[BIOCHEM]LAB_201_LIPIDS-FATTY-ACID-AND-TRIACYLGLYCEROL-METABOLISM.pdf

(001) LIPIDS – FATTY ACID AND TRIACYLGLYCEROL METABOLISM DR. HADLOC | 12/01/2020 OUTLINE...

(001) LIPIDS – FATTY ACID AND TRIACYLGLYCEROL METABOLISM DR. HADLOC | 12/01/2020 OUTLINE ○ Continue heating until saponification is complete I. LIPIDS-FATTY ACIDS AND TRIGLYCERIDE as indicated by miscibility of 1-2 drops of saponified METABOLISM solution in 3 ml water. II. EXPERIMENTS ○ Add now to the saponified mixture 30 ml of distilled A. Extraction of Total Lipids water. B. Test for Fatty Acids ○ Remove any insoluble (unsaponifiable) portion that 1. Solubility may separate out. Transfer the saponified solution 2. Translucent Spot Test into an evaporating dish. 3. Iodine Absorption Test ○ Heat again until the alcohol is driven off. Evaporate III. ESSENTIAL FATTY ACID DEFICIENCY A. Symptoms to a viscous consistency. Finally, add 50 ml of B. Biochemical explanation water and divide the solution into: IV. SYSTEMIC CARNITINE DEFICIENCY a) 20 ml portion and A. Symptoms b) 30 ml portion B. Biochemical explanation V. QUESTIONS A. Essential Fatty Acid Deficiency B. TEST FOR FATTY ACIDS B. Systemic Carnitine Deficiency - Heat the solution to boiling. Add concentrated HCl drop by drop until no more precipitate forms. (An excess of HCl still dissolves the precipitate. The I. LIPIDS - FATTY ACIDS AND TRIGLYCERIDE precipitate is Fatty acid. The fatty acids may METABOLISM appear as an oily layer while the solution is still hot - Fatty acids exist free in the body (that is, they are nonesterified) and but will precipitate upon cooling. Perform the tests as fatty acyl esters in more complex molecules such as triacylglycerols on fatty acids. (Reserve the aqueous portion for (TAG). Low levels of free fatty acids (FFA) occur in all tissues, but test on glycerol). substantial amounts can sometimes be found in the plasma, particularly during fasting. Fatty acids are also precursors of the 1. Solubility hormone-like prostaglandins. Moreover, esterified fatty acids, in the form of TAG stored in white adipose tissue (WAT), serve as the major -Test the solubility of the fatty acids using water energy reserve of the body. and CHCl3 - This test explains to us that the lipids in their II. EXPERIMENT hydrophobically associated form are extracted to be able to get the lipids through the use of non- polar solvents such as chloroform because even if A. EXTRACTION OF TOTAL LIPIDS the fatty acids have amphipathic form (hydrophobic 1. Cut about 30 gms of pig’s brain and homogenize the material state and hydrophilic state), most of the time in a blender with ether-alcohol mixture for 10 minutes. Filter (especially for long chain fatty acids), the the material. The ether-alcohol extract contains total lipids. hydrophobic state is more prominent. -This also explains that the fatty acid, especially the 2. Divide the ether-alcohol extract into two parts: very long chain of fatty acid, are soluble in non- ○ 2/3 portion - this will be used for the rest of the polar solvent (solvents that are hydrophobic). An experiments example of the non-polar solvent is the ○ 1/3 portion chloroform. When fatty acid is added to chloroform, it becomes soluble in that solvent. 3. Isolate Triglycerides While if fatty acid is added to water, the ○ Take the first 2/3 portion and evaporate the extract hydrophobic state will be prominent and it will not over a hot water bath (without flame) or over a hot be soluble with water. plate to a syrupy consistency. - Results: ○ Add 10 ml of alcoholic KOH. Stir and transfer In water: polar insoluble In CHCL3: non polar soluble mixture into an Erlenmeyer flask. Place a few glass beads to prevent bumping during the heating. ○ Cover the mouth of the flask with a funnel and put 2. Translucent Spot Test - Place a small amount of the precipitate on a piece the flask over low heat for 30 minutes with of clean ordinary writing paper. Note the semi- occasional shaking. Add distilled water if transparent spot formed. The spot remains even necessary to replace lost volume. after long standing. Page 1 of 4 PREPARED BY: CMED 1F  (001) LIPIDS – FATTY ACID AND TRIACYLGLYCEROL