Enzymes are organic catalyst that functions to enhance the rates of biochemical reactions from 106 to1012 times those of uncatalyzed reactions.
Characteristics of an enzyme
1. They are protein in nature.
2. They will only act on specific or particular substance or on closely related substance.
3. They are active only within a limited pH range.
4. They are destroyed easily by heat, acids or alkali.
5. They are present in small amount in the blood; therefore they cannot be measured in the usual terms of mg/100 ml but must be measured in special unit.
6. They speed or hasten the chemical reactions without being used up.
7. They act in several ways affecting hydrolysis, oxidation, reduction, etc.
8. They cannot be standardized efficiently as they are hard to obtain in pure forms.
9. Blood enzymes continue to act at room temperature even at refrigerator temperature so that the blood sample should be analyzed as soon as possible.
10. They may be safely stored through freezer.
System of naming enzymes
1. Most enzymes obtained their names by adding the suffix “ase” to the substrate that they act on.
e.g. lipase for fat
amylase acts on starch
protease acts on protein
sucrose acts on sucrose
uricase acts on uric acid
2. An enzyme is also named after its function rather than its substrate
e.g. hydrogenase – brings about the removal of hydrogen
oxidase – bring about oxidation
3. Some enzymes still maintain their original non–descriptive names
e.g. pepsin
ptyalin
trypsin
papain
Terms associated with enzymes
1. Holoenzymes – an active substance formed by combination of coenzyme and apoenzyme.
a. Apoenzyme – the protein portion is subject to denaturation, in which the enzyme loses its activity.
b. Cofactors – these are non–protein substances added in the enzyme–substrate complex before enzyme activity can be manifested.
(1) Coenzyme – an organic molecule that hastens enzymatic reaction but undergoes a change or is consumed to another product; the dialyzable portion of the haloenzymes.
Examples: NAD – nicotinamide adenine dinucleotide
NADP – nicotinamide adenine dinucleotide phosphate
Vitamins
(2) Activators – an inorganic ion that modifies reaction catalyzed
Examples:
(a) Amylase – Cl– , Br–
(b) LDH – Zn2+
(c) Lipase – Ca2+
(d) CPK / ALP – Mg2+
2. Isoenzymes – enzymes present in an individual with similar enzymatic activity but differ in their physical, biochemical and immunologic characteristics.
3. Metalloenzymes – enzymes whose metal ions are intrinsically part of the molecule
4. Proenzymes – inactive precursors of enzymes, also referred to as zymogen .
5. Substrates – substances acted upon by enzymes which are specific for each of their particular enzymes.
Enzyme kinetics
1. The amount of energy required to energize the substrate is known as the energy of activation (Ea). In many laboratory procedures, this energy is supplied by heat. However, since heat beyond normal body temperature (37oC) is injurious to cells, organisms must use a catalyst in the form of an enzyme to provide the Ea sufficient to accelerate the reaction.
2. nzymes function as biochemical catalyst by lowering the energy of activation, thus allowing the reaction to proceed at normal body temperature and at pace compatible with life.
3. Ezymes accomplish this task by attaching to the reacting substrate molecules, forming an enzyme–substrate (ES) complex.
4. This ES complex brings the substrate molecules into proper alignment with the enzymes so that its catalytic activity can be exerted and the product can be formed. Once catalysis occurred, the enzyme remains unchanged and is free to catalyze other reactions.
The general reaction can be written as,
E + S ------------> ES -----------> P + E
Where,
E = Enzyme
S = Substrate
ES = Enzyme–substrate complex
P = Product
5. One of the unique features of enzymes is its ability to exhibit substrate specificity.
Types of specificity enzyme exhibit
a. Absolute specificity – catalyzes only one specific reaction with one specific substrate
b. Group specificity – broader number of substrates or similar structural group can react with the enzyme
c. Bond specificity – enzyme which act on certain types of bonds such as peptide bonds of proteins or glyocosidic bonds of carbohydrates.
d. Stereospecific – react only with certain optical isomer.
Theories of enzyme specificity
a. Emil Fisher’s Lock and Key theory
It is based on the rigid enzyme molecule into which the substrate fits. The shape of the key (substrate) must fit into the lock.
b. Kochland’s induced fit theory
It is based on the attachment of a substrate to the active site of an enzyme, which then causes conformational changes in the latter. This is the more acceptable theory since the protein molecule is flexible enough to allow conformational changes and it somehow explains the influence of hormones on enzymatic activity.
Factors affecting enzyme activity
1. Substrate concentration
The more substrate molecules available, the more likely they are able to attach to the active sites of the enzymes, thus enabling the enzymes to exert its activity at maximum velocity. However, eventually a maximum rate is reached and any further increase in substrate concentration will not increase the velocity. At maximum velocity, the substrate concentration is sufficiently high so that all enzyme molecules have their active site engaged. As soon as a product exits the active site, another substrate molecule enters. The enzyme at maximum velocity is said to be saturated.
Types of reaction order:
a. First order kinetics – velocity is directly proportional to substrate
b. Zero order kinetics – maximum velocity is reached and any additional increase in substrate will not alter the velocity.
Michealis Menten hypothesis
An enzyme–substrate complex can either dissociates back to E +S or breakdown to product (P) or free the enzyme.
2. Enzyme concentration
The velocity of reaction is increased with an increased enzyme concentration and is decreased with lower enzyme concentration
3. Temperature
Reaction rate increases with temperature up to the optimum temperature which gives its maximal reaction. With every 10oC increase, the activity may increase 50 – 100. Some enzymes double their activity for every 10oC increase. Animal enzymes are destroyed above 60oC.
4. Hydrogen ion concentration (pH)
At its optimum pH, the enzyme will be most active. Deviation from this level will lower the reaction, inactivate or destroy the enzyme. Buffers are incorporated into the reaction mixture when measuring enzyme activity to prevent extreme fluctuation in pH during the enzymatic reaction.
5. Activators
Substances known as activators increase the rate of an enzymatic reaction. Activators are usually small molecules or ion such as metal ion. Common enzyme activators include ions such as Zn2+, Mg2+, Fe2+ and Mn2+. One mechanism of action of activators is to provide an electropositive active site that attracts negatively charged groups of substrate.
