Experiment 1: Total and Direct Serum Bilirubin Determination PDF

EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION OUTLINE ○ The liver of newborns is not fully developed at birth I. INTRODUCTION and cannot...

EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION OUTLINE ○ The liver of newborns is not fully developed at birth I. INTRODUCTION and cannot convert unconjugated bilirubin into II. METHODS OF BILIRUBIN DETERMINATION conjugated bilirubin i. Erlich / Diazo Reaction Hence, unconjugated bilirubin is an important ii. Van den Bergh and Muller measure for newborns. iii. Malloy-Evelyn iv. Jendrassik-Grof METHODS OF BILIRUBIN DETERMINATION v. Direct Spectrophotometric Methods vi. Enzymatic Methods 1. ERLICH / DIAZO REACTION vii. Wako DB Method Bilirubin reacts with Diazotized sulfanilic acid viii. Walter and Gerarde Method DIRECT REACTION ix. King and Coxon; Haslewood and King; McNee ○ Does not use Accelerator and Keefer INDIRECT REACTION x. Transcutaneous Measurement of Bilirubin ○ Requires an Accelerator III. EXPERIMENT 1A: TOTAL SERUM BILIRUBIN ACCELERATOR i. Principle ○ Allows reaction of unconjugated bilirubin with diazo ii. Materials and Equipment reagent iii. Reagents ○ Makes it water soluble iv. Sample Considerations v. Assay Conditions 2. VAN DEN BERGH AND MULLER vi. Procedure Simple direct Diazo Reaction a. Calculation Conjugated bilirubin reacts with Diazotized Sulfanilic Acid b. Linearity ○ Produces pigment Azodipyrroles c. Reference Values Reddish-purple at neutral pH IV. EXPERIMENT 1B: DIRECT SERUM BILIRUBIN Blue at high or low pH i. Principle Indirect Reaction: ii. Materials and Equipment iii. Reagent iv. Sample Considerations v. Assay Conditions vi. Procedure a. Calculation b. Linearity c. Reference Values 3. MALLOY-EVELYN V. QUESTIONS FOR RESEARCH Bilirubin reacts with Diazo Reagent at acidic pH (1.2) VI. FAQs AND NOTES Accelerator: 50% Methanol This method is virtually abandoned today Produces red-purple Azobilirubin INTRODUCTION ○ Max absorbance of 560 nm Interferences: BILIRUBIN ○ Methanol can cause turbidity due to protein Yellow bile pigment formed from hemoglobin during RBC precipitation destruction or senescence. ○ Hemoglobin causes a negative interference ○ Around 6g of hemoglobin is released daily. (increases the Bilirubin concentration) Formed EXTRA-HEPATICALLY (outside liver) ○ Has a longer reaction time ○ Hemoglobin enters the reticuloendothelial system ○ Serum is preferred sample (spleen, liver, bone marrow) ○ 1 mole of hemoglobin = 1 mole of bilirubin 4. JENDRASSIK-GROF ○ The bilirubin is in its unconjugated or FREE FORM Quantitatively measures unconjugated, monoconjugated, Conjugated HEPATICALLY (within liver) diconjugated, and delta bilirubin ○ Bilirubin is linked to a transporter called albumin Accelerator: aqueous solution of Caffeine and Sodium SOLUBLE FORM (Bilirubin glucuronate) OR Benzoate INSOLUBLE FORM (Calcium salts in gallstone) ○ Displaces the unconjugated bilirubin from its Bilirubin is removed as a waste product association sites on albumin by: ○ Increase in the formation or retention of bilirubin leads Formation of H-bond between bilirubin and to jaundice caffeine → becomes water-soluble → facilitates ○ Hyperbilirubinemia its reaction with diazo reagent. Can either be pre-hepatic, hepatic, or Formation and disruption of bilirubin internal post-hepatic (obstructive jaundice) hydrogen bonds.  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION Considerations: 7. WAKO DB METHOD ○ Not affected by pH changes = more stable rxn Oxidation method using Vanadate as an oxidizing agent pH can increase the amount of unconjugated Procedure: bilirubin that is measured as direct bilirubin. ○ Sample is mixed with reagent containing Vanadate at ○ Insensitive to a 50-fold variation in protein pH 3.0 concentration of the sample; ○ Bilirubin in sample is oxidized to biliverdin ○ Not affected by hemoglobin concentration up to 50 ○ Causes the yellow color, which is specific to bilirubin, mg/dL and above; to decrease ○ Maintains optical sensitivity even at very low bilirubin ○ Measure concentration of direct bilirubin concentrations. Measure absorbance before and after the Modifications: oxidation of Vanadate. ○ Reaction of bilirubin with diazo reagent in an Alkaline Condition 8. WALTER AND GERARDE METHOD After the formation of azobilirubin, Ascorbic Acid Coupling of bilirubin with diazotized sulfanilic acid in the is added to destroy the diazo reagent presence of Ethylene Glycol and Dimethyl Sulfoxide Tartrate solution is added to intensify the blue Produces a diazo dye color of azobilirubin Intensity of color = directly proportional to bilirubin 9. KING AND COXON; HASLEWOOD AND KING; concentration at 600 nm. MCNEE AND KEEFER 5. DIRECT SPECTROPHOTOMETRIC METHOD Involve a diazo reaction Accelerator: Ethanol Total Bilirubin determination using Two-component Considerations: system ○ Protein is precipitated ○ Measures absorbance at two wavelengths Azobilirubin is lost on the protein precipitate ○ Solving a system of two simultaneous equations ○ Volume of colored supernatant fluid or filtrate varies based on the spectral absorbance from test to test Considerations: ○ Used in the serum of neonates Only unconjugated bilirubin is present 10. TRANSCUTANEOUS MEASUREMENT OF ○ Not used for adults BILIRUBIN Presence of carotenoid compounds that give a Easy, painless, time-saving alternative for bilirubin positive interference. measurement among neonates ○ Correction for oxyhemoglobin is necessary ○ Since total serum bilirubin requires venous or heel prick blood samples, which are invasive and painful 6. ENZYMATIC METHODS Uses a device that directs white light into the skin Based on the oxidation of bilirubin with bilirubin oxidase ○ Measures the intensity of the specific wavelengths ○ Produces biliverdin and molecular Oxygen that are transmitted Considerations: Not suitable for laboratory determinations ○ pH near 8 Useful in determining if a blood draw is necessary to treat ○ Sodium cholate and sodium dodecyl sulfate a jaundiced infant. ○ All four bilirubin fractions are oxidized to biliverdin Oxidized to a purple and colorless product Results: ○ Decrease in absorbance at 425 nm or 460 nm is proportional to concentration of total bilirubin ○ At pH 3.7 to 4.5 Unconjugated bilirubin is not measured ○ At pH 10 Monoconjugated and diconjugated bilirubin are oxidized 5% of unconjugated bilirubin is measured as conjugated bilirubin Figure 1. For legal purposes, this is a joke. EXPERIMENT 1A: TOTAL SERUM BILIRUBIN DETERMINATION PRINCIPLE MATERIALS AND EQUIPMENT PHOTOMETRIC