Biochem Lab - Mitochondrial Electron Transport - PDF

(010) Mitochondrial Electron Transport and Oxidative Phosphorylation Metabolism of Dietary Lipids LABORATORY DR. HADLOC 11/24/2020 OUTLINE...

(010) Mitochondrial Electron Transport and Oxidative Phosphorylation Metabolism of Dietary Lipids LABORATORY DR. HADLOC 11/24/2020 OUTLINE hazard of abruptly lowering the blood pressure is causing hypotension and subsequent ischemia to the brain or heart. I. CYANIDE POISONING In other words, the very treatment designed to prevent end- A. Case organ disease may cause the problem. To avoid B. Clinical Correlation precipitous hypotension, agents that induce a smooth fall in blood pressure are preferable, such as sodium C. Discussion nitroprusside, a titratable intravenous agent used for D. Comprehension Question malignant hypertension. Its desirable properties include the II. HYPERTRYGLYCERIDEMIA ability to precisely increase or decrease the infusion to A. Case affect the blood pressure. One side effect of sodium nitroprusside is that its metabolite is thiocyanate, and with B. Clinical Correlation prolonged use, cyanide poisoning may result, which inhibits C. Discussion the electron transport chain. Thus, in clinical practice, short- D. Comprehension Questions term nitroprusside is used, or serum thiocyanate levels are drawn. III. STEATORRHEA IV. TEST YOUR KNOWLEDGE Approach to Electron Transport System (ETS) and Cyanide Objectives: I. CASE: CYANIDE POISONING A. A 68-year-old female in a hypertensive crisis is being treated 1. Know about the function of the electron transport chain in the intensive care unit (ICU) with intravenous nitroprusside (ETC). for 48 hours. The patient’s blood pressure was brought back down to normal levels; however, she was complaining of a 2. Understand what factors may inhibit the ETC. burning sensation in her throat and mouth followed by 3. Be familiar with the biochemical process by which the nausea and vomiting, diaphoresis, agitation, and dyspnea. therapy for cyanide poisoning works. (Nitrates, The nurse noticed a sweet almond smell in her breath. An Thiosulfate, Oxygen) arterial blood gas revealed a significant metabolic acidosis. 4. Recognize other ETC sites and agents of inhibition. A serum test suggests a metabolite of nitroprusside, thiocyanate, is at toxic levels.  What is the likely cause of her symptoms? C. Discussion: Cyanide poisoning from toxic dose of The electron transport chain (ETC) or electron transport nitroprusside system (ETS) shown below is located on the inner  What is the biochemical mechanism of this problem? membrane of the mitochondrion and is responsible for the Cyanide inhibits mitochondrial cytochrome harnessing of free energy released as electrons travel from oxidase, blocking electron transport and more reduced (more negative reduction potential, E¢ 0 ) to preventing oxygen utilization. Lactic acidosis more oxidized (more positive E¢ 0 ) carriers to drive the results secondary to anaerobic metabolism. phosphorylation of ADP to ATP. Complex I accepts a pair  What is the treatment for this condition? of electrons from NADH (Eʹ 0 = −0.32 V) Supportive therapy, gastrointestinal (GI) decontamination, oxygen, and antidotal therapy with amyl nitrite, sodium nitrite, and sodium thiosulfate. B. Clinical Correlation: Hypertensive emergencies are defined as episodes of severely elevated blood pressure, such as systolic levels of 220 mm Hg and/or diastolic blood pressures exceeding 120 mm Hg with patient symptoms of end-organ dysfunction. These symptoms may include severe headache, neurological deficits, chest pain, or heart failure symptoms. Hypertensive emergencies require immediate lowering of Schemic diagram of Electron Transport chain ATP synthase and the blood pressure to lower (but not necessarily to normal) levels. In contrast, hypertensive urgencies are ATP/ADP translocase. circumstances of markedly elevated blood pressures in the absence of patient symptoms; lowering the blood pressure and passes the electron pair through the intervening over 24 to 48 hours is reasonable in these cases. One carriers to complex IV, which passes the electrons to one PREPARED BY GROUP 2 IN TRANS MAKING  (010) Mitochondrial Electron