Other activators serve a structural function and aid in stabilizing the tertiary and quaternary structure of the enzyme. Regardless of their mechanism, activators must be present for those enzymes with such a requirement to enable their optimum enzymatic activity.
6. Inhibitors
In contrast to activators, inhibitors selectively bind to different sites of the enzyme molecule producing a varying effect on the velocity of the reaction. It has three types:
a. Competitive inhibitors
(1) These are similar to normal substrate molecule and compete with the substrate for binding to the active site of a specific enzyme.
(2) Can be reversed by increasing the concentration of the substrate.
(3) Example: sulfonamides
b. Non–competitive inhibitors
(1) Occurs when a substance binds to the enzymes at a site other the active site. This binding causes a conformational change in the structure of the enzyme in such a way that the active site is altered and is no longer receptive to the substrate.
(2) It can be reversible depending on the type of bond formed.
(3) Example: Lead and Mercury
c. Uncompetitive inhibitors
(1) It occurs when an inhibitor binds to the ES complex to form an enzyme– substrate–inhibitor complex that does not yield products.
(2) Increasing the substrate concentration actually increases the inhibition by providing more complexes to which the inhibitor can bind.
Measurement of enzyme activity
A. Reaction rate method
1. End poind method / Fixed incubation / Two–point assay
a. The reaction is allowed to incubate for pre–determined period. An absorbance reading is taken at the end of that time from which the enzyme activity is calculated.
Disadvantage:
a. Based on the assumption that the reaction progress is linear and follows zero – order kinetics. With no means of verifying that the reaction is linear, an error is more likely to go undetected, resulting in erroneous values.
2. Multiple point method
a. Makes several absorbance readings during the progress of the reaction, enabling the verification of a linear reaction. This means of analysis is more practical with the availability of microprocessor in modern computerized chemical analyzers. Any deviation from linearity can be immediately determined.
3. Continuous monitoring method
a. Uses a recording spectrophotometer to trace the progress of the reaction over a period of several minutes. The slope of the linear portion of the curve is used to determine enzyme activity. Any deviations from linearity can be immediately observed from the trace recording.
B. Progress curve in enzyme activity
1. Lag phase
a. Termed as the period of equilibrium, wherein serum enzymes and reaction mixture where brought together.
b. Enzyme activity should not be measured at this phase since the reaction rate is not yet in zero–order kinetics.
2. Initial rate
a. In this phase, the rate of product formation is constant and product concentration increases linearly with respect to time. The enzymatic reaction now exhibits zero–order kinetics and represents the phase during which the enzyme activity should be measured.
3. Reduced velocity
a. A progressive decline in product formation occurs at this stage as it enters first order kinetics.
b. Factors resulting in reduced velocity:
(1) Decreasing substrate concentration
(2) Establishment of equilibrium
(3) Progressive inactivation of the enzyme as the buffer becomes insufficient to control the pH.
c. This phase is also unsuitable for measurement of enzyme activity because in first order kinetics, the enzyme is not functioning at maximum efficiency.
C. Units for measuring enzyme activity
1. International Units (I.U. or U)
Equivalent to the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minutes under controlled condition.
2. Katal
Equivalent to the amount of enzyme that catalyzes the conversion of 1 mole of substrate per second under controlled condition.
Classification of enzymes
In an attempt to establish a systematic process for enzyme nomenclature, the Commission on Enzymes was establish in 1956 under the direction of International Union of Biochemistry. From the work of this original committee and subsequent recommendations, a completely revised version of enzyme nomenclature was published in 1972.
The numerical designation for each enzyme consist of four numbers separated by periods, e.g. 1.9.3.1
The first number defines the class to which the enzyme belongs. The enzymes are assigned to one of the 6 classes characterized by the type of reaction they catalyze.
The second figure gives more specific information about the enzyme. In the tranferase group, the second figure indicates the nature of the group transferred.
The third figure of the code gives more specific details about such things as the type of acceptor molecules for an oxidoreductase of the precise chemical type of group transferred by a transferase.
The fourth figure is the serial number of the enzyme in the class as indicated by the third element.
A. Oxidoreductase – catalyze the oxidation–reduction reactions between two substrates.
1. Oxidase
a. Cytochrome oxidase EC 1.9.3.1
2. Dehydrogenase
a. 3–hydroxybutyrate dehydrogenase EC 1.1.1.30
b. Lactate dehydrogenase EC 1.1.1.27
c. Glucose–6–phosphate dehydrogenase EC 1.1.1.49
d. Malate dehydrogenase EC 1.1.1.37
e. Isocitrate dehydrogenase EC 1.1.1.42
f. Glutamate dehydrogenase EC 1.4.1.2
B. Transferase – catalyze the transfer of a group other than hydrogen between two subunits.
a. Gamma glutamyl transferase EC 2.3.2.2
b. Aspartate aminotransferase EC 2.6.1.1
c. Alanine aminotransferase EC 2.6.1.2
d. Creatinine kinase EC 2.7.3.2
e. Ornithine–carbamoyl transferase EC 2.1.3.3
C. Hydrolase – catalyze the hydrolytic cleavage of compounds
1. Esterase
a. Acid phosphatase EC 3.1.3.2
b. Alkaline phosphatase EC 3.1.3.1
c. Cholinesterase EC 3.1.1.8
d. Triacylglycerol acylhydrolase (lipase) EC 3.1.1.3
2. Peptidase
a. Leucine aminopeptidase EC 3.4.1.1
b. Trypsin EC 3.4.21.4
c. Pepsin EC 3.4.23.1
d. 5’–nucleotidase EC 3.1.3.5
3. Glycosidase
a. Alpha–amylase EC 3.2.1.1
b. Amylo–1–6–glucosidase EC 3.2.1.33
c. Glucosidase EC 3.2.1.20
d. Galactosidase EC 3.2.1.23
D. Lyases – catalyze the removal of groups from substrates without hydrolysis, leaving double bonds in the product.
a. Aldolase EC 4.1.2.13
b. Glutamate decarboxylase EC 4.1.1.15
c. Tryptophan decarboxylase EC 4.1.1.28
E. Isomerase – catalyze the interconversion of isomers.
a. Glucose–6–phosphase isomerase EC 5.3.1.9
b. Ribose–5–phosphate isomerase EC 5.3.1.6
F. Ligases – catalyze the joining of two molecule coupled with the hydrolysis of a pyrophosphate bond in ATP or similar component.
a. Carbamoyl–phosphate synthetase EC 6.3.5.5
b. Acetyl–CoA carboxylase EC 6.4.1.2
****** LACTATE DEHYDROGENASE ******
A hydrogen transfer enzyme catalyzing the oxidation of L–lactate to pyruvate with the mediation of NAD as the hydrogen acceptor. The reaction is reversible, which strongly favors the reverse reaction which is the reduction of pyruvate to lactate. Specifically, it is important in the Embden–Meyerhoff metabolic pathway of glycolysis.