COLORIMETRIC METHOD Venipuncture set, 70 % ethyl alcohol, cotton balls by JENDRASSIK-GROF 13 x 100 mm and 12 x 75 mm test tubes Direct and Indirect Bilirubin reacts with Diazotized Cuvettes Sulfanilic Acid in the presence of an accelerator Pasteur and serological pipettes with rubber pipettol ○ Forms a purple azobilirubin Test tube rack Read spectrophotometrically at 540 nm. Labeling tape; piece of paper to cover the sample tube Calibration is done using Pure Aromatic Amine Bilirubin reagent kit ○ This couples with the diazo reagent to yield a colored Centrifuge complex similar to serum bilirubin. G. DIZON, A. DELA CRUZ Page | 2  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION REAGENT COMPOSITION Serum bilirubin is stable for: Total Bilirubin reagent ○ 1-2 days at room temperature ○ 0.032 M sulfanilic acid ○ 3 weeks at 2-8 C ○ Accelerator and stabilizer added ○ Up to 3 months in freezing (-20 C) temperature Bilirubin activator ○ Provided that they are kept in the dark. ○ 60 mM Sodium Nitrite ○ Stabilizer added ASSAY CONSIDERATIONS Bilirubin Calibrator ○ 0.346 mM N-1-naphthylethylenediamine Wavelength 540 nm ○ Stabilizer added Optical Path 1 cm Temperature 20-25 C SPECIMEN CONSIDERATIONS Measurement Against sample blank Fresh, hemolysis-free serum or (without nitric reagent) Heparinized plasma. Carefully protect the sample from light until use. ○ Carbon paper may be used to cover the sample tube. PROCEDURE Table 1: Procedure for Total Serum Bilirubin Determination 1 Perform venipuncture observing correct patient identification, proper aseptic technique, and patient care. 2 Prepare serum from the blood collected. Avoid exposure of the sample to light by covering with carbon paper. 3 Label tubes Reagent Blank Standard Patient Sample Control 4 Pipet Total Bilirubin Reagent 1.0 mL 1.0 mL 1.0 mL 1.0 mL 5 Pipet Bilirubin Activator 0.04 mL 0.04 mL 0.04 mL 0.04 mL 6 Cover with parafilm and mix by inversion 7 Pipet Distilled Water 0.1 mL -- -- -- 8 Pipet Calibrator Reagent -- 0.1 mL -- -- 9 Pipet Patient Serum -- -- 0.1 mL -- 10 Control -- -- -- L1 0.1 mL L2 0.1 mL 11 Cover with parafilm and mix by inversion. Let stand for 90 seconds at room temperature. 12 Zero the spectrophotometer at 540 nm with Reagent Blank. 13 Read absorbances of the standard, patient sample, and control. Record the readings. CALCULATION OF RESULTS REFERENCE VALUES Total Bilirubin (Laboratory Manual) 𝑚𝑔 𝐴𝑏𝑠 𝑜𝑓 𝑈𝑛𝑘 At birth Up to 5 mg/dL Up to 85.5 μmol/L 𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑈𝑛𝑘 ( 𝑑𝐿 ) = 𝐴𝑏𝑠 𝑜𝑓 𝑆𝑡𝑑 𝑥 𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑆𝑡𝑑 5 days old Up to 12 mg/dL Up to 205.0 μmol/L 1 month old Up to 1.5 mg/dL Up to 25.6 μmol/L Concentration of Standard = 5.0 mg/dL Adults Up to 1.1 mg/dL Up to 18.8 μmol/L mg/dL to SI units (μmol/L): result x 17.1 Total Bilirubin (Bishop, 7th Ed.) LINEARITY Adults 0.2-1.0 mg/dL 3-17 μmol/L The assay is linear up to 20 mg/ dL. Premature Infants For results that exceed 20 mg/dL: - At 24 hours 1-6 mg/dL 17-103 μmol/L ○ Dilute 1 mL serum with 4 mL physiologic saline - At 48 hours 6-8 mg/dL 103-137 μmol/L Physiologic Saline: 9 g/L NaCl - At 3-5 days 10-12 mg/dL 171-205 μmol/L This creates a 1:5 dilution of serum and saline Full-term Infants Repeat the assay and multiply the result by 5. - At 24 hours 2-6 mg/dL 34-103 μmol/L - At 48 hours 6-7 mg/dL 103-120 μmol/L - At 3-5 days 4-6 mg/dL 68-103 μmol/L EXPERIMENT 1B: DIRECT SERUM BILIRUBIN DETERMINATION Calibration is done using Pure Aromatic Amine PRINCIPLE ○ This couples with the diazo reagent to yield a colored complex similar to serum bilirubin. PHOTOMETRIC COLORIMETRIC METHOD by JENDRASSIK-GROF MATERIALS AND EQUIPMENT Conjugated Bilirubin reacts with Diazotized Sulfanilic Venipuncture set, 70 % ethyl alcohol, cotton balls Acid without an accelerator since it is water soluble 13 x 100 mm and 12 x 75 mm test tubes ○ Forms a colored complex Cuvettes Read spectrophotometrically at 540 nm. Pasteur and serological pipettes with rubber pipettol G. DIZON, A. DELA CRUZ Page | 3  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION Test tube rack SPECIMEN CONSIDERATIONS Labeling tape; piece of paper to cover the sample tube Fresh, hemolysis-free serum or Bilirubin reagent kit Heparinized plasma. Centrifuge Carefully protect the sample from light until use. ○ Carbon paper may be used to cover the sample tube. REAGENT COMPOSITION Serum bilirubin is stable for: Direct Bilirubin reagent ○ 1-2 days at room temperature ○ 0.032 M sulfanilic acid ○ 3 weeks at 2-8 C ○ Accelerator and stabilizer added ○ Up to 3 months in freezing (-20 C) temperature Bilirubin activator ○ Provided that they are kept in the dark. ○ 60 mM Sodium Nitrite ○ Stabilizer added ASSAY CONSIDERATIONS Bilirubin Calibrator ○ 0.346 mM N-1-naphthylethylenediamine Wavelength 540 nm ○ Stabilizer added Optical Path 1 cm Temperature 20-25 C Measurement Against sample blank (without nitric reagent) PROCEDURE Table 2: Procedure for Direct Serum Bilirubin Determination 1 Perform venipuncture observing correct patient identification, proper aseptic technique, and patient care. 2 Prepare serum from the blood collected. Avoid exposure of the sample to light by covering with carbon paper. 3 Label tubes Reagent Blank Standard Patient Sample Control 4 Pipet Direct Bilirubin Reagent 2.0 mL 2.0 mL 2.0 mL 2.0 mL 5 Pipet Bilirubin Activator 0.04 mL 0.04 mL 0.04 mL 0.04 mL 6 Cover with parafilm and mix by inversion 7 Pipet Distilled Water 0.1 mL -- -- -- 8 Pipet Calibrator Reagent -- 0.1 mL -- -- 9 Pipet Patient Serum -- -- 0.1 mL -- 10 Control -- -- -- L1 0.1 mL L2 0.1 mL 11 Cover with parafilm and mix by inversion. Let stand for exactly 3 minutes at room temperature. 12 Zero the spectrophotometer at 540 nm with Reagent Blank. 13 Read absorbances of the standard, patient sample, and control. Record the readings. CALCULATION OF RESULTS Direct Bilirubin: REFERENCE VALUES 𝑚𝑔 𝐴𝑏𝑠 𝑜𝑓 𝑈𝑛𝑘 Direct Bilirubin (Laboratory Manual) 𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑈𝑛𝑘 ( 𝑑𝐿 ) = 𝐴𝑏𝑠 𝑜𝑓 𝑆𝑡𝑑 𝑥 𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑆𝑡𝑑 Adults and Infants over 1 month 𝐾 𝑚 Zero order High [𝑆 ] reaction ([𝑆]+𝐾𝑚 ) for legal purposes, this is a joke. S.S. DE SAGUN, J. LICAY, G. DIZON, A. DELA CRUZ, S. HAMADAIN Page | 3  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION MEASUREMENT OF ENZYME ACTIVITY Enzyme assays MUST be performed ○ Because a constant amount of activity can be In most enzymatic procedures, the reaction rates are not determined constant with time. They may depend on: ○ Measurement does not start at zero time but begins ○ increase in product concentration after the lag phase has occurred ○ decrease in substrate concentration the measurements can be made at any time during this ○ decrease in coenzyme concentration phase up to the substrate depletion phase ○ increase in the concentration of an altered coenzyme at a specific wavelength NOTE: Measurement of enzyme activity is ideally performed during the linear phase. 