[BIOCHEM]LAB_202_LIPIDS-PHOSPHOLIPID-METABOLISM-AND-SPHINGOLIPID-METABOLISM.pdf

(002) LIPIDS– PHOSPHOLIPID METABOLISM AND SPHINGOLIPID METABOLISM DR. MARIA ALOIZA HADLOC| 12/11/2020 OUTLINE...

(002) LIPIDS– PHOSPHOLIPID METABOLISM AND SPHINGOLIPID METABOLISM DR. MARIA ALOIZA HADLOC| 12/11/2020 OUTLINE Results: In Water: Soluble I. EXPERIMENT In CCl3: Insoluble A. Extraction of Total Lipids So, for your solubility test, your glycerol is actually a B. Isolate Triglycerides 1. Test on Glycerol polyol compound which is colorless, odorless C. Isolation and Test on Lecithin viscous liquid that is sweet-tasting and non-toxic. 2. Tests on Lecithin You may consider that glycerol is hydrophobic II. RESPIRATORY DISTRESS AND THE ROLE OF because it is usually attached to your fatty acids most LIPIDS IN LUNG SURFACTANT of the time but in reality, your glycerol has three III. QUESTIONS hydroxyl groups which makes glycerol miscible with water, meaning it is soluble with water. This the reason now for the result of the first test, which proves to us that glycerol is soluble in water because I. EXPERIMENT of the three hydroxyl groups in its structure. A. Extraction of Total Lipids ii. Acrolein Test 1. Cut about 30 grams of pig’s brain and homogenize Place about 0.5 gm of powdered KHS04 in a the material in a blender with ether-alcohol mixture clean, dry test tube. Add about 10 drops of the for 10 minutes. Filter the material. The ether- glycerol solution. Heat the solution cautiously at first alcohol extract contains total lipids. and then more strongly. Note the odor produced. 2. Divide the ether-alcohol extract into two parts: Results: Peculiar order/ Burnt grease a) 2/3 portion- this will be used for the rest of When glycerol is heated with KHSO4, it will the experiments undergo dehydration eventually causing the b) 1/3 portion peculiar odor, burnt grease. B. Isolate Triglycerides Acrolein test is used to detect the presence 1. Take the first 2/3 portion and evaporate the extract of glycerol or fat. When fat is treated strongly in the over a hot water bath (without flame) or over a hot presence of a dehydrating agent like potassium plate to a syrupy consistency. Add 10 ml of alcoholic bisulphate (KHSO4), the glycerol portion of the KOH. Stir and transfer mixture into an Erlenmeyer molecule is dehydrated to form an unsaturated flask. Place a few glass beads to prevent bumping aldehyde, acrolein that has a pungent irritating during the heating. Cover the mouth of the flask with odour. a funnel and put the flask over low heat for 30 minutes with occasional shaking. Add distilled water if necessary to replace lost volume. Continue iii. Benedict’s Test heating until saponification is complete as indicated Test your solution with litmus paper for any by miscibility of 1-2 drops of saponified solution in 3 acidity. Neutralize if acidic with a few drops of dilute ml water. Na2C03. Add 5 drops of the glycerol solution into a 2. Add now to the saponified mixture 30 ml of distilled test tube containing 5 ml of Benedict’s reagent. Boil water. Remove any insoluble (unsaponifiable) the mixture for 2 to 3 minutes. Observe the results. portion that may separate out. Transfer the Compare with the results on sugars. saponified solution into an evaporating dish. Heat Results: (+) Brick Red’s precipitate again until the alcohol is driven off. Evaporate to a viscous consistency. Finally, add 50 ml of water and The copper sulfate (CuSO4) present in divide the solution into: Benedict's solution reacts with electrons from the a) 20 ml portion; and aldehyde or ketone group of the reducing sugar. b) 30 ml portion Reducing sugars are oxidized by the copper ion in solution to form a carboxylic acid and a brick red Remember that your glycerophospholipid precipitate of copper (I) oxide. has glycerol for its backbone. So, we have three tests for your glycerol test – your Solubility test, Acrolein test and Benedict’s test. Isolation and Test on Lecithin Place the remaining 1/3 portion, from extraction of total lipids, in an evaporating dish and Test on Glycerol (use the 30ml portion) heat until concentrated to a syrup consistency. Add Place the aqueous layer reserved from b in about 15ml of ether and stir the mixture. an evaporating dish. Heat until the solution is reduced Pour the ether extract into about 15ml of to about 20 ml. Use the concentrate for the following acetone and stir again. The precipitate is mostly tests: lecithin. Filter off the precipitate and reserve the i. Solubility filtrate for the test on cholesterol. Add a few drops of the solution into 3 ml portions of water and CHCl3 Page 1 of 3 PREPARED BY: CMED 1F 2024  (002) LIPIDS– PHOSPHOLIPID METABOLISM AND SPHINGOLIPID METABOLISM DR. MARIA ALOIZA HADLOC| 12/11/2020 Tests on Lecithin a) Acrolein Test (See procedure in acrolein - Surfactants to infants test under test on glycerol) Result: Burnt odor a. Pre term: reduced to less than 10mg/kg  RESPIRATORY DISTRESS SYNDROME(RDS) b) Iodine-Absorption Test (See procedure o present shortly after birth with apnea, in iodine-absorption test under test on fatty acids – cyanosis, grunting, inspiratory stridor, nasal Week 12) flaring, poor feeding, and tachypnea Result: Discoloration of Iodine o There may also be intercostal or subcostal II. RESPIRATORY DISTRESS AND THE retractions ROLE OF LIPIDS IN LUNG o radiological findings include a diffuse SURFACTANT reticulogranular “ground glass” appearance (resulting from alveolar atelectasis) with Pulmonary surfactant superimposed air bronchograms - Complex mixture of phospholipids (PL) and proteins b. Full term: estimated pool size of 100 mg/kg. (SP) - Reduce surface tension at the air-liquid interface of the - Treatment to RDS: Prenatal corticosteroids and alveolus, thus preventing its collapse during end- postnatal surfactant replacement therapy exhalation. significantly reduce the incidence, severity and mortality - It is made up of about 70% to 80% PL, mainly associated with RDS, and surfactant therapy has dipalmitoylphosphatidylcholine (DPPC), 10% SP-A, become the standard of care in management of preterm B, C and D, and 10% neutral lipids, mainly cholesterol. infants with RDS. - Synthesized and secreted by Type II alveolar - Lung maturity of the fetus can be gauged by epithelial cells (a.k.a pneumocytes), which differentiate determining the ratio of DPPC to sphingomyelin, usually between 24 and 34 weeks of gestation in the human. written as the L (for lecithin)/S ratio, in amniotic fluid. - Lung first appears as a ventral bud off the esophagus. A ratio of two or above is evidence of maturity, because - Canalicular stage of lung development is between 16 to it reflects the major shift from sphingomyelin to DPPC 26 weeks. Components of the surfactant system begin synthesis that occurs in the pneumocytes at about 32 to appear as well weeks of gestation. - Alveolarization occurs from 36 weeks preterm to 36 - RDS due to an insufficient amount of surfactant can also months postnatal in humans. occur in adults whose surfactant-producing - Alveolus is the primary site of gas exchange with the pneumocytes have been damaged or destroyed, for blood in the lungs. example, by infection or trauma. - Two types of epithelial cells are found in the alveoli: a. Type I cells ROLE OF LIPIDS IN LUNG SURFACTANT o make up 95% of the alveolus o form the alveolar wall. - Lipids are ultimately responsible for surface tension b. Type II cells. reduction to low value during inhalation and expiration. o while the Type II cells account for the - DPPC, made and secreted by Type II pneumocytes, is remaining 5%. the major lipid component of lung surfactant—the o synthesize and secrete surfactant. extracellular fluid layer lining the alveoli. - Alveoli have an innate tendency to collapse because - a. spherical shape III. QUESTIONS: b. small size c. contribution of water vapor to surface tension 1) What are the two types of epithelial cells found in the - Surfactant helps to lower surface tension thus alveoli? preventing collapse. - Primary surface-active material in surfactant is the Type I pneumocytes comprises 95% cells in phospholipid, DPPC alveoli and is responsible for gas exchange while the Type II pneumocytes/septal cells, are o A pure film of DPPC on a surface balance is responsible for surfactant production. capable of lowering the surface tension to near 0 mN/m under dynamic compression. 2) What is the significance of the lung surfactant? o The close packed nature of the saturated palmitic acids allows the DPPC molecules to The lung surfactant produced by the type II organize into a two-dimensional surface gel at pneumocytes or septal cells reduces the surface 37 °C, giving it stability. tension in the alveoli. When we inhale, our alveoli o This ultimately helps in lowering surface are inflated while when exhale, they are deflated. tension and preventing collapse of the alveoli. The surfactant prevents the collapse of the alveoli - Maintaining adequate surfactant pools within the air and helps them get inflated faster/more efficiently. space is essential for lung function. Page 2 of 3 PREPARED BY: CMED 1F 2024  (002) LIPIDS– PHOSPHOLIPID METABOLISM AND SPHINGOLIPID METABOLISM DR. MARIA ALOIZA HADLOC| 12/11/2020 Surfactant serves to decrease the surface tension of the fluid layer, reducing the pressure needed to reinflate alveoli, thereby preventing alveolar collapse (atelectasis). 3) What are the components of the lung surfactant? 90% lipids, which are mainly DPCC, and proteins Surfactant is a complex mixture of lipids (90%) and proteins (10%), with DPPC being the major component for reducing surface tension. 4) What is the pathophysiology of Respiratory Distress Syndrome? Respiratory distress syndrome (RDS) in preterm infants is associated with insufficient surfactant production and/or secretion, and is a significant cause of all neonatal deaths in Western countries. RDS due to an insufficient amount of surfactant can also occur in adults whose surfactant-producing pneumocytes have been damaged or destroyed, for example, by infection or trauma. 5) What is the AOG considered to be critical as the lung surfactant is still not developed, thus causing the preterm high risk for RDS? Less than 28 weeks age of gestation 6) If you have a patient which is 26 weeks age of gestation, what can you do to prevent the possibility of RDS? Administer glucocorticoids such as hydrocortisone to the mother, to facilitate production of DCCP Lung maturation can be accelerated by giving the mother glucocorticoids shortly before delivery. Administration of natural or synthetic surfactant (by intratracheal instillation) is also used in the prevention and treatment of infant RDS. REFERENCES: Ferrier, D. R. (2014). Lippincott's Illustrated Reviews: Biochemistry. Philadeplhia: Lippincott Williams & WIlkins. Practical Biochemistry. (n.d.). Retrieved from https://www.bu.edu.eg Qualitative Analysis of Oils and Fats. (2015). Retrieved from OLABS: amrita.olabs.edu.in Swanson T, K. S. (2010). Biochemistry, Molecular Biology, and Genetics. Philadelphia: Lippincott Williams & WIlkins. Page 3 of 3 PREPARED BY: CMED 1F 2024