Inhibitors
1. Sulfhydryl reagents – e.g. mercuric ions
2. Excess pyruvate
3. Excess lactate
Tissue sources
LD is ubiquitous cytoplasmic enzyme found in nearly all cells of the body with highest activity in brain, erythrocytes, white blood cells, kidney, liver, lung, lymph nodes, myocardium, platelets and skeletal muscles.
LD occurs as a tetramer consisting of four polypeptide chains. Each chain or subunit maybe one of the two types and is designated H (heart) or M (muscle). Combinations of these subunits can result in any one of the five isoenzymes:
LD –1 – HHHH
LD–2 – HHHM
LD–3 – HHMM
LD–4 – HMMM
LD–5 – MMMM
LD–x – xxxx
Distribution of LD isoenzymes
a. LD–1 and LD–2 – fast moving fractions and are heat stable, found mostly in the myocardium and erythrocytes; also found in renal cortex.
b. LD–3 – found in a number of tissues predominantly in the white blood cells and brain
c. LD–4 and LD–5 – slow moving and are heat labile, found mostly in the liver and skeletal muscle; also seen in ileum and skin.
d. Each isoenzyme is broken down as follows:
(1) LD – 1 – 22 – 36%
(2) LD – 2 – 35 – 46%
(3) LD – 3 – 13 – 26%
(4) LD – 4 – 3 – 10%
(5) LD – 5 – 2 – 9%
Method of quantitation
1. Total LD
a. An optimum pH is required to catalyze the inteconversion of NAD+ and NADH at 340 nm. The basic reaction can thus proceed:
Forward: (L–lactate --------------------> pyruvate) pH 8.3 – 8.9
Reverse: (P–pyruvate -----------------> lactate) pH 7.1 – 7.4
b. Wrobleuski – La Due method
The method employs the pyruvate–lactate reaction and is followed by measuring the rate absorbance decrease at 340 nm and at 25oC as NADH is oxidized to NAD.
c. Cabaud – Wrobleuski
Pyruvate remaining in the solution after incubation in the presence of NADH by reacting it with 2, 4,dinitrophenylhdrazine to form the corresponding phenylhydrazone which has a golden–brown color at alkaline pH.
d. Nachlas – Raabo
NADH is coupled to the reduction of a tetrazolium salt (2–p–iodophenyl–3–p– nitrophenyl–5–phenyl tetrazolium chloride) with phenazine methosulfate (PMS) serving as intermediate electron carrier forming bright red color with maximum absorption at 500 nm.
2. LD isoenzymes
a. Chemical methods
(1) Substrate – product relationship
(2) Coenzyme affinity
(3) Differential chemical inhibition of LD activity
b. Physical methods
(1) Heat denaturation at 60oC for 30 minutes.
(2) Ion exchange chromatography using diethylaminoethyl (DEAE)
(3) Hydroxybutyrate substrate substitution
(4) Immunoprecipitation
(5) Electrophoresis
Specimen collection and handling
1. Serum is the specimen of choice however; heparinized plasma may also be used.
2. Oxalates inhibit LD activity
3. Ascorbic acid can also decrease LD activity.
4. Increased values can be obtained from hemolyzed samples and if not immediately separated from cells.
5. Serum for LD analysis should be kept at 25oC and should be analyzed within 24 hours of collection.
6. LD–4 and LD–5 are sensitive to cold exposure.
Clinical significance
A. Highest LDH value is seen in
1. Megaloblastic anemia
2. Extrinsic carcinomatosis
3. Severe shock
4. Anoxia
B. Moderate elevations (2 – 4 fold) is seen in
1. Myocardial infarction
2. Pulmonary infarction
3. Leukemia
4. Hemolytic anemia
5. Infectious mononucleosis
C. Elevated LDH activity have been reported in the urine of patients with the following disease:
1. Carcinoma of kidney or bladder
2. Malignant hypertension
3. Glomerulonephritis
4. Lupus nephritis
5. Acute tubular
Reference range:
1. Forward reaction: 100 – 226 U/L
2. Reverse reaction: 90 – 320 U/L
3. Newborn to 2 y/o: 450 U/L above
****** GLUCOSE–6–PHOSPHATE DEHYDROGENASE ******
Glucose–6–phosphate dehydrogenase (G–6–PD) is an oxidoreductase that catalyzes the first step in the pentose phosphate pathway of glucose metabolism. Specifically, it catalyzes the oxidation of glucose–6–phosphate to 6–phosphogluconate with the production of NADPH. NADPH is necessary to reduce glutathione, an important reducing substance that protects hemoglobin from oxidative denaturation.
Tissue sources:
1. Highest activity is seen in immature erythrocytes and decrease with cell maturation.
2. Minimal activity is seen in serum, adrenal cortex, lymph nodes, thymus and spleen.
3. Adrenal glands and mammary gland (especially during lactation) also has highest activity.
Inhibitors:
1. All heavy metals except cupric and zinc ions.
2. Inhibition can be counteracted by EDTA but not by sulfhydryl agents like cysteine.
Activators:
1. Magnesium
2. Calcium
Method of quantitation
1. Bishops
2. Ellis and Kirkman
3. Motulsky
4. Bentler and Mitchells
Clinical significance
Glucose–6–PD deficiency is a genetically determined disorder that results in an inadequate supply of NADPH and ultimately inadequate levels of reduced glutathione. Beutler Test is a screening procedure for G–6–PD deficiency.