METABOLISM DR. HADLOC | 12/01/2020 - In this test, you actually just need to compare which eicosapentaenoic acid and docosahexaenoic acid, among the two (fatty acid or glycerol) is transparent synthesized from EFAs, are important components of the and which one is translucent (translucent=semi- brain and retina. transparent, whitish). - Linoleic and α-linolenic acids are essential fatty acids (EFA) Results: required for membrane fluidity and synthesis of eicosanoids. Positive - For EFA deficiency to develop, dietary intake must be very Fatty acid – translucent low. Even small amounts of EFAs can prevent EFA Glycerol – transparent deficiency. Cow’s milk has only about 25% of the linoleic acid in human milk, but when ingested in normal amounts, it has 3. Iodine Absorption Test enough linoleic acid to prevent EFA deficiency. Total fat - Dissolve small amounts of precipitate in 5 mL of intake of people in many developing countries may be very CHCl3. Add one drop of Hubl’s iodine reagent and shake. The iodine reagent will decolorize if low, but the fat is often vegetable based, with large amounts unsaturated fatty acids are present. This is due to of linoleic acid and enough linolenic acid to the absorption of the iodine by the double bonds of prevent EFA deficiency. the fatty acids. Shaking of CHCl3 with one drop of - Babies fed formula low in linoleic acid, such as a skim-milk the iodine reagent should do a control test but to formula, can develop EFA deficiency. EFA deficiency used which no fatty acid has been added. to result from long-term total parenteral nutrition (TPN) if fat - Hubl’s iodine is a method to know if the fatty acid is unsaturated. was not included. But now, most TPN solutions include fat - In this test, if fatty acid is added, the Hubl’s reagent emulsions to prevent EFA deficiency. In patients with fat is colored pink. If the fatty acid is unsaturated (it malabsorption or increased metabolic needs (eg, because of has a lot of double bonds), it will decolorize. surgery, multiple trauma, or burns), laboratory evidence of Decolorizing of Hubl’s iodine reagent, it is the EFA deficiency may be present without clinical signs. positive reaction (the fatty acid added to Hubl’s Dermatitis due to EFA deficiency is generalized and scaly; in iodine reagent which is pink, is an unsaturated fatty infants, it can resemble congenital ichthyosis. The dermatitis acid). Results: increases water loss from the skin. - Diagnosis of fatty acid deficiency is usually clinical; however, HubI’s Iodine in CHCl3 with dissolved fatty laboratory assays are now available in large research acid precipitate: centers. Treatment of fatty acid deficiency consists of dietary Decolorized (Cloudy white) EFAs, reversing the deficiency. HubI’s Iodine in CHCl3 with no fatty acid - Humans are unable to make linoleic and linolenic fatty acids. precipitate (control): Consequently, these fatty acids are essential in the diet. Iodine Decolorized (Pink) - Plants provide us with these essential fatty acids. III. ESSENTIAL FATTY ACID DEFICIENCY - Essential fatty acid (EFA) deficiency is rare, occurring most often in infants fed diets deficient in EFAs. A. SYMPTOMS - Signs include scaly dermatitis, alopecia, thrombocytopenia, and, in children, intellectual disability. Diagnosis is clinical. Figure 1. Structure of a fatty acid Dietary replenishment of EFAs. - EFA deficiency, caused primarily by fat absorption, is IV. SYSTEMIC CARNITINE DEFICIENCY characterized by scaly dermatitis as a result of the depletion - Systemic carnitine deficiency is a rare metabolic disorder in of skin ceramides with long-chain fatty acids. which the body cannot properly process fats into energy. Carnitine (3-hydroxy-4-trimethylaminobutyric acid) is mostly B. BIOCHEMICAL EXPLANATION derived from dietary intake, but can also be synthesized by lysine and methionine in the liver and kidney. - The EFAs linoleic and linolenic acid are substrates for the - Carnitine functions to carry fatty acids obtained through diet endogenous synthesis of other fatty acids that are needed to the energy centers in muscle cells (mitochondria). for many physiologic