[BIOCHEM]LAB_206_AMINO-ACID-CONVERSION-TO-SPECIALIZED-PRODUCTS.pdf

(006) AMINO ACIDS- CONVERSION TO SPECIALIZED PRODUCTS DR. FAHAD...

(006) AMINO ACIDS- CONVERSION TO SPECIALIZED PRODUCTS DR. FAHAD ABDUL RAZAK | 01/19/21 OUTLINE I. PREPARATION OF AMINO ACID SOLUTION II. XANTHOPROTEIC TEST A. Materials B. Principle C. Method III. HEAT COAGULATION TEST A. Materials B. Principle Concentrated nitric acid cause nitration of the activated C. Method benzene rings of tyrosine and tryptophan IV. FOUCHET’S TEST A. Materials B. Principle C. Method V. CASE NO. 1 A. Discussion B. Degradation of heme VI. CLINICAL CORRELATION A. Jaundice in newborns I. PREPARATION OF AMINO ACID SOLUTION ➔ Nitrated activated benzene is yellow in color 1. EGG SOLUTION - Mix 25 mL of whole egg (egg white and yolk) in 500 mL of ➔ Turns orange in alkaline medium water (make sure to use within 24 hours) 2. PROTEIN POWDER Although phenylalanine contains benzene ring, the ring - To make a 0.5% solution of mix 2.5 gm od casein in 20 mL is not activated, thus will not undergo nitration 40% NaOH solution - Add above reagent to 500 mL of distilled water II. XANTHOPROTEIC TEST This test is for the detection of aromatic amino acids (Tyrosine, Tryptophane) A. MATERIALS -Bunsen burner -2test tubes -Distilled water -Concentrated HNO3 On adding alkali to these nitro derivative salts, the color -40% NaOH solution or prepare 40 gm NaOH in 100 mL change from yellow to orange distilled water B. PRINCIPLE Xanthoproteic test is used to detect amino acids containing in an aromatic nucleus tyrosine and tryptophan protein solution which gives yellow color nitro derivatives on heating with conc HNO3 This test is used to differentiate aromatic amino acids which gives positive result from other amino acid Page 1 of 8 CMED 1E/F  (006) AMINO ACIDS- CONVERSION TO SPECIALIZED PRODUCTS DR. FAHAD ABDUL RAZAK | 01/19/21 C. METHOD -When coagulable protein is heated at isoelectric pH, it is irreversible because of uncoiling and break down of peptide 1.Add 1 mL test solution to a test tube A bonds. 2.Add 1 ml distilled water to test tube B -Core hydrophobic regions of denatured albumin can form intermolecular associations and cause precipitation. Thus, 3.Add 1 ml concentrated (HNO3 to test tubes A and B (HNO3 in order to precipitate proteins like albumin, two conditions is a strong oxidizing agent and corrosive) are required. 1) Bring albumin to its pI (5.4) by adding few 4.Heat A and B x 1 – 2 minutes under Bunsen burner drops of 1% acetic acid. 2)Heat the solution 5.Cool tubes under tap water C. METHOD 6.Add 1 ml 40% HNO3 to test tubes A and B 1. Add 3/4test solution to a test tube (bottom will act as control) 7.Record Observations (yellow color developed = Protein present) 2. Heat top half of test tube under Bunsen burner till precipitate seen Xanthoproteic test is a biochemical test for the detection of amino acids containing phenolic or indolic groups like phenylalanine, 3. Add 2 to 4 drops of 1% acetic acid tyrosine, and tryptophan (aromatic amino acids). The test is named Xanthoproteic test due to the formation of a yellow precipitate of 4. White precipitates are formed, reheat the solution. xanthoproteic acid. 5. Record observation II. HEAT COAGULATION TEST Interpretation: 1. A dense coagulum will be formed (upper part of solution) This test is used to detect the presence of protein in a given which shows presence of albumin or globulin. solution. 2. For gelatin, proteases, polypeptides, which are non- coagulable proteins no coagulation is formed. A. MATERIALS -1% acetic acid: 1 ml acetic acid up to 100 ml with distilled water Heating a protein in acidic medium results in denaturation of protein -1 Test tube due to the breaking of certain bonds responsible for the tertiary and -Bunsen burner quaternary structure of proteins. However, in the case of coagulable proteins, when these proteins are heated at their isoelectric pH, the B. PRINCIPLE polypeptide chains become uncoiled and matt with each other to form an insoluble mass. The mass formed doesn’t dissolve back to -Proteins have net zero charge at their iso-electric pH (pI); meaning, molecules have minimum repelling force. Thus, the liquid. The process of coagulation is maximum at the isoelectric proteins are easily precipitated at pI. point, and