Transport and Oxidative Phosphorylation Metabolism of Dietary Lipids LABORATORY DR. HADLOC 11/24/2020 atom of molecular oxygen (Eʹ 0 = +0.82 V) to form water Each of the cytochromes has a heme cofactor but they vary with hydrogen ions (H+) from the medium. The transport of slightly. The b-type cytochromes have protoporphyrin IX, electrons through the carriers is highly coupled to the which is identical to the heme in hemoglobin. The c-type formation of ATP from ADP and Pi through the formation cytochromes are covalently bound to cysteine residue 10 in and relaxation of the proton gradient formed across the the protein. The a-type cytochromes have a long isoprenoid inner mitochondrial membrane by electron transport. Each [(CH2 –CH=C(CH3 )–CH2 )n ] tail bound at one side-chain time electrons are transported between complexes I and III, position. Inhibition of the electron transport chain in coupled between complexes III and IV, or between complex IV and mitochondria can occur at any of the three constituent oxygen, protons are extruded from the mitochondrial matrix functional processes; electron transport per se, formation of across the inner membrane to the intermembrane ATP, or antiport translocation of ADP/ATP (Table 16-1). space/cytosol. (The outer membrane poses no barrier to The best-known inhibitor of the ADP/ATP translocase is proton passage.) In other words, the energy gained from atractyloside in the presence of which no ADP for these electron transfers is used to pump protons from the phosphorylation is transported across the inner membrane mitochondrial matrix side to the cytosol side. Because the to the ATP synthase and no ATP is transported out. In the mitochondrial membrane is impermeable to protons, there absence of ADP phosphorylation, the proton gradient is not is a gradient that develops with a higher concentration of reduced allowing other protons to be extruded into the protons outside the matrix. The protons then come through intermembrane space because of the elevated [H+], and the ATP synthase complex through proton pores, and as thus electron transfer is halted. Likewise, the antibiotic they come back into the mitochondrial matrix, ADP is oligomycin directly inhibits the ATP synthase, causing a phosphorylated to ATP. Thus, because the process of cessation of ATP formation, buildup of protons in the electron transport is tightly coupled to ADP intermembrane space, and a halt in electron transfer. phosphorylation, ADP must be present for electron Similarly, a blockade of complex I, III, or IV that inhibits transport to proceed, and therefore the ADP/ATP electron flow down the chain to O2 would also stop both translocase must be able to exchange a molecule of ADP ATP formation and ADP/ATP translocation across the inner in the cytosol for a molecule of ATP (newly made) in the mitochondrial membrane. Cyanide ion (CN−) is a potent matrix of the mitochondria. When these various processes inhibitor of complex IV the cytochrome c oxidase operate in concert the mitochondria are said to exhibit component of the electron transport system in the oxidized coupled respiration. The components of the electron state of the heme (Fe3+). It can be delivered to tissue transport chain have various cofactors. Complex I, NADH electron transport systems as a dissolved gas after dehydrogenase, contains a flavin cofactor and iron sulfur breathing HCN or ingested as a salt such as KCN or as a centers, whereas complex III, cytochrome reductase, medication leading to the formation of CN− such as contains cytochromes b and c1. Complex IV, cytochrome nitroprusside. Cyanide ion competes effectively with oxidase, which transfers electrons to oxygen, contains oxygen for binding to cytochrome c oxidase at the oxygen- copper ions as well as cytochromes a and a3. The general binding site. Cyanide binding and therefore cyanide structure of the cytochrome cofactors is shown in the figure poisoning is reversible if treated properly and early. The below. treatment strategy depends on dissociation of cyanide from cytochrome a/a3 (Fe3+). Increasing the percentage of oxygen breathed will increase the competition of oxygen over cyanide for the cytochrome a/a3 (Fe3+). Two other medications