof.Dr. H.D.El-Yassin 2011

 

  41 intestinal obstruction: obstruction: abdominal trauma: salivary gland disorders: mumps: salivary calculi: Sj # # gren's g   ren's syndrome: severe glomerular glo merular dysfunction (may be markedly ra raised); ised); myocardial infarction (occasionally): acute alcoholic intoxication: diabetic ketoacidosis ket oacidosis (may be markedly raised) raised);; macroamylasaemia.

Pancreatic Pancreat ic pseudocy pseudocy s t.  If the th e plasma amylase activity fails to fall after an attack of acute  pancreatitis there may may be leakage of pancre pancreatic atic fluid into the lesser sac sac (a pancreatic  pseudocyst). Urinary amylase amylase levels are high, high, different differentiating iating it from m macroam acroamylasaem ylasaemia. ia. This is one of the few indications in dications for estimating urinary amylase amylase activity, w which hich is inappropriately low relative to the plasma activity if there is glomerular impairment or macroamylasaemia

 Macr oamylas aemia. In some patients a high plasma amylase activity is due to a low renal excretion of the enzyme, despite normal glomerular function. The condition is symptomless; it is thought that either the enzyme is bound to a high molecular weight plasma component such as protein, or that the amylase molecules form large polymers that cannot pass through the glomerular membrane. This harmless condition may be confused with other causes of hyperamylasaemia

Prof.Dr. H.D.El-Yassin 2011

 

  42

  2. Lipase: (EC 3.1.1.3; triacylglycerol acylhydrolase; LPS) .is a single & chain chain glycoprotein with molecular weight of 48 kDa. For full catalytic activity and greatest specificity the presence of bile salts and a cofactor called colipase, which is secreted by the pancreas, is required. LPS is a small molecule and is filtered through the glomerulus. It is totally reabsorbed by the renal tubules, and it is not normally detected in urine.

Clinicall S ig nificance Clinica nificance Normal values: 40-200 U/L Plasma lipase levels are elevated in acute pancreatitis and carcinoma of the pancreas. Note: serum amylase is increased in mumps, pancreatic disease or due to some other cause, where as lipase is increased only in pancreatitis. Therefore, the determination of both amylase and lipase together helps in the diagnosis of acute pancreatitis.

3. Trypsin: ( E C 3.4.21. 3.4.21.4; 4; no sy stem stemic name; name; TR Y)  is a serine proteinase that hydrolyze the peptide bonds formed by the carboxyl groups of lysine arginine with other amino acids.

Clinicall S ig nificance Clinica nificance Normal values of trypsin: 25 ' '  5.3 5.3 µg/L Increased in pancreatic disease. But as there is no distinct role of trypsin estimation in the routine management of patients with acute pancreatitis, this test is therefore considered of limited clinical value.

hymotrypsin ( E C 3.4.21.1; no sy stemic stemic name; name; CHY )   4. C hymotrypsin

5. E lastas-1 lastas-1 ( EC 3.4.21.36; 3.4.21.36; no sys temic temic nam name; E 1)  

Prof.Dr. H.D.El-Yassin 2011

 

  43

Liver enzymes: The assay of serum enzymes is very useful for the differential diagnosis and monitoring of various heptobiliy disorders. There are three types of enzymes:

1. Enzymes which are normally present inside the hepatocytes released into the blood markers ers of hepatocellul hepatocellular ar damag damag e. when there is a hepatocellular damage= mark  2. Enzymes which are primar p rimary y membrane bound (plasma membrane or side of hepatocytes) = markers markers of cholestasis cholestasis .

 3. Enzymes which are synthesize ates es dis turbances turbances i n the the s ynthesized d in the hepatocyte= indic at hepatocellular synthesis . 

1.Markers of hepatocellular damage. 1.  A minotran min otranss aminas es   The transaminases are enzymes involved in the transfer of an amino group from a 2-amino- to a 2-oxoacid: they need the cofactor, pyridoxal phosphate for optimal activity. They are widely distributed in the body. The 2-oxoglutarate/L-glutamate couple serves as one amino group acceptor and donor pair in all amino-transfer reactions; the specificity of the individual enzymes derives from the particular amino acid that serves as the other donor of an amino group. Thus AST catalyzes the reaction:

Prof.Dr. H.D.El-Yassin 2011

 

  44  ALT catal catalyze yzes s the analogo analogous us reac reaction tion::

The reactions are reversible, but the equilibrium of AST and ALT reactions favor formation of aspartate and alanine respectively.

  Location:

o

   AST present present in cytos cytosol ol a and nd mitocho mitochondr ndria ia

o

   ALT loca located ted in c cytos ytosol ol o off liver liver

o

  In the liver, the concentration of ALT per unit weight of the tissue is more than AST.

o

  These enzymes are more important in assessing and monitoring the degree of liver cell

o

inflammation and necrosis.   The highest activities of ALT are found in hepatocytes and muscle cells.

o

   Aga  Again in the the hepat hepatocyte ocytes sh have ave very high high activity activity of AL ALT. T.

o

  Therefore elevations in serum ALT are considered to be relatively specific for liver disease.

o

   AST may may be eleva elevated ted in o other ther form forms s of tissue dam damage, age, such as myocar myocardial dial infarcti infarction, on,

o

muscle necrosis and renal disorders.   In liver disease, the ALT level is increased markedly compared to AST.

o

  In acute viral hepatitis there is a 100-1000 times increase in both ALT and AST but ALT

o

level is increased more than that of AST

Prof.Dr. H.D.El-Yassin 2011

 

  45

  Table Tab le:: A minotransferas minotransferase e activities activities in human human tiss ues, rel r elat ative ive to serum as unity uni ty

a. A spartate spartate Transaminase (E C 2.6.1.1; L-a L -ass pa parta rtate te:2 :2-oxog -oxoglut lutara arate te aminot minotransferase; ransferase; AS T) Clinicall S ig nificance Clinica nificance Normal values of AST:

male:

 µkat/L