Metabolism of Ethanol Biochemistry Lecture PDF 2023

Metabolism of Ethanol Lecture 39 Reference: Lieberman and Peet, Chapter 33 Bluefield University – VCOM Campus MABS Program Biochemistry Jim Mahaney, PhD 1 Lecture Objectives a. Recall the two phases of ethanol metabolism in the liver. b. Compare and contrast the function of alcohol dehydrogenase...

Metabolism of Ethanol Lecture 39 Reference: Lieberman and Peet, Chapter 33 Bluefield University – VCOM Campus MABS Program Biochemistry Jim Mahaney, PhD 1 Lecture Objectives a. Recall the two phases of ethanol metabolism in the liver. b. Compare and contrast the function of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) enzymes in ethanol metabolism. c. Recall the function of the microsomal ethanol oxidizing system (MEOS) and when it is most active in ethanol metabolism. d. Recall the fundamental mechanism of ethanol metabolism by the MEOS cytochrome P450 enzyme. Recall the two potentially harmful products produced by the MEOS. e. Compare and contrast the energy yield from ethanol metabolism by ADH/ALDH versus the microsomal ethanol oxidizing system in the liver. f. Interpret the acute (reversible) effects of ethanol metabolism on fatty acid oxidation, ketone body formation, glucose metabolism and lactate metabolism by the liver. g. Recall the chronic (irreversible) effects of ethanol on liver tissue and function. h. Relate how acetaldehyde can be toxic to liver and other tissues, including alcohol induced hepatitis. 2 2 Objective A Ethanol Metabolism • Dietary fuel: 7 kcal / g • Ethanol is lipid and water soluble • Absorbed by passive diffusion in GI tract • Majority of ethanol is metabolized in the liver Fig 33.1 • Normally metabolized to acetate in the liver (2 steps) • Alcohol dehydrogenase in cytoplasm à acetaldehyde • acetaldehyde dehydrogenase in mitochondria à acetate • NADH is produced, which enters ETC à ATP synthesis • Acetate enters blood and is taken up by muscle and other tissues • Acetyl CoA enters TCA cycle • Acetyl CoA synthetase converts acetate to acetyl CoA for use in tissues • Acetaldehyde may also enter the blood • Toxic intermediate, low levels okay…high, chronic levels bad 3 3 Objective B ADH and ALDH • Alcohol Dehydrogenases (ADH) • Family of cytoplasmic enzymes with various specificities for different alcohols • Class I is most common: highest affinity for ethanol • Class I located in liver = most metabolism in liver. • Ethanol oxidized to acetaldehyde, producing 1 NADH • Acetaldehyde Dehydrogenase (ALDH) (mitochondria) • ALDH2 oxidizes 80% of acetaldehyde, producing 1 additional NADH. • Some people have an allelic variant that has greatly reduced affinity…BAD. • ALDH1 is cytoplasmic and can pick up excess acetaldehyde if necessary • Note: NADH is only weakly inhibitory towards ADH and ALDH. Fig 33.2 4 4 Objective C Microsomal Ethanol Oxidizing System: MEOS • About 10-20% of ethanol is oxidized by MEOS. • ER based enzyme system • cytochrome P450 mixed-function oxidase isozyme (CYP2E1) • Electrons from ethanol and NADPH used to reduce O2 to water (note 4 e- so 2 H2O). • Acetaldehyde produced w/o NADH generation • Acetaldehyde oxidized by ALDH1 (cytoplasmic) • MEOS is a low affinity, high-capacity system. When EtOH concentration is high, the enzyme will process it quickly. At lower EtOH levels, the enzyme is not as active. • Very efficient system that contributes to ethanol metabolism under a high ethanol load with few side effects. • Problem: Chronic ethanol metabolism leads to increased ROS generated and increased cytoplasmic acetaldehyde: both are BAD. Fig 33.3 5 5 Review: Cytochrome P450 Key Points: • Fe2+ binds O2 forming bound Fe3+O2− • Superoxide attacks