1. Enzyme Activity is Obtained by the Measuring any of its Succeeding Reactions SUBSTRATE-DEPLETION PHASE For example, in Aspartate Aminotransferase (AST) Assay: the time late in an enzyme assay when substrate ○ The rate of decrease in the concentration of NADH is concentration is falling measured, which is proportional to the enzyme assay is NOT following the zero-order kinetics activity in the sample. little change in absorbance per unit time, thus enzyme ○ the reaction measured in this assay in the decrease assays MUST NOT be performed in the concentration of the coenzyme - which is if enzyme activity is too great, substrate concentration NADH may occur before the measurements have been completed 2. Some Enzymes do not form Substrates that can be ○ Corrective measures: Measured Directly dilute the sample to the point that the diluted activity is within range of the enzyme assay Thus, it would require more than one enzyme to be added in excess as a reagent so that multiple reactions NOTE: remember that dilution is not applicable if inhibitors are catalyzed are present For example, in the AST Assay: ○ In this reaction, the first products of the reaction (glutamate and oxaloacetate) are not measured. ○ However, the products of the first reaction becomes the substrates for an intermediate reaction, which is catalyzed by an intermediate auxiliary enzyme ○ The product of this intermediate reaction then becomes the substrate for the final reaction, which is catalyzed by an indicator enzyme. The indicator enzyme commonly involves the conversion of NAD to NADPH or vice versa This final reaction is measured ○ The initial enzyme usually is coupled to a second indicator enzyme reaction that does not contain a substrate to make a convenient assay. METHODS FOR MEASURING ENZYMATIC ACTIVITY 3. Not all enzyme reactions involve coupling of multiple enzymes FIXED TIME ASSAY For example, Gamma-glutamyl transferase (GGT) Test measures enzyme concentration in fixed periods of time ○ Here, the product of the initial reaction is directly Sequential Process: measured Reactants are combined → Reactants proceed for a designated time → PHASES OF ENZYME ACTIVITY reaction is stopped → a measurement is made from the amount of reaction that has occurred. LAG PHASE NOTE: the LARGER the reaction, the MORE ENZYMES The early time in an assay are present ○ when temperature and kinetic equilibrium is the reaction is linear over the reaction time established MAJOR PROBLEMS: there is little change in the absorbance per unit time ○ when there is a sample with a high activity ○ thus NOT recommended for measuring enzymatic ○ when substrate depletion occurs; activity ○ then the rate of reaction drops off part way through the assay; LINEAR PHASE ○ thus, the true activity of the enzyme is underestimated When assay is following zero order kinetics ○ Produces constant amount of product per unit time COMMON FIXED TIME ASSAY METHOD There is constant absorbance change per unit time due done using a microplate reader to a constant rate of product formation ○ to read multiple solution concentrations S.S. DE SAGUN, J. LICAY, G. DIZON, A. DELA CRUZ, S. HAMADAIN Page | 4  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION multiple dilutions for substrate and dilutions for enzymes FACTORS AFFECTING ENZYME MEASUREMENT / are prepared RATE OF ENZYME REACTION ○ while solutions are placed onto the well sequential process: 1. Substrate ↑ substrate concentration = ↑ reaction Concentration velocity until it reaches a maximum Reaction starts → solutions are incubated for a fixed period ○ This assumes that the enzyme of time → a stop solution is added to stop enzyme and concentration is constant substrate reaction After reaching the maximum, increases in the substrate concentration will not this type of assay was introduced in most of the enzymes increase the velocity assay performed in Biochemistry laboratory 2. Enzyme To study effect of increasing enzyme Concentration concentration, the substrate must be CONTINUOUS MONITORING ASSAY present in an excess amount ○ In other words, the reaction must Multiple measurements are made during the reaction be independent of the substrate ○ usually absorbance change concentration) During reaction, the enzyme activity is being measured ○ This means that as long as the ○ In the form of absorbance substrate concentration exceeds Adequately verifies the linearity of the reaction the enzyme concentration, the ○ deviation from linearity is readily observed velocity of the reaction is Common cause of deviation: proportional to the enzyme □ when enzyme is so elevated concentration □ all substrate is used early in the reaction time ↑ enzyme concentration = faster ○ because multiple data points were obtained reaction rate since more enzymes are Optimum pH: determined before conducting a continuous present to bind with the substrate enzyme assay 3. pH pH affect enzymes since it is a factor in DISADVANTAGE: their stability ○ only one reaction can be measured at a time interval Changes in pH may denature an enzyme or influence its ionic state UNITS OF ENZYME ACTIVITY ○ This results in structural changes or changes in the charge on an The quantification of enzymes is based on their catalytic amino acid residue in the active activity. site Results of enzyme determination are expressed as: Each enzyme has a region of pH ○ an activity unit in terms of the amount of product optimal stability formed per unit of time under specific conditions for a ○ Optimum pH: most favorable pH given volume of sample value at which the enzyme is most Thus, 1 unit of enzyme activity might be the amount of active enzyme that would (under certain conditions) cause the ○ Extremely high or low pH: cause formation of milligram (mg) of the product per minute when complete loss of activity for most 1 ml of the sample was used. enzymes 4. Temperature Temperature Effect to Enzymes INTERNATIONAL UNIT OF ENZYME ACTIVITY (IU) amount of enzyme that would convert 1 micromole of 25, 30, 37 C Enzymes are active substrate per minute under standard conditions (such as 37 C Optimum temp for temperature, pH, substrate concentration) to produce enzymatic reaction maximal catalytic activity of the enzyme 40-50C Denaturation 60-65 C Inactivation of SI UNIT (KATAL) enzymes reflects amount of enzyme that would convert 1 mole of Low Temp. Enzymes reversibly substrate per second under standard conditions (such as temperature, pH, substrate concentration) inactive This is the recommended unit of enzyme activity by the Every 10 C Two-fold increase in IFCC and IUPAC increase enzyme activity ○ but, many find this as an inconveniently small unit of Generally, ↑ temperature = ↑ rate of activity so they continue to use IU chemical reaction because more energy is available for the reaction. 