foster this competition. Nitrite ion (NO2 −) is administered to convert some oxyhemoglobin [HbO2 (Fe2+)] to methemoglobin [met HbOH(Fe3+)], another competitor for cyanide binding. Heme active center of cytochromes a, b, and c components of ETC PREPARED BY GROUP 2 IN TRANS MAKING  (010) Mitochondrial Electron Transport and Oxidative Phosphorylation Metabolism of Dietary Lipids LABORATORY DR. HADLOC 11/24/2020 A. Cytochrome c reductase B. Cytochrome oxidase C. Coenzyme Q reductase D. NADH dehydrogenase E. Succinate dehydrogenase ANSWER: B The culprit here is cyanide produced from acetonitrile. Cyanide inhibits the electron transport chain of cytochrome oxidase. 2. Which of the following, best describes the reason for the latency of acetonitrile toxicity and why prompt treatment would have prevented this child’s respiratory distress and death? A. Acetonitrile crosses the mitochondrial membrane slowly. B. Acetonitrile induces hemolysis by inhibiting glucose 6- phosphate dehydrogenase. C. Acetonitrile is only poorly absorbed by the intestinal The cyanide adduct of methemoglobin is formed releasing system. cytochrome oxidase in the Fe3+ form ready to bind oxygen D. Complex IV of the electron transport system binds and disinhibit the electron transport chain. To remove the acetonitrile weakly. cyanide, adduct in a nontoxic fashion, thiosulfate ion is E. Cytochrome P450 enzymes oxidize acetonitrile and administered. The mitochondrial enzyme rhodanese slowly release cyanide. catalyzes the conversion of cyanide and thiosulfate to thiocyanate and sulfite. Thiocyanate is incapable of inhibiting cytochrome oxidase and is excreted. The ANSWER: E methemoglobin can be reconverted to oxyhemoglobin by Acetonitrile itself is not the toxicant but undergoes metabolism and NADH and methemoglobin reductase. Other sites of the produces cyanide, which is the toxic agent here electron transport chain can be targets of inhibitors based on similarity in structure to enzyme components or to substrates of the various components. For instance, the fish poison rotenone resembles the isoalloxazine ring of the 3. Inhibition of oxidative phosphorylation by cyanide ion leads to increases in which of the following? FMN cofactor of complex I, the NADH CoQ reductase. Rotenone binds the enzyme quite avidly and prevents A. Gluconeogenesis to provide more glucose for transfer of electrons from NADH to coenzyme Q through metabolism the iron sulfur centers and thus inhibits oxidation of NADH B. Transport of ADP into the mitochondria and subsequent reduction of oxygen to water. On the other hand, carbon monoxide resembles molecular oxygen and C. Utilization of fatty acids substrates to augment glucose utilization binds with a higher affinity than oxygen to complex IV, the cytochrome oxidase component, inhibiting transfer of D. Utilization of ketone bodies for energy generation electrons to oxygen. E. Lactic acid in the blood causing acidosis D. Comprehension Questions: A 16-month-old girl was found to have ingested approximately 30 mL of an acetonitrile based cosmetic nail remover when she vomited 15 minutes post ingestion. The poison control center was contacted, but ANSWER: E no treatment was recommended because it was confused with an Gluconeogenesis requires ATP, which is in short supply, turning up acetone-based nail polish remover. The child was put to bed at her the catabolism of glucose to lactate in the absence of an intact normal time, which was 2 hours post ingestion. Respiratory distress electron transport chain. ADP cannot be transported into the developed sometime after the child was put to bed, and she was mitochondrion because ATP, its antiporter partner, is not made by found dead the next morning. oxidative phosphorylation as a result of cyanide inhibition of cytochrome oxidase. Metabolism of fatty acids and ketone bodies requires a functional electron transport chain for their metabolism, and these possibilities are also ruled out 1. Inhibition of which of the following enzymes was the most likely cause of this child’s death? PREPARED BY GROUP 2 IN TRANS MAKING  (010) Mitochondrial Electron Transport and Oxidative Phosphorylation Metabolism of Dietary Lipids LABORATORY DR. HADLOC 