processes, including maintaining the - A deficiency of carnitine results in accumulation of fats in the integrity of skin and cell membranes and synthesizing liver, muscle, and heart. When fat cannot be utilized, glucose prostaglandins and leukotrienes. For example, Page 2 of 4 PREPARED BY: CMED 1F  (001) LIPIDS – FATTY ACID AND TRIACYLGLYCEROL METABOLISM DR. HADLOC | 12/01/2020 is consumed without regeneration via gluconeogenesis V. QUESTIONS resulting in hypoglycemia. - In addition, fat released from adipose tissue accumulates in the liver, skeletal muscle, and heart resulting in hepatic A. ESSENTIAL FATTY ACID DEFICIENCY steatosis and myopathy. - Carnitine can be obtained from the diet, where it is found 1. Draw the structure of the fatty acid primarily in meat products. It can also be synthesized from the amino acids lysine and methionine by an enzymatic pathway found in the liver and kidneys but not in skeletal or cardiac muscle. - Carnitine deficiencies: Such deficiencies result in decreased ability of tissues to use LCFA as a fuel. - Primary carnitine deficiency is caused by defects in a membrane transporter that prevent uptake of carnitine by cardiac and skeletal muscle and the kidneys, causing carnitine to be excreted. Treatment includes carnitine supplementation. - Secondary carnitine deficiency occurs primarily as a result of defects in fatty acid oxidation leading to the accumulation 2. What are the 2 essential fatty acids and where do we usually of acylcarnitines that are excreted in the urine, decreasing get them? carnitine availability. a. Linoleic acid (omega-6) - Vegetable oils (corn, sunflower, safflower, soybean, seaweed, olive, A. SYMPTOMS canola), fish and shellfish, poultry fat, nuts, seeds In infants: b. Alpha-Linolenic Acid (omega-3)- present in plant oils 1. Poor feeding including soybean, canola oils and flaxseed. 2. Tiredness 3. What are the symptoms of essential fatty acid deficiency? 3. Irritability Symptoms may include scaly dermatitis, alopecia (hair loss), 4. Low blood sugar (hypoglycemia) thrombocytopenia (very low platelet). Symptoms in children Carnitine deficiency can occur particularly in the newborn include intellectual disability. Patients also present visual and especially in preterm infants owing to inadequate abnormalities. biosynthesis or renal leakage. Losses can also occur in 4. What fatty acids are found in significant quantities in milk? hemo-dialysis. The fatty acids found in milk are saturated fatty acids (no In childhood, double bonds), such as myristic, palmitic, and stearic make 1. Heart abnormalities up two thirds of milky fatty acids. The most abundant 2. Muscle abnormalities unsaturated fatty acid in milk is the oleic acids with one double bond. B. BIOCHEMICAL EXPLANATION B. SYSTEMIC CARNITINE DEFICIENCY - CDSP is caused by mutations in the SLC22A5gene and inherited as an autosomal recessive genetic condition. 1. What is “systemic carnitine deficiency (SCD)? What is Introduction CDSP is treatable by the daily use of L-carnitine the genetic reason behind it? supplements. Without early detection and treatment, an affected child may experience low blood sugar - Systemic carnitine deficiency is a rare metabolic disorder in (hypoglycemia), seizures, heart muscle weakness which the body cannot properly process fats into energy. (cardiomyopathy) that can be life-threatening. Newborn - CDSP is caused by mutations in the SLC22A5gene and screening is available for CDSP. inherited as an autosomal recessive genetic condition. - Treatment is by oral supplementation with carnitine. 