1 IU = 1 umol/min = 16.67 nmol/s In most enzyme reactions, for every 10 16.67 nanokatal = 16.67 nmol/s degree increase in temperature, the rate of reaction doubles until it is Therefore, 1 IU = 16.67 nanokatal denatured. 5. Buffer Acts as an acceptor IU/L ○ Example, in ALP, a buffer serves Currently being used in expressing enzyme activity as an acceptor of the phosphate amount of enzyme that would cause the formation of a group removed from the substrate milligram (mg) of the product per minute per liter of sample Changes in its concentration and example: 40 IU/L AST activity structure may affect enzyme reactions S.S. DE SAGUN, J. LICAY, G. DIZON, A. DELA CRUZ, S. HAMADAIN Page | 5  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION ○ When the buffer to substrate ratio is very large, the buffer may compete with the substrate for the enzyme. ○ It may also make the enzyme activity appear to be related to substrate concentration in a nonlinear way. 6. Cofactors These are non-protein substances needed by the enzyme to perform its catalytic function In the absence of cofactors, enzyme 3. COMPETITIVE INHIBITORS activity may not occur; in excess, it may Binds to the enzyme-substrate complex, thus no free also inhibit enzyme activity enzyme is released 7. Inhibitors They alter the catalytic action of the Increasing the substrate concentration results in more enzyme and slow down or stop the enzyme-substrate complexes (to which the inhibitor binds), catalysis thus increasing the inhibition They may also act by removing an Does not yield a product activator or by binding to the enzyme Inhibition of enzymes may be reversible or irreversible ○ Irreversible Inhibition = a covalent bond is formed between the inhibitor and the enzyme Thus, enzyme activity cannot be restored by dissociation of the inhibitor ○ Reversible Inhibition = a hydrogen bond is formed between the inhibitor and the enzyme Thus, the enzyme activity may be restored after removal of the inhibitor THREE COMMON TYPES OF INHIBITORS FACTORS AFFECTING REFERENCE VALUES OF 1. COMPETITIVE INHIBITORS ENZYMES Bind to the active site of the enzyme; compete with substrate for the active site 1. Sampling enzymes do not undergo significant This type of inhibition is reversible when there is a circadian rhythm Time significantly higher substrate concentration compared to the ○ Thus, sampling time with respect inhibitor concentration. to time of the day is unimportant ○ This is because the substrate is more likely to bind for the determination of enzyme the active site than the inhibitor reference intervals BUT, sampling time may be important for detection of a variety of acute and chronic conditions ○ This may affect values of enzymes necessary for observation and diagnosis of diseases Example: in myocardial infarction, CK-MB levels begin to rise within 4-8 hrs and peak at 12-24 hrs; then they return to normal within 48-72 hrs. 2. Age Example: alkaline phosphatase level is higher among children due to 2. COMPETITIVE INHIBITORS increased osteoblastic activity Bind an enzyme at a site other than the active site associated with bone growth. Since the inhibitor binds independently from the substrate, Thus, in most labs, reference values increasing the substrate concentration will not reverse the for ALP varies with different population inhibition 3. Sex Differences in sex are related to Some substances may reversibly bind to the enzyme, but muscle mass, exercise, or hormone most commonly, binding of this inhibitor may abolish concentration enzyme activity Example 1: since creatinine kinase is This inhibitor may also rarely bind to the enzyme-substrate related to muscle mass, activity is complex, thus abolishing the reaction higher in males than in females S.S. DE SAGUN, J. LICAY, G. DIZON, A. DELA CRUZ, S. HAMADAIN Page | 6  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION Example 2: alcohol dehydrogenase level in gastric mucosa is higher in males than in females; ○ Thus, males metabolize ethanol more rapidly than females 4. Race Example: creatinine kinase is higher among blacks than in whites, due to attribution with muscle mass. 5. Exercise Example: creatinine kinase is higher among ambulatory patients than patients in a prolonged bed rest. CHANGES IN KM AND VMAX IN PRESENCE OF INHIBITORS Given the following types of inhibitions, determine what will happen to the Km and Vmax. Would it increase, decrease or will not be affected? Type of inhibition Change in Km Change in Vmax Competitive Increased Unaffected Noncompetitive Reduced Reduced Uncompetitive Unaffected Reduced S.S. DE SAGUN, J. LICAY, G. DIZON, A. DELA CRUZ, S. HAMADAIN Page | 7  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION QUESTIONS FOR RESEARCH e. SALT AND PROTEIN CONCENTRATION ☺ Increase ionic strength = decrease in activity 1. Explain fully the specificity of enzyme reaction. Increase in protein concentration = increase enzyme activity Enzymes bind only to its designated substrate and react only to the reaction they are supposed to catalyze f. COFACTORS An enzyme has an active site that only its substrate can Non protein molecules that need to bind to an react and bind with. enzyme before a reaction occurs ○ ACTIVATORS = inorganic cofactors that Enzyme Specificity Mechanism: modify the spatial configuration of enzymes ○ COENZYME = organic cofactors that act as Covalent bond on substrate interacts with reactive side chain a 2nd substrate moieties of active site → covalent bond weakens → decreased activation energy for chemical reax → weakened bonds → breaks g. ANTICOAGULANTS the covalent bond → allows new bonds to form → product loses the HEPARIN = may inhibit amylase and ALT same affinity for the active site CITRATE = falsely lowers CK and ALP h. STORAGE 2. Explain the Michaelis-Menten kinetics of enzyme -20 C = preservation for a long period of time reaction. 