11/24/2020 4. Which of the following procedures best describes the abdominal pain is located in the epigastric region and radiates emergency intervention for cyanide poisoning? to his back. He has had several episodes of similar pain in the past but none quite as severe. His parents deny fever/chills A. Decrease the partial pressure of oxygen and change in bowel habits. In the ER, the patient is afebrile B. Treatment with nitrites to convert hemoglobin to and in moderate distress. Both the liver and spleen appear to methemoglobin. be enlarged and he has epigastric tenderness. Several small C. Treatment with thiosulfate to form thiocyanate yellow-white papules were noted on his back and buttocks. Laboratory tests reveal elevated amylase and lipase levels. D. Use of Mucomyst (N-acetylcysteine) taken orally On further questioning, the father reports having high ANSWER: B triglyceride levels and several members of the mother’s family Increased oxygen competes with cyanide bound to cytochrome have had early heart disease. Laboratory tests performed oxidase displacing it. Nitrites bind to hemoglobin, converting it to after hospitalization revealed elevated triglyceride levels and methemoglobin, which binds cyanide more tightly than reduced lipoprotein lipase activity. cyanhemoglobin and pulls cyanide from cyanhemoglobin to form  What is etiology of the boy’s abdominal pain? cyanomethemoglobin. Thiosulfate is used to displace cyanide from Acute pancreatitis cyanomethemoglobin to form thiocyanate, which can be excreted, a happy ending for cyanide poisoning. N-acetylcysteine is used for acetaminophen toxicity, not cyanide toxicity How to diagnose Acute pancreatitis? 5. An unskilled worker in a water garden/plant nursery was 1. Identify if the symptoms of the patient is suggestive sent to sweep up a spill of a white powder in the storage shed. Later of acute pancreatitis he was found with labored breathing and convulsions. On further - Epigastric pain for the past 24 hours examination, the white powder was identified as rotenone. - Moderate to severe abdominal pain Respiratory distress is induced on rotenone exposure because it - Pain is also characterize as radiating to the inhibits the complex that catalyzes which of the following? back A. Electron transfer from NADH to coenzyme Q - Accompanied with anorexia; nausea - Did you have alcohol binge drinking in the B. Oxidation of coenzyme Q past few hours? C. Reduction of cytochrome c  Alcohol intake is considered the second D. Reduction of cytochrome c most common cause of acute E. Electron transfer from cytochrome a1/a3 to oxygen pancreatitis ANSWER: A - Do you have any history of gallbladder stones? Rotenone binds avidly to the flavoprotein NADH CoQ reductase,  Gallbladder stones are considered to be complex I (also called NADH dehydrogenase). The central portion of the first common reason of acute the rotenone structure resembles the isoalloxazine ring of the FMN pancreatitis molecule, and when it binds to complex I, rotenone prevents the - Any history of dyslipidemia specifically transfer of electrons from NADH to coenzyme Q. hypertriglyceridemia in the family? 6. The major metabolic consequence of perturbation of the Most common causes of acute pancreatitis: Gallbladder stones, electron transfer in mitochondria is which of the following? Alcohol intake and Hypertriglyceridemia A. Increased production of NADPH B. Increased oxidation of NADH C. Increased reduction of O2 to H2 O 2. Request for serum amylase lipase in the laboratory D. Decreased regeneration of NAD+ Amylase and lipase – If positive, the patient is having acute E. Decreased reduction of FAD pancreatitis. It is more than times three of the upper limit of the ANSWER: D normal value. Inhibition of the electron transport chain shuts down the major pathway of regenerating NAD+ from the NADH produced in intermediary metabolism. This forces the cytosolic conversion of 3. Radiographic imaging of acute pancreatitis pyruvate to lactate to regenerate NAD+ so that glycolysis can - CT scan continue in the absence of a functioning electron transport system. III.CASE: HYPERTRIGLYCERIDEMIA (LIPOPROTEIN  What is the likely underlying biochemical disorder? LIPASE DEFICIENCY) Disorder