the mass of the coagulum might differ with the particle size and the concentration of proteins in the sample -When proteins are heated, weak bonds like hydrogen-bonds, ionic bonds and Van-der-Waal forces are broken → denatured. II. FOUCHET’S TEST - When protein is heated in acidic medium, its primary structure retained but it denatured – reversible. This test is used to detect bile pigments in urine. Page 2 of 8 CMED 1E/F  (006) AMINO ACIDS- CONVERSION TO SPECIALIZED PRODUCTS DR. FAHAD ABDUL RAZAK | 01/19/21 A. MATERIALS Observation – pista green or bluish green colored precipitate on filter -Fouchet’s reagent prepared by mixing: paper which indicates the presence of bile pigments in urine – bilirubin ▪ 25gms trichloroacetic acid and biliverdin ▪ 10ml 10% ferric chloride ▪ 100ml distilled water Positive test indicates conjugated bilirubin in urine -If no available Fouchet solution may use yellow colored Diseases with increase in bilirubin in urine HNO3 1.Obstructive jaundice 2.Hepatocellular jaundice -Fresh Urine Other abnormal constituents of urine: Glucose, -10% barium chloride ketone bodies, bile salts, albumin and blood. -Filter paper -Funnel glass -Measuring cylinder or beaker A color test for the presence of bilirubin in the urine. A green color is produced when the sample is treated with iron (iii) chloride B. PRINCIPLE solution and trichloroacetic acid - Fouchet's test is an oxidizing reagent or test. Barium chloride precipitates the sulphate radicals present in urine to form precipitate of barium sulphate. If bile pigments are present in urine, they adhere to these molecules. Ferric chloride present in Fouchet’s reagent then oxidizes yellow bilirubin, in the presence of trichloroacetic acid to green biliverdin. - Therefore, the development of green color due to the formation of biliverdin indicates the presence of bilirubin (bile) in urine. - Bile pigment present in urine adsorb on the barium sulphate precipitate. TCA releases adsorbed bilirubin from precipitate and ferric chloride oxidizes bilirubin to green colored biliverdin. CASE NO. 1 C. METHOD You are the resident physician on duty. A 4-day old premature infant 1. Place 5 ml of fresh urine in a test tube was brought to your clinic with a chief complaint of yellowish discoloration of the skin as stated by the mother. The patient was born 2. Add 2.5 ml of 10% of barium chloride, and mix well until to a 25-year-old G1P1(0101) mother with no noted comorbidities via a precipitate form NSD (normal spontaneous delivery). Hepatitis B and BCG vaccines were given at the hospital. Birth weight was 1.9 kgs with Apgar score 3. Filter the precipitate on a filter paper through measuring noted at 8, 9 at 1 and 5 minutes of life. cylinder/beaker On the 3rd day of life, the patient was noted to have yellow 4. Add 1 drop of Fouchet’s reagent to precipitate discoloration of the skin later progressing to the eyes and body. 5. Record observations Physical examination revealed an active infant with jaundice up to the upper chest area. No noted abdominal enlargement or bleeding/hematomas noted. Good suck noted. What is the most likely diagnosis in the above case? - Physiologic jaundice What is the suggested mechanism of jaundice in this case? - Decreased amounts of Beta glucuronidase leads to decreased in amounts of conjugated bilirubin (not efficiently secreted in bile) What would you advise the mother? - Reassurance since jaundice is physiologic but continue to monitor patient. Page 3 of 8 CMED 1E/F  (006) AMINO ACIDS- CONVERSION TO SPECIALIZED PRODUCTS DR. FAHAD ABDUL RAZAK | 01/19/21 Test Observation Inference Xanthoprot Yellow to orange color Presence of eic test develops on test tube A tyrosine/tryptophan in solution Heat white coagulation forms Albumin noted to be Coagulatio at the top half of the test denatured at its n test tube while the bottom isoelectric point. half remains relatively clear. Fouchet’s Note of greenish Formation of bilverdin Test discoloration once (green color) solution added to indicates that urine precipitate contains bilirubin. A. DISCUSSION Bilirubin is a brownish-reddish substance formed from the catabolism of heme. This is important as it gets rid of waste products from destroyed RBCs (whether from abnormal hemolysis or normal aging). Normal aging of RBCs is