2-8 C = ideal for storing substrates & coenzymes ROOM TEMP = ideal for storing LD enzymes It is a visual representation that describes their kinetic (lactate dehydrogenase) activity Represents the relationship of the substrate i. INHIBITORS concentration to the velocity rate. Competitive inhibition Low substrate conc.: Uncompetitive inhibition ○ the rate is dependent on the substrate and follows the Noncompetitive inhibition First order Kinetics High substrate conc.: 5. What are the reasons for the abnormal levels of ○ Reaction no longer increases and remains constant enzymes in blood? ○ Follows the Zero order kinetics a. SAMPLING TIME 3. What is the Lineweaver-Burke equation? b. AGE c. SEX Lineweaver-Burke equation is the double reciprocal plot of the d. RACE Michaelis-Menten curse. e. EXERCISE Allows the determination of the Vmax (maximum velocity) and Km (substrate concentration) 6. Describe briefly: ○ Vmax = reciprocal of the x-intercept of the straight line ○ Km = negative reciprocal of the x-intercept of the same line a. FIXED TIME REACTION Reactants are combined → 4. Give and briefly describe the various factors that Reaction proceeds for a designated time → affect enzyme assays. Reaction is stopped by inactivating the enzyme with a weak acid → a. SUBSTRATE CONCENTRATION Measurement of the amount of reaction occurred The substrate readily binds to a free enzyme at The reaction is linear low-substrate concentrations ○ The larger the reaction, the more enzyme present The reaction rate steadily increases as more substrates are added b. CONTINUOUS MONITORING (aka Kinetic Assay) If the substrates reach a maximal value, adding Multiple measurements are made during the reaction more substrates no longer results in increased ○ Done at specific time intervals (e.g., every 30 or rate of reaction due to enzyme saturation. 60 seconds); or b. ENZYME CONCENTRATION ○ Done continuously with a continuous-reading If the enzyme concentration/level is high: spectrophotometer ○ Reaction is fast since more enzymes are Advantages over Fixed Time: available for reaction ○ The linearity of a reaction may be more adequately verified. c. TEMPERATURE Measurement of absorbance at different Higher temperature = faster reaction since it intervals increases the accuracy of linearity allows faster movement of molecules assessment. Two fold increase in enzyme activity for every 10 C increase in temperature c. MULTI-ENZYME METHODS A profile of enzymes that catalyzes a sequence of d. PH reactions in a biochemical pathway Most reactions occur in pH: 7.0-8.0 ○ The product of the first enzyme reaction becomes Extreme pH: the substrate of the second enzyme reaction and ○ Denatures enzymes (structural); or so on until the target substrate is produced ○ Influences the ionic states of amino acids on ○ The amount of target substrate is measured the active site. The rate of reaction depends on the enzyme and substrate concentration. S.S. DE SAGUN, J. LICAY, G. DIZON, A. DELA CRUZ, S. HAMADAIN Page | 8  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION 7. Answer briefly: HYDROLASE Esterase a. What are isoenzymes? How do they differ from each Acid phosphatase 3.1.3.2 other? Isoenzymes are enzymes similar in enzymatic activity Alkaline phosphatase 3.1.3.1 but different in terms of their physical, biochemical, and Cholinesterase 3.1.1.8 immunological properties. Triacylglycerol acylhydrolase (lipase) 3.1.1.3 Slight different molecular structure due to amino acid Peptidase differences Leucine aminopeptidase 3.4.1.1 Trypsin 3.4.21.4 b. Describe some techniques to measure or Pepsin 3.4.23.1 differentiate isoenzymes. Electrophoresis 5’–nucleotidase 3.1.3.5 ○ Different molecular structure = different mobility Glycosidase in electrophoresis Alpha–amylase 3.2.1.1 Resistance to heat denaturation Amylo–1–6–glucosidase 3.2.1.33 ○ Larger structure = more resistance Glucosidase 3.2.1.20 Galactosidase 3.2.1.23 c. What is the clinical relevance of isoenzyme studies? LYASES In plasma, the pattern of isoenzymes may serve as a means of identifying the site of tissue damage. Aldolase 4.1.2.13 Example: Glutamate decarboxylase 4.1.1.15 ○ Plasma levels of creatine kinase (CK) are Tryptophan decarboxylase 4.1.1.28 commonly determined in the diagnosis of ISOMERASE myocardial infarction (MI). Glucose–6–phosphase isomerase 5.3.1.9 ○ They are particularly useful when the electrocardiogram (ECG) is difficult to interpret Ribose–5–phosphate isomerase 5.3.1.6 such as when there have been previous episodes LIGASES of heart disease. Carbamoyl–phosphate synthetase 6.3.5.5 ○ Lippincott*** Acetyl–CoA carboxylase 6.4.1.2 8. Define: REFERENCES a. International unit of enzyme activity CC2 Laboratory Supplementary Module International unit (IU) Bishop, M. L., Fody, E. P., & Schoeff, L. E. (2013). Clinical chemistry: Equivalent to the amount of enzyme that catalyzes principles, techniques, and correlations. 7th ed. the conversion of 1 umol of substrate per minute Lecture Notes in Medical Technology under specified conditions of pH, substrates and http://mt-lectures.blogspot.com/search?q=enzyme activators. https://bit.ly/3BKJjyQ Harvey, R. & Ferrier, D. (2011). Lippincott’s Illustrated Reviews: b. Katal (mol/s) Biochemistry (5th ed.). Philadelphia : Lippincott William & Wilkins. Equivalent to the amount of enzyme that catalyzes the conversion of 1 mole of substrate per second under controlled conditions. 9. How are enzymes classified? Give specific enzymes in each classification. OXIDOREDUCTASE Oxidase Cytochrome oxidase 1.9.3.1 Dehydrogenase 3–hydroxybutyrate dehydrogenase 1.1.1.30 Lactate dehydrogenase 1.1.1.27 (^ 3 ^) Glucose–6–phosphate dehydrogenase 1.1.1.49 Malate dehydrogenase 1.1.1.37 Isocitrate dehydrogenase 1.1.1.42 Glutamate dehydrogenase 1.4.1.2 TRANSFERASE Gamma glutamyl transferase 2.3.2.2 Aspartate aminotransferase 2.6.1.1 Alanine aminotransferase 2.6.1.2 Creatinine kinase 2.7.3.2 Ornithine–carbamoyl transferase 2.1.3.3 S.S. DE SAGUN, J. LICAY, G. DIZON, A. DELA CRUZ, S. HAMADAIN Page | 9  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION Recommended name CA SA EC Code Systematic name OXIDOREDUCTASES Lactate dehydrogenase LDH LDH 1.1.1.27 L-Lactate:NAD oxidoreductase Glucose-6-phosphate dehydrogenase G-6-PDH G-6-PD 1.1.1.49 D-Glucose-6-phosphate:NADP1-oxidoreductase Glutamate dehydrogenase GLD GLD 1.4.1.3 L-glutamate:NAD(P) oxidoreductase, deaminase TRANSFERASES Aspartate amino-transferase GOT AST 2.6.1.1 L-Aspartate:2-oxaloglutarate aminotransferase Alanine amino-transferase GPT ALT 2.6.1.2 L-Alanine:2-oxaloglutarate aminotransferase Creatine kinase CPK CK 2.7.3.2 ATP:creatine N phosphotransferase y-Glutamyl-transferase GGTP GGT 2.3.2.2 (5-Glutamyl)peptide: amino acid-5- Glutathione-S-transferase a-GST GST 2.5.1.18 Glutathione transferase Glycogen phosphorylase GP GP 2.4.1.1 1,4-a-D-Glucan: orthophosphate a-D-glucosyltransferase Pyruvate kinase PK PK 2.7.1.40 Pyruvate kinase HYDROLASES Alkaline phosphatase ALP ALP 3.1.3.1 Orthophosphoric monoester phosphohydrolase (alkaline optimum) Acid phosphatase ACP ACP 3.1.3.2 Orthophosphoric monoester phosphohydrolase (acid optimum) a-Amylase AMY AMS 3.2.1.1 1,4-D-Glucan glucanohydrolase Cholinesterase PCHE CHE 3.1.1.8 Acylcholine acylhydrolase Chymotrypsin CHY CHY 3.4.21.1 Chymotrypsin Elastase-1 E1 E1 3.4.21.36 Elastase 5-Nucleotidase NTP NTP 3.1.3.5 5’-Ribonucleotide phosphohydrolase Triacylglycerol lipase LPS 3.1.1.3 Triacylglycerol acylhydrolase Trypsin TRY TRY 3.4.21.4 Trypsin LYASES Aldolase ALD ALD 4.1.2.13 D-D-Fructose-1, 6-bisdiphosphate D-glyceraldehyde- 3-phosphate -lyase ISOMERASES Triosephosphate isomerase TPI TPI 5.3.1.1 Triose-phosphate isomerase LIGASE Glutathione Synthetase GSH-S GSH-S 6.3.2.3 Glutathione synthase S.S. DE SAGUN, J. LICAY, G. DIZON, A. DELA CRUZ, S. HAMADAIN Page | 10  EXPERIMENT 3: ASPARTATE AMINO TRANSFERASE (AST) DETERMINATION OUTLINE I. INTRODUCTION II. METHODS FOR AST ACTIVITY DETERMINATION i. Karmen method This coenzyme undergo regeneration and can be ii. Reitman-Frankel method used again for another reaction. iii. Reaction with diazonium salt iv. Chemiluminescence v. Chromatography METHODS FOR AST ACTIVITY DETERMINATION III. SEPARATION AND QUANTITATION OF AST ISOENZYMES 1. KARMEN METHOD i. Electrophoresis Kinetic assay that involves coupling of AST with malate ii. Immunoprecipitation IV. SAMPLE AND ASSAY CONSIDERATIONS dehydrogenase in the presence of NADH. V. EXPERIMENT 3: AST DETERMINATION (UV-KINETIC Basis for International Federation of Clinical Chemistry METHOD (IFCC) kinetic method. i. Principle Optimal pH reaction is 7.3 to 7.8 pH ii. Materials and Equipment iii. Reagent iv. Interfering Substances v. Specimen Considerations vi. Assay Conditions vii. Procedure i. Calculation ii. Temperature Correlation 2. REITMAN-FRANKEL METHOD iii. Detection Limit iv. Reference Values Also include the transamination from L-aspartate to VI. QUESTIONS FOR RESEARCH alpha-ketoglutarate, catalyzed by AST. VII. FAQs Reaction of ketoacid formed with dinitrophenylhydrazone in an alkaline medium. o Formation of blue-colored complex measured at 505 nm INTRODUCTION o Nonspecific, because it may react with any ketoacid. ASPARTATE AMINOTRANSFERASE (AST) TEST Cellular enzyme normally found in the heart muscle, the cells of the liver, the cells of the skeletal muscle. o Also found in the kidneys, pancreas, RBCs, spleen, and lungs but in low concentration. o Help facilitate fundamental biological processes in these organs and tissues. 3. REACTION WITH DIAZONIUM SALT Formerly known as serum glutamic-oxaloacetic transaminase (SGOT). Colorimetric assay of serum AST activity using a stabilized diazonium salt This liver enzyme test is NOT SPECIFIC of a hepatic o Reacts specifically with oxaloacetic acid, yielding a disease, and is usually associated with other enzymes such red colored compound. as ALT and ALP to verify the course of hepatic diseases. § Color reaction is more sensitive and specific; Catalyzes the transfer of an amino group from aspartate to thus, reagent blank must be minimized. alpha-ketoglutarate, forming glutamate and oxaloacetate: o Oxaloacetate produced is further reduced to malate, by malate dehydrogenase and NADH. 4. CHEMILUMINESCENCE Catalytic activity of AST present in the sample is Involves the conversion of L-aspartate to oxaloacetate, proportional to the rate of decrease in concentration of forming L-glutamate. NADH, which is measured photometrically at 340 nm. o Is then catalyzed by glutamate oxidase, producing o Pyridoxal-5’-phosphate hydrogen peroxide A coenzyme involved in the reaction which accepts § Which can oxidize luminol, resulting in amino acid from the substrate, aspartate, to form chemiluminescence pyridoxamine-5’-phosphate, and first reaction product oxaloacetate. Highly sensitive, with low background signal level, as well as wide range of measurable concentrations. Then transfers its amino group to either alpha- o However, superoxide dismutase and N-nitro-L- ketoglutarate or oxoglutarate, which results to the arginine methyl ester hydrochloride, are existing second product, glutamate. inhibitors of chemiluminescence  EXPERIMENT 3: ASPARTATE AMINO TRANSFERASE (AST) DETERMINATION 5. CHROMATOGRAPHY Based on the direct detection of enzymatically formed products xanthine and glutamate. Accurately and simultaneously measure AST and guanase activity, as low as 0.1 and 5 U I-1, respectively. o Direct measurement of the enzyme activity as micromoles of glutamate § formed within a known period of time without any coupled reaction. Identification of all components of reaction mixture allows the reaction course to be controlled and possible side-reaction be monitored. SEPARATION & QUANTITATION OF AST ISOENZYMES 1. ELECTROPHORESIS Cytoplasmic is ionic Mitochondrial is cationic 2. IMMUNOPRECIPITATION Using antibodies against both mitochondrial and cytoplasmic fraction SAMPLE AND ASSAY CONSIDERATIONS Non-hemolyzed specimen o Hemoglobin level exceeding 45 mg/dl, may also falsely increase AST activity Non-lipemic/non-icteric specimen o Bilirubin and triglyceride level should not exceed 19 mg/dl and 650 mg/dl, respectively. Avoid using diluent water contaminated with microbial growth. o Pyridoxal phosphate may be present, and elevate AST values by activating the apoenzyme form of the transaminase. Pyruvate levels exceeding 0.2 mmol/L, may falsely increase AST activity. Anabolic steroids, chloramphenicol, and aspirin also falsely increases AST activity Serum AST must be stored at 2-8 degrees to be stable o May last for 7 days Plasma can be used since most anticoagulants do not affect AST activity. A. AGUSTIN, D. MOLANO, J. MALVAR Page | 2  EXPERIMENT 3: ASPARTATE AMINO TRANSFERASE (AST) DETERMINATION EXPERIMENT 3: AST DETERMINATION (UV-KINETIC METHOD) PRINCIPLE INTERFERING SUBSTANCES Pyridoxal phosphate à elevates AST values o by activating the apoenzyme form of the transaminase o may be found on diluent water contaminated with microbial growth High level of pyruvate à also interferes with the assay SPECIMEN CONSIDERATION AST catalyzes the transfer of an amino group between L- aspartate and 2-oxoglutarate Hemolysis-free serum samples The oxaloacetate formed in the first reaction is then AST in serum remains stable at 4 degrees Celsius for reacted with