Lack of adequate levels of reduced glutathione results in oxidative destruction of hemoglobin and various components of the cell membrane, rendering the red cell susceptible to hemolysis
Reference range:
1. Serum: 0 – 0.18 U/L
2. RBC: 0.117 – 0.143 U/L
****** SORBITOL DEHYDROGENASE ******
a. Found mainly in the liver.
b. Involved in the conversion of sorbitol to fructose
c. Normal serum contains little SDH but levels are markedly elevated in sera of patients with acute hepatitis.
d. Little SDH activity has been found in sera of patients with obstructive jaundice or in patients with acute myocardial infarction.
e. Determination of SDH should therefore provide valuable diagnostic assistance in the differences between hepatitis on one hand and obstructive jaundice and myocardial infarction on the other hand.
****** AMINOTRANSFERASE ******
The aminotransferase, commonly referred to as transaminase, constitute a class of enzymes that catalyze the transfer of an amino group from an alpha–amino to an alpha– keto acid forming a new amino acid and a new keto acid.
Two important members of this group are:
1. Aspartate aminotransferase – formerly known as Serum Oxaloacetic Acid Transferase (SGOT)
2. Alanine aminotransferase – formerly known as Serum Pyruvic Acid Transferase (SGPT)
The aminotransferase are relatively stable enzyme. They will maintain their activity for several days if the serum is stored in a refrigerator.
A. Aspartate aminotransferase
AST catalyzes the transfer amino group from L–glutamate or L–aspartate to alpha oxaglutarate or oxaloacetate.
L–aspartate + alpha–oxaglutarate --------------> oxaloacetate + L–glutamate
Although the reaction is reversible, the equilibrium favors the formation of aspartate. The coenzyme pyridoxal–5’–phosphate (P5P) binds to AST and thus serves as a necessary prosthetic group for full catalytic activity.
This enzyme is found in great concentration in the following organs and tissues:
1. Cardiac
2. Hepatic
3. Skeletal muscles
4. Kidneys
5. Brain
6. Pancreas
7. Spleen
8. Lungs
Method of quantitation
1. Spectrophotometric method
Serum is incubated with aspartic acid and alpha glutamic acid. Oxaloacetic acid, as it is formed is made to react with malic dehydrogenase in the presence of NADH. Oxaloacetic acid is transformed to malic acid by malic dehydrogenase and the NADH is oxidized to NAD. NADH shows strong absorption of ultraviolet rays at 340 nm while NAD has no specific absorption.
2. Colorimetric method (Reitman and Frankel)
The method measures the amount of oxaloacetic acid produced under fixed condition by the reddish–brown hydrazone it forms with 2,4 dinitrophenylhydrazine in alkaline solution.
Advantages of colorimetric method over the spectrophotometric methods are:
1. Procedure is simple.
2. Small amount of serum is needed.
3. Does not require a special kind of spectrophotometer.
4. Easier to control temperature
Temperature is a problem in spectrophotometric method. AST increases by 7% from each 1oC rise in temperature
Clinical significance
1. Myocardial infarction
2. Acute liver disease
a. Viral hepatitis
b. Toxic liver damage
3. Moderate increase occurs in other liver disease like
a. Late cirrhosis
b. Neoplasm of the liver
4. Certain forms of muscular dystrophy
5. Crush muscle injury
6. Dermatomyositis
B. Alanine aminotransferase
ALT along with P5P catalyzes the transfer of an amino group from L–glutamate or L –alanine to alpha ketoglutarate or pyruvate
Alanine + alpha – oxoglutarate --------> pyruvate + L – glutamate
Although the reaction is reversible, the equilibrium of the ALT reaction favors formation of alanine. Both reactions proceed to the right in vivo to provide a source of nitrogen for the urea cycle.
This enzyme is distributed in skeletal muscle, kidney, heart, pancreas, lungs, spleen and erythrocytes but is predominantly a liver–specific enzyme. Although a mitochondrial ALT exists in tissue, it has not been demonstrated in normal human serum.
Methods of determination
1. Spectrophotometric method
The pyruvic acid produced by the substrate alanine and alpha – ketoglutarate acid are converted to lactic acid by lactic dehydrogenase in the presence of NADH. The conversion of NADH to NAD is followed by a decrease in absorbance at 340 nm.
2. Colorimetric method (Reitman and Frankel)
The pyruvic acid formed in the reaction is measured by the reddish–brown hydrazone it forms with 2,4,dinitrophenyl–hydrazine in alkaline solution.
Principle:
AL T in serum is incubated with buffered substrate, DL alanine and alpha– ketoglutaric acid at pH 7 at 37oC for 30 minutes. The pyruvic acid reacts with with DNPH to form pyruvate dinitrophenylhydrazone which presents a brown color upon addition of an alkaline solution.
The procedure is similar to AST except for
a. The substrate used.
b. Incubation period which is ½ hour instead of 1 hour.
c. The substance that is formed is pyruvic acid which reacts with DNPH.
3. Enzymatic method
Measures the disappearance of NADH at 340 nm and proceeds as follows
Alanine + alpha–ketoglutarate -------> pyruvate + glutamate
pyruvate + NADH -----------------------> lactate + NAD+
4. Fluorescence method (Henry Pollard)
The disappearance of NADH in the proceeding coupled enzymatic reaction is monitored as a decrease in fluorescence
Clinical significance
1. Primarily used for the diagnosis of acute liver injuries like viral hepatitis
2. Maybe elevated in myocardial infarction
****** CREATININE KINASE ******
CPK catalyzes the reversible phosphorylation of creatine by adenosine triphosphate. The equilibrium position for the reaction is dependent on the hydrogen ion concentration.
Creatinine + ATP -------------> creatinine phosphate + ADP
Activators
1. Magnesium ions – required for maximum activity
2. Calcium – to a lesser degree.
3. Manganese
Inhibitors
1. Zinc ions
2. Cuprous ions
3. Silver ions
4. Mercurous ions
5. L–thyroxine
6. Iodoacetate
7. Fluorides
8. Citrates
9. EDTA
Tissue distribution
1. The largest distribution of CK is found in skeletal muscle. The remaining tissue sources in order of descending activity are brain, rectum, stomach, bladder, colon, uterus, prostate, small intestine and kidney. Negligible amounts can be detected in the liver, placenta and thyroid tissue.