substrate, RH, resulting in ROH and H2O formation • Electrons needed for the reaction come from NADPH NH3 • Possible O2− can be released from active site, but low probability normally. • Cell stress / pathology greatly increases ROS production by P450s 6 6 Objectives D MEOS: Cytochrome P450 Mixed-Function Oxidase Isozyme (CYP2E1) • CYP2E1 has much lower affinity for ethanol than ADH • Low level of ethanol? MEOS not very active • High levels of ethanol? MEOS much more active. • Other cytochrome P450 isozymes metabolize ethanol. EtOH Acetaldehyde + 2 H2 O • MEOS is a collective term for all ethanol metabolism by cytochrome P450s • Ethanol already has an –OH group. CYP450 oxidizes -OH to C=O without adding an oxygen atom to ethanol. TWO water molecules are created: “oxidase” function that pulls two e- out of EtOH and two come from NADPH to make the two H2O. • Chronic alcohol use in high volume can lead to Cyt P450 induction and increased superoxide and high levels of acetaldehyde – two very harmful products. 7 7 Objective E Energy Yield of Ethanol Oxidation • ATP yield per EtOH varies based on pathway used for oxidation • ADH and ALDH • • • • 2 NADH formed (+ 5 ATP) acetate activated to acetyl CoA using 2 ATP (-2ATP) Acetyl CoA enters TCA producing 9 ATP + 1 GTP Net yield? 12 ATP + 1 GTP per EtOH oxidized • MEOS • • • • • 1 NADPH consumed (-2.5 ATP) 1 NADH produced by ALDH (+2.5 ATP) acetate activated to acetyl CoA using 2 ATP (-2ATP) Acetyl CoA enters TCA producing 9 ATP + 1 GTP Net yield? 7 ATP + 1 GTP per EtOH oxidized • Much better off energetically metabolizing small amounts of EtOH 8 8 Objective F, G Effect of Ethanol Metabolism on Overall Cell Metabolism: Acute versus Chronic Effects • Acute Effects: increased NADH / NAD+ ratio • TCA cycle is inhibited by NADH, Fatty acid catabolism à ketone bodies à ketogenesis à ketoacidosis • FA oxidation inhibited as NADH levels rise, fats driven to TG à VLDL àhyperlipidemia • Pyruvate driven to Lactate à gluconeogenesis inhibited à Lactic acidosis • Chronic Effects: Alcohol-induced liver disease • • • • Hepatic steatosis (fatty liver) Alcohol-induced hepatitis Cirrhosis Increased acetaldehyde and free radicals • Both damage liver cells: proteins, lipids, DNA, etc. 9 9 Objective F, G Ethanol Metabolism Affects Other Pathways • Alcohol-based liver disease has two phases: • Reversible effects: • Inhibition of FA oxidation and stimulation of TG synthesis, leading to fatty liver. • Inhibition of glycolysis by high NADH – if patient is consuming carbs, accumulated glycolysis intermediates will inhibit glucose imported into cells: hyperglycemia • If the patient is not eating carbs – High NADH inhibits gluconeogenesis: hypoglycemia • Irreversible effects: • Acetaldehyde and free radicals can cause alcohol-induced hepatitis (liver inflammation), where liver cells become necrotic and die. • Cirrhosis, caused by fibrosis, which obstructs blood flow. Loss of liver function and hepatic failure. 10 10 Effect of Ethanol Metabolism on Other Pathways in the Liver Fig 33.6 Described on following slides Objective F 11 11 Objective F Alcohol-induced Ketoacidosis • Normal: Ketone bodies synthesized from acetyl CoA when FA are being oxidized as major fuel (fasting). TCA cycle is down regulated (due to high NADH and high acetyl CoA) and excess acetyl CoA is available. Liver makes KBs and tissues use KB under these conditions and blood KB levels are normal. • Ethanol metabolism produces sufficient NADH to convert oxaloacetate to malate, which prevents acetyl CoA from entering TCA cycle. Acetyl CoA levels rise sharply. • Very high acetyl CoA leads to increased ketone body synthesis. • Problem: acetate (produced by ethanol metabolism) is