about 120 days. B. DEGRADATION OF HEME 1. Formation of bilirubin - first step is catalyzed by the microsomal heme oxygenase system of the reticuloendothelial cells (macrophages) - green pigment biliverdin is produced as ferric iron and CO are released - Carbon monoxide later acts as a signaling molecule and vasodilation - Biliverdin is reduced, forming the red-orange bilirubin - Bilirubin and its derivatives are collectively termed bile pigments 2. Uptake bilirubin by liver - Bilirubin then transported to liver as a complex with albumin - Unconjugated bilirubin (B1 or indirect) insoluble in plasma 3. Formation of bilirubin diglucuronide/conjugated bilirubin (B2 or direct) - Facilitated by glucoronyl transferase Fig 1. Shows how bilirubin is formed from Heme (take note of the enzymes) Page 4 of 8 CMED 1E/F  (006) AMINO ACIDS- CONVERSION TO SPECIALIZED PRODUCTS DR. FAHAD ABDUL RAZAK | 01/19/21 CLINICAL CORRELATION A. JAUNDICE IN NEWBORNS Excess bilirubin gets deposited into tissues including the skin. → Jaundice. Newborn at increased risk - Glucoronyl transferase will only reach normal at 4 weeks 2 TYPES OF JAUNDICE IN NEWBORNS 1. Physiologic - Occurs after 24 hours of life until 2 weeks - Increased unconjugated bilirubin - Usually remains asymptomatic - Total Levels ≤ 12mg/dL (depends on age as well) 2. Pathologic - Onset < 24 hours or > 2 weeks - May have increased B1 or B2 - Increased risk of being symptomatic - Total Levels ≥ 12 mg/dL (depends on age as well) - May be due to hemolysis/obstruction or even sepsis CLINICAL MANIFESTATIONS Yellow discoloration of the skin Poor suck Kernicterus - Deposition of bilirubin at the basal ganglia - Permanently affects infant - Levels depend on age group but ~30mg/dL almost universal - Lethargy, seizures or movement disorders DIAGNOSTIC appendix1 Gold Standard is transcutaneous bilirubin measurement Visual estimation done using Kramer scoring 4. Secretion of bilirubin into bile - inaccurate as it is subjective - Still being used as it describes location of jaundice - actively transported to bile canaliculi - rate limiting step TREATMENT Treat underlying cause if pathologic 5. Formation of urobilinogen in the intestines and excretion Reassurance if physiologic and no increased risk of kernicterus If increased risk for kernicterus due to ↑ B1 - Reduced to urobilinogen (colorless) by bacteria - May do phototherapy Isomerization of B1 to soluble forms → easier to - Fates of urobilinogen: excrete Not invasive and usually first step a. Then oxidized to stercobilin (red brown) → feces - May do exchange transfusion b. Urobilinogen can enter enterohepatic urobilinogen Immediate reduction in total bilirubin by ~ 50% cycle Invasive procedure Retaken by liver → bile c. Remainder transported to kidney Form urobilin (yellow) → urine Page 5 of 8 CMED 1E/F  (006) AMINO ACIDS- CONVERSION TO SPECIALIZED PRODUCTS DR. FAHAD ABDUL RAZAK | 01/19/21 2. δ-Aminolevulinic acid synthase activity: A. in liver is frequently decreased in individuals treated with drugs, such as the barbiturate phenobarbital. B. catalyzes a rate-limiting reaction in porphyrin biosynthesis. C. requires the coenzyme biotin. D. is strongly inhibited by heavy metal ions such as lead. E. occurs in the cytosol 3. The catabolism of hemoglobin: A. occurs in red blood cells. B. involves the oxidative cleavage of the porphyrin ring. C. results in the liberation of carbon dioxide. D. results in the formation of protoporphyrinogen. E. is the sole source of bilirubin 4. A 50-year-old man presented with painful blisters on the backs of his hands. He was a golf instructor, and indicated that the blisters had erupted shortly after the golfing season began. He did not have recent exposure to poison ivy or sumac, new soaps or detergents, or new medications. He denied having previous episodes of blistering. He had partial complex seizure disorder that had begun about three years earlier after a head injury. The patient had been taking phenytoin—his only medication—since the onset of the seizure disorder. He admitted to an average weekly ethanol intake of about eighteen 12-oz cans of beer. The patient’s urine was reddish orange. Cultures obtained from skin lesions failed to grow organisms. A 24- hour urine collection showed elevated uroporphyrin (1,000 mg; normal,