2. It has the following isoenzymes
a. CK–1 or CK–BB – found predominantly in the brain and CNS; however, small quantities are found in numerous tissues, including the lungs, prostate, uterus and gastrointestinal tract. It migrates most rapidly toward the anode on electrophoresis.
b. CK–2 or CK–MB – is confined almost exclusively to cardiac muscle tissue. It is the second most rapid moving isoenzyme.
c. CK–3 or CK–MM – is the predominant isoenzyme in both skeletal and cardiac muscle.
d. CK–mito – rarely seen in normal human serum but its presence signals a grave prognosis for the patient because it indicated extensive tissue damage with release of mitochondrial content
e. CK–IgG complex or CK type I
f. CK–IgA complex or CK type II
Method of quantitation
1. Total CK
a. Forward reaction: pH 9.0
Creatinine ----------------> creatinine phosphate
b. Reverse reaction: pH 6.8
Creatinine phosphate ----------------> creatinine
Rosalki and oliver method (reference method)
Tanzer and Gilvarg method
a. ADP + PEP -----------> ATP + Pyruvate
b. Pyruvate + NADH ------------> pyruvate + NAD+
Sax and Moore
Creatinine reacts with ninhydrin (triketohydrinoene hydrate) solution to produce fluorphoe
Hughes method
Creatinine is made to react with diacetyl and alpha napthol to produce a pink end product.
Colorimetric method
a. ATP + creatinine --------->ADP + phosphocreatinine
b. Phosphocreatinine ----------> creatinine + inorganic phosphorous
2. CK isoenzymes
a. Electrophoresis
(1) CK bands are separated by applying an electromotive force at pH 8.6
(2) The relative amount of CK activity present in each band is determined by incubating the gels with a substrate mixture.
(3) The fluorescence NADPH generated by each band is proportional to the amount of CK activity in each band.
(4) To detect the amount of NADPH generated colorimetrically, addition of phenazine methosulfate and tetranitrozolium dye is coupled in the reaction and purple formazan is produced.
(5) To determine the relative percentage of CK activity in each band, the gels are then scanned by a densitometer.
Advantages:
(1) Rapid, has good sensitivity, requires only a very small amount of sample.
(2) Allows the technician to visualize the band and any isoenzyme variant.
Disadvantage:
(1) A semiquantitative method
b. Ion exchange chromatography – considered as the reference method
c. Immunoinhibition
d. Radioimmunoassay
Precaution in the assay
1. CK is heat labile, when serum is heated at 56oC for 15 minutes, there is total loss of activity.
2. CK is unstable in serum so that 45% of the original activity is lost in 4 hours at room temperature. Full activity can be restored by the addition of suitable sulfhydryl compound like cysteine and Cleland’s reagent.
****** ORNITHINE CARBAMOYL TRANSFERASE ******
This enzyme catalyzes the reversible conversion of ornithine to citrulline which is intimately involved in the synthesis of urea. The enzyme is found mostly in the liver and only 1% of this is found in the intestine.
The enzyme activity is markedly increased in acute viral hepatitis and other form of hepatic necrosis
Slight elevation of OCT is observed in
1. Obstructive jaundice
2. Cirrhosis
3. Metastatic carcinoma
4. Heart failures
5. Delirium Terence
6. Cholecystitis
7. Intestinal obstruction
Assay methodology
Ornithine + carbamoyl phosphate ------------>citrulline + H3PO4
The citrulline formed is measured colorimetrically
****** GAMMA GLUTAMYL TRANSFERASE ******
Gamma glutamyl transferase (GGT) is an enzyme belongings to the class of transferases. It is membrane–associated enzyme whose important function involves the transfer of the gamma glutamyl residue from glutathione and other gamma glutamyl peptides to amino acids or small peptides to form the gamma glutamyl amino acids and cysteinyl–glycine.
Gamma–glutamyl–p–nitronilide + glycylglycine ------> gamma–glutamyl–glycylglycine + p– nitroaniline
Tissue sources
1. Liver
2. Kidney
3. Intestine
4. Pancreas
Clinical significance
1. It has 85–93% sensitivity in the differential diagnosis of hepatic versus biliary disorders.
2. Five times above the upper reference range seen in biliary tract disorder in which cholestasis is the primary pathologic process such as biliary obstruction.
3. Two to five times above the reference range is seen in hepatic disease owing to hepatocellular dysfunction such as viral hepatitis and cirrhosis.
4. Because GGT is a microsomal enzyme, its synthesis is induced by alcohol and other drugs, making it a sensitive indicator of alcohol abuse.
5. Many drugs also induced the synthesis of GGT. These include phenobarbital, antidepressants, anticonvulsants such as phenytoin and some contraceptive pills containing estrogen.
Assay methodology
1. Spectrophotometric method
Gamma–glutamyl–p–nitroaniline -----------> p–nitroaniline
The absorbance is measured at 405 nm
Normal value:
Male: 6 – 45 U/L
Female: 5 – 30 U/L
****** PHOSPHATASE ******
These are a group of related enzymes, low specificity are characterized by their ability to hydrolyze a large variety of organic phosphate esters with the formation of an alcohol and phosphate ion. They attack only monoesters of phosphoric acid.
Clinically, there are three types of phosphatase:
1. Alkaline phosphatase (of serum, liver, bone, lungs, spleen, leukocytes and intestines) – with optimal activity at pH 9.8
2. Acid phosphatase (of serum, prostate, liver) – with optimal pH at 4.9 – 5.0.
3. Red cell phosphatase – with optimal pH 5.9 – 6.0.
Probable function of the phosphatase is the transfer of the phosphate groups from a color substrate to an acceptor compound containing an OH group.
A. Serum alkaline phosphatase
Serum phosphatase is the generic name for a group of enzymes that display maximum activity at pH 9.0 to 10.5. ALP functions to liberate inorganic phosphate from an organic esters with the concomitant production of alcohol.
H2O + R–HPO4 ---------------> R–OH + H2PO4
Activators: Mg++, Mn++
Inhibitors: PO4, Zn++, Ba++, SO4, oxalates
Tissue sources
1. Liver – the rapidly moving isoenzymes; 50 – 70% inactivated by heat; 0 – 10% inactivated by L – phenylalanine; strongly inhibited by urea.