NADH in the presence of the presence of minimum of 7 days malate dehydrogenase (MDH) to form NAD Hemolyzed samples should not be used as erythrocytes AST activity is determined by measuring the rate of contain fifteen times the AST activity of serum oxidation of NADH at 340 nm Lactate dehydrogenase is included in the reagent to ASSAY CONDITIONS convert endogenous pyruvate in the sample to lactate during the lag phase prior to measurement Wavelength 340 nm Optical path 1 cm MATERIALS AND EQUIPMENT Temperature 37°C Venipuncture set 13 x 100 mm test tubes PROCEDURE 12 x 75 mm test tubes Cuvettes Pasteur pipettes Reconstitute reagent according to instructions on vial label. Serologic pipettes (1ml, 0.2ml, 0.01 ml) Test tube rack Labelling tapes Parafilm strip Zero spectrophotometer at 340 nm Applicator sticks with distilled water. Centrifuge Spectrophotometer Water Bath Label tubes (cuvettes) according to patients and controls. REAGENTS When reconstituted as directed, the reagent AST contains the following: Pipette 1 ml of reagent into the tube and allow to equilibrate at 37 degrees o 2-oxoglutarate, 12 mM Celsius. o L-Aspartic Acid, 200 mM o NADH 0.19 mM o LDH 800 U/L Add 0.1 ml (100 uL) of specimen test o MDH 600 U/L samples and L1 and L2 controls to o Buffer 100 mM reagent and mix gently. o pH 7.8 +/- 0.1 Reagent Storage and Stability o Store dry reagents at 2-8 degrees Celsius Maintain solution at 37 degrees o Stability: Celsius. After one minute, measure § for 8 hours at room temperature (15-30C) the absorbance at 340 nm. § for 21 days when refrigerated immediately Reagent Deterioration Take three additional absorbance o Discard reagent if: readings at one (1) minute interval. § initial absorbance read against water 340 nm is Calculate the mean absorbance below 0.800 change per minute (▲ A/min). § reagent fails to meet stated parameters of performance Multiply the change in absorbance per minute by 1768 to calculate IU/L of AST activity. A. AGUSTIN, D. MOLANO, J. MALVAR Page | 3  EXPERIMENT 3: ASPARTATE AMINO TRANSFERASE (AST) DETERMINATION CALCULATIONS In the kinetic method of AST determination, the analyte’s concentration or activity is determined by measuring the 12%+ %34/ min 8 #. :. 8 1000 rate of change in absorbance** at 340 nm over a fixed-time !"# %&'()('* (+ ,-// = interval € x S. V. X L. P. Rate of change in absorbance à directly proportional to = 12%+ %34=>3%+&2 ?(@@2>2+&2/ min 8 1768 the AST activity in the sample Where: **rate of change in absorbance/absorbance reduction is the Mean Abs/ min = mean absorbance difference per minute consequence of the NADH oxidation; so, in other words, the method T.V. = total assay volume (1.1 mL) determines AST activity by measuring the oxidation rate of NADH 1000 = conversion of IU/ mL to IU/ L € = millimolar absorptivity of NADH (6.22) 3. What are the conditions affecting the reaction? S.V. = sample volume (0.1 mL) L.P. = light path (1 cm) Table 1: Factors and conditions affecting the reaction Factor Effect ** To convert to SI units (nkat/ L) multiply the IU/L value by Hemolyzed specimens Falsely increase AST activity 16.67. and hemoglobin levels exceeding 45 mg/dl TEMPERATURE CORRELATION Lipemic or icteric samples Falsely increase AST activity Pyridoxal phosphate Elevate AST If the reaction is performed at 30 degrees Celsius but is to High levels of pyruvate (> Falsely increase AST activity be reported at 37 degrees Celsius, simply multiply the 0.2 mmol/L) result obtained at 30 degrees Celsius by the factor 1.43 to Anabolic steroids, Falsely increase AST activity obtain the correct value. chloramphenicol, and If the assay is performed at 37 degrees Celsius but is to aspirin be reported at 30 degrees Celsius, multiply the results Chronic alcoholism Increases AST activity by 0.7. Race/gender 5-10% variation in African- American men DETECTION LIMIT Exercise Threefold increase with The reagent is linear up to 500 IU/L. strenuous exercise Sample that has AST values greater than 500 IU/L should Muscle injury Significant increase in AST be diluted 1:1 with saline, re-assayed and the results activity multiplied by 2. REFERENCE VALUES 4. What are the advantages of the kinetic method over the colometric method? 30 degrees Celsius Up to 28 IU/L 37 degrees Celsius Up to 40 IU/L The spectrophotometric procedure has an important advantage over colorimetric procedures in which the QUESTIONS FOR RESEARCH oxaloacetate produced in reaction 1 is allowed to react with a chromogenic reagent such as 2,4- 1. Describe the principle of AST Kinetic method dinitrophenyl-hydrazine or a diazonium reagent Oxaloacetate is a potent inhibitor of AST and in the spectrophotometric procedure is removed as fast as it is formed, whereas in the colorimetric techniques it is allowed to accumulate The colometric method may lacks specificity because it may react with any ketoacid (Reitman-Frankel) AST catalyzes the reversible transamination of L- FAQs aspartate and 2-oxoglutarate to oxaloacetate and L- glutamate What is linearity of the method? o The capability to show results that are directly The oxaloacetate formed is then reduced to malate in the proportional to the concentration of the analyte in the presence of malate dehydrogenase (MDH) and the sample coenzyme NADH is oxidized to NAD o Often measured within a given range § AST activity within 0-500 IU/L is considered 2. What is meant by the kinetic method of AST linear determination? o Manifested by a straight line in the calibration curve o In most cases, enzyme activity exceeding the Kinetic methods make use of the rate of a chemical or linearity of the method may lead to machine errors physical reaction to determine an analyte’s § Machine may not detect concentration/activity, concentration/activity thus making it impossible to report quantitatively. A. AGUSTIN, D. MOLANO, J. MALVAR Page | 4  EXPERIMENT 3: ASPARTATE AMINO TRANSFERASE (AST) DETERMINATION o Sample dilution may be done as intervention § This will help lower the concentration of the substance being measured, and will be set within detectable range of the method. § Dilution factor should be taken in account in obtaining the final result. What is the purpose of the one-minute incubation after adding the sample? o This procedure is considered as the lag phase o To ensure the complete NADH-dependent reduction of endogenous oxoacids in the sample, before transaminase reaction § ONLY Oxoacid reduction due to transamination should be considered § Thus, other sources of reduction should be eliminated. References Why is lactate dehydrogenase added in the Supplemental