of lipoprotein metabolism  What is the role of lipoprotein lipase? A. 35 A 9-year-old boy is brought to the ER by his parents after 2 days of worsening nausea/vomiting and abdominal pain. The PREPARED BY GROUP 2 IN TRANS MAKING  (010) Mitochondrial Electron Transport and Oxidative Phosphorylation Metabolism of Dietary Lipids LABORATORY DR. HADLOC 11/24/2020 Hydrolysis of triglycerides from VLDL and cholesterol and fat-soluble vitamins. The free fatty acids chylomicrons and monoglycerides are absorbed by the microvilli of the intestinal epithelial cells. In the epithelial cell, the fatty acids B. Clinical Correlation: and monoglycerides are reformed into triglycerides, which Lipoprotein lipase (LPL) is an enzyme found on the are packaged with phospholipids, cholesterol, and capillary endothelial surface of adipose tissue, heart, and apolipoprotein B-48 into chylomicrons. The newly skeletal muscle and is required, along with apoC-II, for the synthesized chylomicrons are secreted into the lymph and hydrolysis of triglycerides. ApoC-II, found on the surface of enter the bloodstream via the thoracic duct. In the chylomicrons and VLDL, serves as an activator of LPL. A bloodstream, the chylomicron particles obtain proteins from deficiency in LPL results in elevated levels of triglycerides high-density lipoproteins (HDL), including apoC-II and (VLDL and chylomicrons). LPL deficiency is inherited in an apoE, which are important for the function of the autosomal recessive pattern. Patients with LPL deficiency chylomicron. often present with recurrent episodes of pancreatitis in their childhood and may have other clinical signs of hypertriglyceridemia such as: xanthomas, hepatosplenomegaly, and lipemia retinalis. Reduced serum LPL activity, after an injection of IV heparin, confirms diagnosis of LPL or apoC-II deficiency. The initial intervention is primarily of diet modification (reduction of fat intake). Approach to Lipid Transport Objectives 1. Describe the metabolism and transport of lipoproteins. 2. Understand the rationale for serum blood test results with the different hypertriglyceridemias. If the patient doesn’t have gall stones and doesn’t have history of binge drinking of alcohol, then consider if the patient has hypertriglyceridemia. Request for serum total cholesterol including triglycerides in order to rule out if the patient has hypertriglyceridemia. The significant value of triglycerides considered to be a risk factor for acute pancreatitis is 1000 mg/dL. C. Discussion: Formation and metabolism of chylomicrons Triglycerides (triacylglycerols, TG) are safely transported in In the capillary beds in adipose tissue, muscle tissue the bloodstream packaged into lipoproteins called (especially cardiac muscle) and in lactating mammary chylomicrons or very-low-density lipoproteins (VLDLs). glands, apoC-II binds to and activates lipoprotein lipase Chylomicrons are formed in the epithelial cells of the (LPL), which is bound to the endothelial surface of the intestine and are responsible for the transport of dietary capillaries by heparan sulfate. LPL hydrolyzes the TG in the lipids. VLDL is synthesized in the liver and transport core of the chylomicron to free fatty acids and glycerol. The endogenously synthesized lipids from the liver to peripheral fatty acids are taken up by the adipose or muscle cells; tissues. Both of these lipoproteins are composed of a core glycerol is recycled back to the liver. In muscle, the fatty composed primarily of TG enveloped by a monolayer acids are oxidized to produce ATP and in adipose they are composed of phospholipids, free cholesterol, and reformed into TG for storage. The TG-depleted chylomicron apolipoproteins. Dietary triglycerides are hydrolyzed by remnant remains in the blood stream until it binds to the pancreatic lipase in the lumen of the small intestine. chylomicron remnant receptor located on hepatocytes, a Colipase, a protein secreted along with pancreatic lipase, process mediated by apoE. The remnants are taken into binds to the TG and the pancreatic lipase and improves the the hepatocytes by endocytosis and degraded in the hydrolytic process. TG is broken down to free fatty acids lysosome to fatty acids, amino