2. Bone – the 2nd fastest moving isoenzyme; the most susceptible to heat inactivation; 0 – 10% inactivated by L – phenylalanine; strongly inhibited by urea.
3. Intestine – the 3rd fastest moving isoenzyme; 50 – 60% inactivated by heat; 75% inhibited by L – phenylalanine; moderately inhibited by urea.
4. Placenta – overlap with bone and liver bands in PAGE electrophoresis; resistant to heat inactivation, 75% inhibited by phenylalanine; moderately inhibited by urea.
5. Regan isoenzyme – also known as carcinoplacental isoenzyme because of its similarities to the placental fraction. It is the result of ectopic production from malignant tissue. It is the most heat resistant of the isoenzymes, resisting heat denaturation at 65oC for 30 minutes, but has low sensitivity since it is detected only in 3 – 15% of cancer patient.
6. Nagao isoenzyme – associated with carcinoma of the pleural cavity, pancreas and bile duct
Clinical significance
1. Increased values
a. Hepatic
(1) Cholestatic liver disease
(2) Hepatobiliary disease
(3) Infectious mononucleosis
(4) Cholestasis
(5) Cholangiolitis
(6) Portal cirrhosis
(7) Primary hepatocellular carcinoma
(8) Secondary metastatic liver carcinoma
b. Non–hepatic
(1) Acute and chronic pancreatitis
(2) Chronic renal failure
(3) Extrahepatic sepsis
(4) Intraabdominal bacterial infection
(5) Thyrotoxicosis
(6) Benign transient hyperphosphatemia
(7) Drugs: estrogens, progesterone, chlorpromazine
c. Osteoblastic
(1) Paget’s disease (osteitis deformans) – excessive bone destruction
(2) Ricketts
(3) Osteomalacia
(4) Hyperparathyroidism
(5) Acromegaly
2. Decreased values
(1) Hypothyroidism
(2) Scurvy
(3) Hypophosphatemia
(4) Kwashiorkor
(5) Cretinism
(6) Severe anemia
Method of quantitation
1. Total ALP
a. Bodansky method
Serum is added to sodium glycerophosphate that has been buffered to pH 8.6 to ensure optimum condition of pH and electrolyte content. The mixture is incubated at 37oC for exactly one hour. The phosphatase in serum decomposes sodium glycerophosphate during this incubation period and liberates phosphorous.
Phosphorous determination is then run on the incubated serum and on non– incubated serum which consists of adding trichloroacetic acid to both specimen to precipitate the proteins, adding molybdic acid to form a phosphate complex, adding a reducing agent to reduce the complex and finally measuring the depth of color in a colorimeter
The alkaline phosphatase units are then determined by subtracting the phosphorous value of the non–incubated specimen form the phosphorous value of incubated specimen.
b. Bessey –Lowry Brock method
Serum is added to p–nitrophenyl phosphate that has been buffered to pH 10.3 to ensure optimum conditions for reaction. The mixture is incubated at 37oC for exactly 30 minutes. During this period, phosphate hydrolyzes p – nitrophenylphosphate and liberates p–nitrophenol. The nitrophenol color intensity is measured in a colorimeter.
p–nitrophenyl phosphate + H2O ----------> p–nitrophenyl + H3PO4
c. Klein, Babson and Reed method
Phenolphthalein monophosphate + H20 --------------> phenolphthalein + H3PO4
Phenolphthalein formed is determined colorimetrically by its red color in alkaline solution
Advantages over p–nitrophenyl phosphate
(1) Absorption peak of phenolphthalein is much further from that of bilirubin and hemoglobin thus interference from this compound is reduced.
(2) For a given enzyme concentration, much more color is produced by phenolphthalein, thus increasing sensitivity of this method.
d. Huggins and Talalay
Phenolphthalein diphosphate is used as a substrate. The substrate gives linear reaction kinetic and is useful and sensitive substrate for assaying substrate.
e. Cornish method
The substrate 4–methyl umbelliferyl phosphate is hydrolyzed to 4–methyl umbelliferone which displays fluorescence at 360 nm.
f. King and Armstrong method
Phenylphosphate is used as substrate. The rate of reaction is followed by measuring the phenol formed. Phenol formed is measured using Folin Ciocalteau or amino antipyrine (King–King) or by reaction with a diazo reagent.
The method employing the Folin Ciocalteau reagents requires 30 minutes incubation period followed by deproteinization of the incubation mixture when antipyrine is used as the chromogenic reagent, deproteinization is not needed and a 15 minute reaction period is sufficient.
2. ALP isoenzyme
a. Electrophoresis
b. Heat inactivation
c. Inhibition by urea
d. Immunochemical method
Precaution in quantitation
1. The type of buffer present affects the rate of enzymatic activity. The buffers used are barbital, glycine, piperazine, MAP, Tris.
2. In the post–absorptive state, elevations of ALP occur in individuals who are of blood group B or O.
B. Acid phosphatase
Acid phosphatase (ACP) is a non–specific heterogenous group of phosphatase that belong to the class of hydrolase enzymes. It catalyzes the same reactions as ALP but functions in an acidic environment. The optimum pH ranges from 4.5 to 7.0
ACP catalyzes the hydrolysis of several orthophosphoric monoesters to yield the corresponding alcohol and inorganic phosphate as seen in the following equation.
H2O + R–HPO4 ---------------> R–OH + H2PO4
Tissue source
ACP has greatest concentration in prostate, liver, kidney, erythrocytes, platelets and osteoclastic cells of bone. Since ACP activity in the prostate is more than 100 times greater than in other tissues, it is primarily for prostate disorders that ACP measurement are requested.
Clinical significance
1. Prostatic
a. Prostatic carcinoma
b. Prostatic hypetrophy
ACP assays suffer from insensitivity in detecting carcinoma that has not metastasized and are not completely specific for the prostate. Measurement of prostate ACP (PAP) levels has been proven to be more sensitive.