notes pp. 31 – 35 reagent? Davis, C.P (2021). Liver Function Tests (Normal, Low, and High Ranges and o To convert endogenous pyruvate to lactate during Results). Retrieved from lag phase. https://www.medicinenet.com/liver_blood_tests/article.htm Marcin, J. (2018). Aspartate Aminotransferase (AST) Test. Retrieved from § Because pyruvate may interfere with AST https://www.healthline.com/health/ast activity, leading to erroneous results. Mayo Clinic. (n.d.). Aspartate Aminotransferase (AST)(GOT), Serum. Retrieved Why is the absorbance trend decreasing? September 10, 2021, from https://www.mayocliniclabs.com/test- catalog/Clinical+and+Interpretive/8360 o The rate of decrease in concentration of NADH is Wilkinson, J. H., Baron, D. N., Moss, D. W., & Walker, P. G. (1972). being measured photometrically at 340 nm. Standardization of clinical enzyme assays: a reference method for aspartate o Includes the measurement of four absorbances. and alanine transaminases. Journal of Clinical Pathology, 25(11), 940–944. doi:10.1136/jcp.25.11.940 How are you going to compute for AST activity? 1. From the four absorbances, determine the change for each interval. 2. Take the average of the change and multiply with the factor 1768 (refer to manual for derivation) What is the normal value for AST? o Up to 40 IU/L, but may vary from each laboratory. A. AGUSTIN, D. MOLANO, J. MALVAR Page | 5  EXPERIMENT 4: ALANINE AMINOTRANSFERASE (ALT) DETERMINATION When used in conjunction with AST: OUTLINE ○ Aids in the diagnosis of I. INTRODUCTION Myocardial Infarction II. METHODS FOR ALT ACTIVITY DETERMINATION - ALT stays within normal limits i. Wroblewski and LaDue Method in the presence of ↑AST ii. Reitman-Frankel Method - Small amount of ALT is found iii. Reaction with Diazonium Salt in cardiac tissue; hence, ALT iv. Chemiluminescence remains normal unless III. SAMPLE ASSAY CONSIDERATIONS subsequent liver damage IV. EXPERIMENT 4: ALT DETERMINATION occurred. (UV-KINETIC METHOD) ALT DOES NOT INCREASE IN i. Principle (unlike AST): ii. Materials and Equipment ○ Muscle Dystrophy ○ Pulmonary Emboli iii. Reagents ○ Acute Pancreatitis iv. Interfering Substances Assay for Enzyme A typical ALT procedure consists of v. Specimen Considerations Activity an enzymatic reaction using Lactate vi. Assay Conditions Dehydrogenase (LD) vii. Manual Procedure ○ Catalyzes reduction of pyruvate a. Calculation to lactate with simultaneous b. Temperature Correlation oxidation of NADH c. Detection Limit Measured at 340 nm d. Expected Values ○ Change in absorbance is directly V. QUESTIONS FOR RESEARCH proportional to ALT activity VI. FAQs INTRODUCTION The reaction uses pyridoxal-5’- ALANINE AMINOTRANSFERASE (EC 2.6.1.2) phosphate as a coenzyme aka Serum Glutamic Pyruvate Transferase (SGPT) ○ Carries one amino group from Properties Transferase that is similar to AST one acid to another acid ○ Catalyzes transfer of alanine to (ketoacid) alpha ketoglutarate ○ Forms: Pyridoxal-5'-phosphate accepts the amino acid - Glutamate from Alanine - Pyruvate ↓ Forms pyridoxamine-5’-phosphate and pyruvate ↓ The pyridoxamine-5’-phosphate transfers its amino group to alpha ketoglutarate (aka oxoglutarate) ↓ Forms glutamate Half-life: 16 hours (AST = 24 hours) Tissue Source High concentrations in the liver More liver-specific than the other transferases. Highest concentration in the liver Sources of Error Stable for 3-4 days at 4°C (Bishop) and kidneys (module; Bishop says Relatively unaffected by hemolysis liver only) (Bishop) Diagnostic Increased in Hepatic diseases: Reference Range 7-45 IU/L at 37°C Significance ○ Hepatitis 0.1-0.8 µkat/L ○ Cirrhosis ○ Hepatic Obstructive Disorders ○ Acute Inflammatory Conditions of the Liver - ALT levels > AST levels - Due to longer half-life of ALT  EXPERIMENT 1: TOTAL AND DIRECT SERUM BILIRUBIN DETERMINATION METHODS FOR ALT ACTIVITY DETERMINATION EXPERIMENT 4: ALT DETERMINATION (UV-KINETIC METHOD) 1. Wroblewski Basis for the International Federation and LaDue of Clinical Chemistry (IFCC) kinetic PRINCIPLE OF REACTION Method method Optimal pH: 7.3-7.8 Involves coupling of ALT with Lactate Dehydrogenase (LD) in the presence of NADH A decrease in NADH concentration is proportional to ALT activity Measured at 340 nm * Note. The principle is the same with the method mentioned earlier. ɑ-ketoglutarate is the same as 2-oxoglutarate. 2. Reitman- ALT catalyzes the transamination of MATERIALS AND EQUIPMENT Frankel L-alanine to alpha-ketoglutarate → Method pyruvate + glutamate 1. Venipuncture set 7. Test tube rack Pyruvate combines with 2. Test tubes: 8. Labeling tapes 2,4-dinitrophenylhydrazine 12x75 and 13x100mm 9. Parafilm strips ○ Forms a blue-colored complex 3. Cuvettes 10. Applicator sticks that is measured at 505 nm 4. Pasteur pipettes 11. Centrifuge Disadvantage: lacks specificity 5. Serological pipettes 12. Spectrophotometer since it can react with any keto acid. 6. Timing device 13. Water bath REAGENTS The ALT reagent contains the following: ɑ-ketoglutarate 13 Mn L-alanine 400 Mm NADH 0.2 Mm * Note: blue-colored complex acc to the LDH 800 U/L module, but brown-color in the picture Buffer 100 mM, pH 7.8 3. Reaction with Presented by Babson Nonreactive stabilizers and preservatives - Diazonium Stabilized diazonium salt reacts Salt specifically with pyruvate The ALT reagent should be discarded if: ○ Forms a red-colored compound ○ Turbidity is observed (sign of contamination) Advantage: the color reaction is ○ Moisture has penetrated the vial and caking occurred specific and sensitive for the reaction ○ Reagent fails to meet linearity or fails to recover ○ Thus, a reagent blank is not control values in the stated range needed ○ The reconstituted reagent has a reagent blank 4. Chemi- Only method out of the four that absorbance less than 0.8 at 340 nm luminescence uses Glutamate instead of Pyruvate The glutamate is catalyzed by INTERFERING SUBSTANCES glutamate oxidase to produce Pyridoxal phosphate hydrogen peroxide (H2O2) ○ Elevates ALT values by activating the apoenzyme ○ H2O2 can oxidize luminol, form of ALT generating chemiluminescence ○ Can be found in the diluent water contaminated with microbial growth High levels of Pyruvate (> 0.2 mmol/L) Advantages: Young et al. gave a list of drugs and substances that ○ Highly sensitive interfere with ALT activity ○ Low background signal level ○ Wide range of measurable SPECIMEN CONSIDERATION concentrations (up to 5 decades Serum is the preferred sample, but plasma can be used of magnitude) since anticoagulants do not affect ALT activity Disadvantages: Hemolyzed sample should not be used since RBCs