acids, cholesterol, glycerol, and 2-monoacylglycerols, which form micelles along with and phosphate. Chylomicrons appear in the blood stream bile salts and other lipid soluble compounds such as shortly after consumption of a meal containing fat. PREPARED BY GROUP 2 IN TRANS MAKING  (010) Mitochondrial Electron Transport and Oxidative Phosphorylation Metabolism of Dietary Lipids LABORATORY DR. HADLOC 11/24/2020 However, the clearance rate for chylomicrons is fast and blood is usually free of chylomicrons following an overnight fast. VLDLs are lipoproteins synthesized in the liver from endogenous TG, cholesterol, and phospholipids, along with several apolipoproteins including, apoB100, apoE, and apoC-II. Following synthesis, the VLDL is secreted into the bloodstream, where it obtains more apoE and apoC-II from HDL. There is hypertriglyceridemia when there is inability for lipoprotein lipase to degrade triglycerides. High amount of triglycerides in the bloodstream, pancreatic lipase will identify this elevated number and will act on them. Eventually, when these triglycerides will be degraded by pancreatic lipase, there will be enormous fatty acids in the bloodstream. These free fatty acids will cause cytotoxic injury to the pancreas causing inflammation and acute pancreatitis. Treatment of acute pancreatitis: 1. Hydrate the patient with 200 ml saline solution - In acute pancreatitis, there is a severe inflammation of the pancreas 2. Pain medication 3. Identify the cause of acute pancreatitis - Ultrasound – to identify if patient has gall stones - Serum amylase and lipase - Lipid profile – hypertriglyceridemia - CBC test - This will tell you if the patient has necrotizing pancreatitis Formation of VLDL and 4. Bowel rest metabolism into LDL - Don’t allow patient to eat fatty foods 5. Antibiotics In a process similar to that of chylomicron degradation, the VLDL apoC-II binds to and activates LPL in the capillary beds of adipose, muscle and mammary tissue. LPL degrades the VLDL TG core, releasing free fatty acids and glycerol. About half of the resulting VLDL remnants bind to receptors in liver cells that recognized apoE and are taken up by endocytosis. The remaining VLDL receptors are further degraded to intermediate-density lipoproteins (IDL), which have their residual TG removed by the action of hepatic lipase to yield low-density lipoproteins (LDL). Hepatic lipase (HL) is synthesized and secreted from liver and is tethered to the external surfaces of liver cells by heparan sulfate. LDL is taken up by receptor-mediated endocytosis upon binding to the LDL receptor in liver and peripheral tissues. Elevated serum TG levels can occur due to a number of factors. Hypertriglyceridemia can be the result of a genetic disorder in one of the proteins involved in lipoprotein metabolism, or it can arise secondarily to a number of other disorders, including diabetes mellitus, obesity, and alcohol abuse, and as a side effect of some PREPARED BY GROUP 2 IN TRANS MAKING  (010) Mitochondrial Electron Transport and Oxidative Phosphorylation Metabolism of Dietary Lipids LABORATORY DR. HADLOC 11/24/2020 medications such as β-blockers, oral estrogens, and some 2) Laboratory results for a patient with uncontrolled diuretics. Genetic deficiencies in LPL, apoC-II, or HL can Type I diabetes mellitus reveal hyperglycemia give rise to elevations in circulating TG, as can over (634 mg/dL) and hypertriglyceridemia (498 mg/dL). The production of apoB100 or increased apoE2 levels. ApoE2 most likely cause of the hypertriglyceridemia in this has a decreased affinity for hepatic receptors than apoE3 patient is which of the following? and chylomicron remnants and VLDL remnants containing apoE2 are cleared more slowly from the circulation. The A. Deficiency in apoprotein C-II most common genetic defect leading to B. Increased hepatic triglyceride synthesis hypertriglyceridemia is a deficiency in LPL, which results in C. Decreased lipoprotein lipase activity increased levels of both chylomicrons and VLDL. Individuals who are homozygous for the defective gene D. Deficiency in LDL receptors usually present with symptoms of chylomicronemia (TG E. Absence of hormone-sensitive lipase levels >2000 mg/dL, abdominal