2. Non–prostatic
a. Gaucher’s disease
b. Neimann–Pick disease
3. Osteoclastic
a. Hyperthyroidism
b. Paget’s disease
c. Multiple myeloma
4. Hematologic
a. Granulocytic leukemia
b. Acute / chronic lymphocytic leukemia
c. Plasma cell leukemia
d. Hairy cell leukemia
e. Polycythemia vera
f. Primary thrombocythemia
Method of quantitation
1. Total ACP
a. Bodansky method
Exactly the same procedure as the Bodansky alkaline phosphatase procedure except that the buffer solution is an acid buffer of pH 5.0 instead of 8.6.
b. King–Armstrong method
The same procedure for ALP except that the buffer solution is acidic (pH 5.0)
c. Bessey–Lowry–Brock method
Same as for ALP except the pH is at 4.8 and serum blanks are not incubated. The p–nitrophenol liberated during the incubation period is measured. The non– incubated serum blanks are also measured.
d. Roy and Hillman method
The substrate thymolphthalein monophosphate is hydrolyzed by the prostate ACP to yield thymol with the formation of blue color which is measured spectrophotometrically at 595 nm.
e. Babson, Read and Philips
The substrate alpha napthyl phosphate is acted upon by ACP to produce alpha napthol which is brown in color and can be determined spectrophotometrically.
2. Prostatic ACP
a. Differential substrate
b. Chemical inhibitors
c. Fluorometric assays
d. Immunologic techniques
(1) RIA
(2) Counterimmunoelectrophoresis
(3) Fluorescent immunoassay
(4) ELISA
Precaution in quantitation
1. ACP is unstable at temperature above 37oC.
2. 50% of the prostatic enzymes are particularly labile and over 50% activity may be lost in one hour at room temperature. To prevent this, 0.01 ml of 20% acetic acid per ml serum is added to stabilize the enzyme.
3. Hemolyzed serum should not be used because RBC contains high level of ACP.
4. Prostatic enzyme is strongly inhibited by tartrate while red blood cell acid phosphatase is inhibited by formaldehyde and cupric ions.
****** CHOLINESTERASE *****
Cholinesterase catalyzes the hydrolysis of choline esters to form choline and fatty acids as shown in the following reactions:
Acetylcholine + H2O ----------------> choline + acetic acid
Two types of cholinesterase
1. True cholinesterase / Acetylcholinesterase (AChE) – found primarily in the red blood cells and central nervous system and function in the hydrolysis of acetylcholine at the neuromuscular junction to allow nerve conduction.
2. Pseudocholinesterase (ChE) / Non – specifc cholinesterase – found primarily in the liver and white matter of the brain and accounts for the enzyme activity measured in serum or plasma. Its physiologic role is not known.
Clinical significance
1. As a measure of exposure of some organophosphorous compounds that are found in many insecticides and nerve gases.
2. Detection of abnormal variants of ChE that cannot hydrolyze succinyl choline, a muscle relaxant administered during the induction of anesthesia for surgical procedures.
3. Indication of synthetic capacity of the liver.
Method of quantitation
1. Base on the following reaction
Butyrylcholine ------------> thiocholine + butyric acid
Thiocholine + 5,5–dithosis–(2–nitrobenzene) --------------> 5–thio–nitrobenzoic acid
The assay is performed with or without dibucaine. The atypical variant of ChE is detected by its greater resistance to inhibition by dibucaine.
****** LIPASE *****
Lipase hydrolyzes glycerol esters of long chain fatty acid tricglycerides. LPS preferentially hydrolyzes the ester bonds of carbon 1 and 3 of the triglyceride molecule.
Triacylglycerol ----------------> 2–monoglyceride + 2 fatty acids
Activators:
1. Sulfhydryl compounds like cystine and thioglycollic acid
2. Bile salts – promote the formation of stable and finely dispersed emulsion of fat in water
3. Calcium – removes fatty acids liberated in the reaction by forming insoluble calcium salts.
Inhibitors
1. Heavy metal ion
2. Acrinine
3. Aldehyde
Tissue source
LPS activity has been observed in pancreas, intestinal mucosa, stomach, leukocytes and adipose tissue. However, only pancreatic lipase is of clinical significance. Its main function is to hydrolyze dietary triglycerides that have been emulsified by bile acids, thus aiding in fat absorption in digestive tract.
Clinical significance
Increase levels are seen in:
1. Acute pancreatitis
2. Acute alcohol poisoning
3. Accidental or surgical trauma to the abdomen
Method of quantitation
1. Cherry–Crandall method
Lipase in incubated serum and in the presence of a phosphate buffer catalyzes the hydrolysis of triolein (olive oil) with subsequent formation of oleic acids. The acids are then quantitated with the standard base using thymolpthalein indicator. The differences between the amount of standard base that neutralize the acid in the test and in the control represent the lipase activity present in serum.
2. Sigma–Tietz
Instead of olive oil as a substrate, a lipase substrate is used and titration is carried to the end point using N/10 NaOH as titrating agent and thymolphthalein as indicator
****** LEUCINE AMINOPEPTIDASE *****
Leucine aminopeptidase is a proteolytic enzyme which catalyzes the hydrolysis of the N – terminal amino acid of peptides during intestinal protein digestion
Activators:
1. Manganese ion
2. Magnesium ion
Inhibitor: EDTA
Tissue sources:
1. Liver
2. Kidney
3. Small intestine
Clinical significance
Increased levels are seen in
1. Acute pancreatitis
2. Carcinoma of the pancrease
3. Carcinoma of the bile duct
4. Viral hepatitis
5. Pregnancy
Method of quantitation
1. Based on the following reaction
a. Leucine–p–nitroanilide ----------> p–nitroaniline
b. L–leucyl–beta–napthylamide + H2O ---------> leucine + beta–napthylamine
The amount of beta–napthylamine is proportional to the amount of LAP activity. The beta napthylamine being colorless is diazotized with nitrous acid and then reacted with a dye base, N–(l–napthyl)–ethylenediamine hydrochloride to form a puplish colored complex azo dye that is proportional to the amount of beta–napthylamine formed.