2. Review the carnitine transport of fatty-acyl CoA. What is - Inherited CPT-I deficiency affects only the liver, resulting in it for? reduced fatty acid oxidation and ketogenesis, with hypo- glycemia. CPT-II deficiency affects primarily skeletal muscle - Most long-chain fatty acids are formed outside of the and, when severe, the liver. The sulfonylurea drugs mitochondria. The mitochondrial membrane is impermeable (glyburide [glibenclamide] and tolbutamide), used in the to CoA and its derivatives. Fatty acids are transported into treatment of type 2 diabetes mellitus, reduce fatty acid the mitochondria by using carnitine as a carrier. oxidation and, there-ore, hyperglycemia by inhibiting CPT-I. Page 3 of 4 PREPARED BY: CMED 1F  (001) LIPIDS – FATTY ACID AND TRIACYLGLYCEROL METABOLISM DR. HADLOC | 12/01/2020 Carnitine palmitoyltransferase I (CPT-I) is present in the catalyzes the gluconeogenesis pathway which is also active outer mitochondrial membrane, this will convert the long- during catabolic states like fasting. chain acyl-CoA into acylcarnitine, which is then able to penetrate the inner membrane and gain access to the beta- - When there is a deficiency in carnitine, the long chain fatty oxidation system of enzymes. Carnitine-acylcarnitine acids (LCFA) cannot be transported to the mitochondrial translocase acts as an inner membrane exchange matrix. Improper utilization of fatty acids during fasting transporter wherein acylcarnitine is transported in, coupled impairs gluconeogenesis that leads to non ketotic or with the transport out of one molecule of carnitine. The hypoketotic (no or minimal ketone body production) acylcarnitine would then react with CoA, which is catalyzed hypoglycemia. by Carnitine palmitoyltransferase II (CPT-II) located on the 7. How can we diagnose such disorder? What laboratory inside of the inner membrane. Acyl-CoA is reformed in the procedures can be used? mitochondrial matrix, and carnitine is freed. - SCD manifests in infants which shows the importance of newborn screening. Newborn screening is a test used to diagnose if an infant has SCD, another diagnostic test that can be done includes plasma free carnitine concentration, urine organic acid analysis and fibroblast carnitine transport. 8. What are the treatment modalities for SCD? - L-carnitine supplementation and avoid prolonged fasting. TEST YOURSELF 1. What are the two essential fatty acids? 2. Where can we find linoleic acid? 3. Where can we find alpha-linolenic acid? 4. What result is expected in Hubl’s Iodine in CHCl3 with dissolved fatty acid precipitate? 3. What are the outstanding manifestations of patients with 5. What are the three tests for fatty acids? SCD. 6. This type of test extracts lipids through the use of non-polar solvent like chloroform. a. Primary SCD – Carnitine concentration is extremely 7. TRUE or FALSE. For Essential fatty acid deficiency to reduced mainly in the heart, skeletal muscles, liver develop, dietary intake must be very high. and blood plasma. 8. What are the symptoms of essential fatty acid deficiency? b. Primary myopathic CD – reduced carnitine but not in the liver, plasma and in the kidney. The molecular basis ANSWERS of this is defect of the carnitine influx carrier causing 1. Linoleic acid (omega-6) and Alpha-Linolenic Acid (omega-3) increase loss of carnitine in the tissue. 2. Vegetable oil, meat, fish, poultry, fats and seeds 3. Plant oils including soybean, canola oils and flaxseed. 4. Decolorized (cloudy white) 4. What can cause accumulation of lipids in the liver of 5. Solubility, Translucent Spot test and Iodine Absorption Test patients with SCD? 6. Solubility Test 7. FALSE Carnitine functions to carry fatty acids obtained through diet 8. Scaly dermatitis, alopecia (hair loss), thrombocytopenia (very low platelet). Symptoms in children include intellectual disability and visual to the energy centers in muscle cells (mitochondria). A abnormalities. deficiency of carnitine results in accumulation of fats in the liver, muscle, and heart. REFERENCES 5. What is the cause of hypoglycemia in patients with 1.Dahash BA, Sankararaman S. Carnitine Deficiency. [Updated SCD? 2020 Aug 10]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from: When fat cannot be utilized, glucose is consumed without https://www.ncbi.nlm.nih.gov/books/NBK559041/ regeneration via gluconeogenesis. It results in 2.Ferrier, Denise R. (2017). Lippincott Illustrated Reviews: hypoglycemia. Biochemistry (7th ed.). Philadelphia: Wolters Kluwer 6. Patients with SCD are unable to produce ketone bodies after a 24-hour fasting. Why? - Acetyl CoA is used for energy and ketone body production. It is also an allosteric activator of pyruvate carboxylase that Page 4 of 4 PREPARED BY: CMED 1F