pain, pancreatitis, xanthomas, lipemia retinalis) in childhood. Clinical ANSWER: C diagnosis of LPL deficiency requires measurement of LPL activity in plasma following intravenous injection of heparin, Decreased lipoprotein lipase activity which displaces the LPL from its heparan sulfate tether. However, heparin also releases HL into the plasma and the The triglyceride components of VLDL and chylomicron are post-heparin plasma must be treated with antibodies hydrolyzed to free fatty acids and glycerol in the capillaries of tissues specific to HL to remove it. Alternatively, LPL can also be such as liver, adipose tissue, and skeletal muscle by the actions of measured in adipose tissue, which has no HL activity. A lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL, also deficiency in apoC-II will also show evidence of decreased called hepatic lipase, HL). Insulin exerts numerous effects on overall postheparin plasma LPL activity. An increase in LPL activity metabolic homeostasis. One of the effects of insulin, which relates to when normal apoC-II is added to the assay indicates that a the symptoms in this patient, is the regulation of the expression of defect in apoC-II is the culprit. A deficiency in HL can also LPL on the surface of endothelial cells. Since type 1 diabetics do not lead to elevated plasma TG levels, however, these synthesize insulin, they do not properly regulate the level of LPL, increases in TG are usually found in VLDL remnants, LDL, which contributes to hypertriglyceridemia in these individuals. and HDL, which all become more buoyant in their densities. Definitive diagnosis of HL deficiency is made by demonstrating the absence of HL activity in postheparin plasma. DIARRHEA is having vowel movement of at least 3 times per day with a quantity of at least 200 grams of stool weight per day. D. Comprehension Questions: - ACUTE DIARRHEA: may be caused by INFECTON, FOOD 1) A teenage boy presents with moderate to severe ALLERGIES, and MEDICATIONS epigastric pain. Physical examination reveals extensive - CHRONIC DIARRHEA: 4 weeks or more eruptive xanthomas and hepatosplenomegaly. A blood After the symptoms are fulfilled: sample reveals milky plasma. Which of the following is the - Request for FECALYSIS which identifies if the diarrhea is most likely lipoprotein to be elevated in this patient’s characterized by WATERY, INFLAMMATORY OR FATTY plasma? (causing Steatorrhea) A. Chylomicrons B. Chylomicron remnants III.STEATORRHEA C. HDL D. IDL Steatorrhea is defined as increase in fat excretion in the E. LDL stools. Patients present with bulky, pale, foul-smelling oily stools. In the early stages, steatorrhea may be asymptomatic. Children may present with growth failure ANSWER: A and delayed puberty. In severe cases, loss of Chylomicrons are assembled in the intestinal mucosa as a subcutaneous fat and muscle wasting may be evident. means to transport dietary cholesterol and triglycerides to Manifestations of fat-soluble vitamin (A, D, E, and K) the rest of the body. Chylomicrons leave the intestine via deficiencies can accompany fat malabsorption. the lymphatic system and enter the circulation at the left The causes of steatorrhea are numerous and subclassify subclavian vein. High levels of chylomicrons in the blood under three broad categories: can give it a milky appearance. The term chyle refers to the milky fluid consisting of lymph and emulsified fats or free 1) Exocrine Pancreatic Insufficiency (EPI) due to chronic fatty acids, which is how the term chylomicron was derived. pancreatitis, cystic fibrosis PREPARED BY GROUP 2 IN TRANS MAKING  (010) Mitochondrial Electron Transport and Oxidative Phosphorylation Metabolism of Dietary Lipids LABORATORY DR. HADLOC 11/24/2020 (CF), and conditions resulting in pancreatic duct obstruction or resection of the pancreas (e.g., pancreatic Sudan III is a red fat-soluble dye that is tumors) utilized in the identification of the presence of lipids, triglycerides and CHRONIC PANCREATITIS: inability of the pancreas to lipoproteins. deliver lipases into the pancreatic duct down into the duodenum so lipids are not digested well, eventually the The Reaction: patients will present fatty diarrhea. Sudan III reacts with the lipids or triglycerides to stain red in colour. 