2. Other suitable substrate are
a. L – leucyl amide
b. L – leucyl glycine
c. L – leucylglyglycine
d. Leucyl napthylamid
e. Alamyl – napthylamide
3. Optimal pH: 7.2 – 7.5
****** 5’ – NUCLEOTIDASE *****
5’–nucleotidase (5’–NT) catalyzes the hydrolysis of most ribonucleoside 5’–monophosphate and deoxynucleoside 5’–monophosphate to the corresponding nucleoside and orthophosphate. It is found predominantly in the liver.
Adenosine–5’–monophosphate + H2O ----------> adenosine + H3PO4
Inhibitor: Nickel ion
Method of quantitation:
1. Based on the following reaction
5’–adenosine monophosphate + H2O ---------> adenosine + H3PO4
Adenosine + H2O -----------> inosine + NH3
************ AMYLASE ***********
Amylases are group of enzymes belonging to class hydrolase which can split polysaccharides like starch and glycogen. It is a calcium–requiring metalloenzymes.
Two types of amylase:
1. Beta–amylase (bacterial amylase) – can act only at the terminal reducing ends of a polyglucan chain splitting off a section of a two glucose limits (maltose) at a time.
2. Alpha–amylase (animal amylase including man) – also referred to as endoamylase because they can attack alpha 1,4 linkage in a random manner anywhere in the polyglucan chain. Large polysaccharide molecules are rapidly broken down into smaller units.
Characteristics of alpha amylase:
1. Active at pH 6.9 – 7.0
2. Active at temperature as high as 50oC but it customarily assayed at 37–40oC.
3. Full activity is displayed only in the presence of a variety of univalent anions like chlorides, bromides, nitrates, chlorates, or monohydrogen phosphatase.
4. Molecular weight of amylase is 45,000.
5. The size is small enough to pass through glomerular filter.
6. Enzyme is stable, negligible activity is lost at room temperature in the course of one week and at refrigerator temperature over a two month period. All common anticoagulants except heparin inhibits amylase activity.
Two isoenzyme of alpha–amylase
1. Salivary amylase – fast moving, secreted also by lungs, bone and thyroid. Sixty percent of activity is derived from this isoenzyme.
2. Pancreatic amylase – slow moving, secreted exclusively in the pancreas. Accounts for 40% activity in serum. The P3 fraction is the most elevated in acute pancreatitis.
Methods of determination
1. Saccharogenic methods
The course of the enzyme reaction is followed by measuring the quantity of reducing sugars formed. Reducing sugars determined on PFF of the of reaction mixture.
2. Amyloclastic methods
Enzyme activity is evaluated by following the decrease in substrate (starch) concentration rather than measuring the product.
a. Chronometric method
The time required for amylase to completely hydrolyze all starch present in a reaction mixture is measured. The end point is reached when there is absence of any material capable of forming the blue starch iodine color.
b. Wohlgemuth
Serial dilution of the enzyme preparation is added to a fixed quantity of starch and the dilution is found which is just able to hydrolyze all the starch present in a fixed time.
c. Amylometric procedure
Measures the amount of starch hydrolyzed in a fixed period of time using the blue starch iodine color as the measure for quantitating starch.
3. Enzyme coupled assay
Maltotetraose + H2O ----------> 2 maltose
Maltose + Pi -------------------> glucose + B–glucose–1–P
B–glucose–1–P -----------------> glucose–6 – P
Glucose–6–P + NAD -----------> gluconate–6 –P + NADH + H+
MP – maltose phosphorylase
B–PGM–B – posphoglucose mutase
G–6–PD – glucose–6–phosphate–dehydrogenase
Precautions in specimen collection
1. Opiates and morphine causes false elevation in value.
2. Elevated triglycerides act as an inhibitor and falsely lower amylase levels when assayed with starch – iodine methods.
Clinical significance
1. Elevated levels (Hyperamylasemia)
a. Acute pancreatitis
b. Perforate peptic ulcers
c. Intestinal obstruction
d. Ruptured ectopic pregnancy
e. Salivary gland disease
f. Adenocarcinoma of the lung, ovary, etc.
Macroamylasemia – occurs when amylase binds with an immunoglobulin (IgA or IgA), forming a complex that is too large to be filtered by the glomerulus.
************ ALDOLASE ************
This enzyme belongs to a class of enzyme called lyases. It is the enzymes that takes part in the intermediary breakdown of glucose at the levels of fructose–1–6–diphosphate and convert it into dihydroxyacetone phosphate and glyceraldehyde–3–phosphate.
fructose–1–6–diphosphate dihydroxyacetone phosphate + glyceraldehyde–3–phosphate
The enzyme shows absolute specificity only for dihydroxyacetone (DHAP). The optimal pH for this enzyme is broad from 7.0 – 9.6. Heavy metals (Cu, Ag, Fe) inhibits its activity and should not be used. Chelating agents like EDTA do not counteract the inhibition. Its optimal activity is at 46oC.
The enzyme is present in all cells of the body but differences are observed in the rates at which the enzymes of various organs act on two substrate: fructose–1–6–diphosphate (FDP) and fructose–1–6–phosphate (F–1–P)
This serum enzyme is quite stable. Activity is unchanged for 48 hours at room temperature and remains unchanged for at least 3 – 4 weeks if the enzyme is refrigerated.
The level in red cells is about 150 times higher than that of serum; therefore hemolyzed serum should not be used for analysis. The enzyme is also found in CSF and serous effusions, but not in urine unless associated with proteinuria.
Clinical significance
1. ALS A – found in highest concentration in muscle. Increase levels is seen in progressive muscular dystrophy (Duchenne type, Erb’s paralysis)
2. ALS B – found predominantly in the liver. Increase levels are seen in acute and chronic hepatitis, cirrhosis, liver cell carcinoma and metastatic liver carcinoma.
3. ALS C – found predominantly in brain tissue. It is useful marker for cell damage within the CNS
Assay methodology
1. Coupled enzymatic procedure
fructose–1,6–diphosphate -------------> dihydroxyacetone phosphate + glyceraldehyde–3–phosphate
glyceraldehyde–3–phosphate------------> dihydroxyacetone phosphate
2 dihydroxyacetone phosphate + 2NADH ------------> 2 glyceraldehyde phosphate + 2NAD
TPI – triosephosphate isomerase
GD – glycerol phosphate dehydrogenase
Absorbance is measured at 340 nm.
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