2) Bile acid deficiency either due to cholestasis (e.g. primary biliary cirrhosis, currently referred as primary Lab results: The oil will stain red with biliary cholangitis (PBC), primary sclerosing cholangitis Sudan III dye since it is a lipid and (PSC)) or inability to absorb bile acids in the distal ileum contains triglycerides. However, since resulting in diminished the oil is less dense than water and bile acid pool (e.g. ileal resection or Crohn disease of insoluble in water, the oil will form a the ileum) or deconjugation of bile acids (e.g., small layer or globules above the water and intestinal bacterial overgrowth (SIBO)) appear as a red layer above the water in the test tube. BILE is produced in the LIVER. It will eventually mix with pancreatic enzymes, and works to emulsify the lipids in the intestines. EMULSIFICATION is a process where by SUDAN III the lipids to be readily absorbed in the intestines. - Get a test tube. Place the stool samples (most of the time CHOLESTASIS: obstruction within the biliary duct. Bile appears watery) in the test tube. Then add the Sudan III dye. from the liver is not delivered adequately down in the common bile duct down to the duodenum. - At first, the dye will appear in the upper most portion of the tube and the fecal sample is on the lower portion of the test tube. BILE ACIDS ARE RECYCLED IN THE TERMINAL - Sudan III is fat soluble. It is used to utilize the presence of lipids, ILEUM. After being delivered in the common bile duct, triglyceride and lipoproteins. The expected reaction, the upper these acids containing bile salts will deliver in the (Sudan III) portion of the tube will react with the fatty stool of the terminal ileum then eventually absorbed and recycled patient causing the entire tube to become red. And this will back into the liver to facilitate its work to emulsify fats. identify if it is a watery or fatty diarrhea. QUANTITATIVE ESTIMATION OF FECAL FAT 3) Diseases affecting proximal small intestines such as celiac disease, tropical sprue, giardiasis, Whipple - A 24-hour stool sample will be collected, and will ask the disease, lymphoma, amyloidosis, SIBO, and HIV laboratory to estimate the presence of fecal fats. enteropathy. - Significant value: exceeding 7 grams of fat present in the 24-hr stool sample - You must have enough fecal elastase in the stool Screening for steatorrhea may be carried out by examining stool samples for the presence of fat by Sudan III staining. - More than 200 microgram per gram of stool = NORMAL fecal However, quantitative fecal fat estimation is required to elastase confirm the diagnosis. Quantitative estimation of fecal fat - less than 100 microgram per gram of stool = ABNORMAL – (exceeding 7 g per 24 hours) is an essential first step for pancreatic insufficiency the diagnosis. The standard method of fecal fat quantification is by calculating the coefficient of fat absorption (CFA). For evaluation of EPI, fecal elastase may be utilized instead of the 72-hour fecal fat testing. A value of > 200 µg/g of stool is normal; 100 to 200 µg/g as indeterminate, and < 100 µg/g is abnormal and indicative of EPI. PREPARED BY GROUP 2 IN TRANS MAKING  (010) Mitochondrial Electron Transport and Oxidative Phosphorylation Metabolism of Dietary Lipids LABORATORY DR. HADLOC 11/24/2020 References: 1F, 2A, 3Liver, 4-24 hour stool, 5C, 6.alcoholism https://schoolbag.info/chemistry/biochemistry/21.html https://www.memorangapp.com/question/3962/ https://www.memorangapp.com/question/3963/ IV.TEST YOUR KNOWLEDGE 1. All are fat-soluble vitamins except a. Vit. A b. Vit D c. Vit K d. Vit E e. All of the above f. None of the above 2. Diarrhea I. 200 g of stool per day II. 300g of stool per day III. vowel movement atleast 3 times/day IV. vowel movement atleast 2 times/day a. I And III b. I and IV c. II and III d. II and IV 3. What is the organ that produces bile? 4. What is the sample for the quantitative estimation of fecal fat? 5. Value for normal fecal elastase a. 100 microgram per gram of stool c. >200 microgram per gram of stool d. >300 microgram per gram of stool 6. 2nd most common cause of acute pancreatitis PREPARED BY GROUP 2 IN TRANS MAKING