a preferred substrate over ketone bodies, so KB not utilized much…ketone body level in blood increases. **Much higher than normal fasting conditions. • Ketone bodies are weak acids: lose H+ and decrease blood pH. 12 12 Objective F Changes in Fatty Acid Metabolism • High NADH / NAD+ ratio inhibits FA oxidation (don’t need more NADH). • Fatty acids accumulate in the liver, and are reincorporated into TGs for storage. • High NADH ensures plenty of glycerol 3-phosphate is available for TG synthesis. • Notice glycerol that normally would go to gluconeogenesis is directed toward triglyceride synthesis • TGs packaged into VLDLs for export (hyperlipidemia), but VLDL also accumulates in liver (hepatic steatosis – fatty liver). • FA come from diet, or from FA synthesis or from FA released from adipose…depends on fed vs. fasted conditions. • Chronic alcoholics often decease food intake, exist in fasting condition. 13 13 Objective F Lactic Acidosis and Blood Sugar • High NADH level pushes pyruvate toward lactate • Lactate is acidic (acidosis) • decreases pyruvate levels needed for gluconeogenesis, which can cause hypoglycemia during fasting times. • Even amino acids (Ala) converted to pyruvate are converted to lactate. • High blood lactate decreases excretion of urea, so urea accumulates as uric acid crystals in joints (gout). (Nitrogen metabolism lectures). • High NADH level inhibits glycolysis, so when consuming dietary carbohydrate can result in a higher blood glucose level than normal (hyperglycemia). 14 14 Aside: Acidosis / Ketoacidosis / Lactic Acidosis • Acidosis: The acid/base status of the body is regulated by the kidneys and the lungs. Acidosis is caused by an accumulation of acid or a significant loss of bicarbonate. The major categories of acidosis are respiratory acidosis (blood buffer system is out of balance) and metabolic acidosis (metabolic pathways are out of balance. • Ketoacidosis: the accumulation of ketones in the blood, leading to acidosis. • Alcoholic ketoacidosis: caused by excessive alcohol consumption. • Diabetic ketoacidosis: a complication of diabetes mellitus caused by the buildup of ketone bodies from fat metabolism, which occurs when glucose is not available as a fuel source for the body. • Ketones eliminated in urine, sweat, breath • characteristic smell of acetone or a “fruity” odor. • Lactic Acidosis: excess amounts of lactate exported to blood, leading to acidosis. • Ethanol metabolism à high NADH, which favors lactate formation from pyruvate, rather than pyruvate conversion to acetyl CoA. • Lactate exported to blood 15 15 Objective H Acetaldehyde Toxicity • Acetaldehyde (bad) is converted to acetate (good) by ALDH • ALDH2 in mitochondria gets most of it • ALDH1 in cytoplasm gets most of the remainder • Some acetaldehyde escapes to the blood. Large single doses of ethanol or chronic consumption of ethanol results in much higher blood acetaldehyde levels. • MEOS makes acetaldehyde in the cytoplasm, increasing cell exposure to acetaldehyde. • Acetaldehyde is very reactive, forming adducts with amino groups, sulfhydryl groups, nucleotides and glycerophospholipids (covered in Toxic Oxygen lecture) 16 16 Objective H Alcohol Induced Hepatitis Key: Acetaldehyde… 1. affects amino acids, inhibits synthesis of key blood proteins synthesized by the liver. 2-3. also inhibits GSH activity, removing the protective effects of glutathione. 4. can damage ETC-OxPhos proteins, leading to increased free radical damage. 5. affects fatty acid metabolism Fig 33.7 6. damages lipid membrane integrity (lipid radicals), cells become leaky 17 17 Objective H Acetaldehyde and Free Radicals Acetaldehyde: • binds to glutathione • Cell loses a primary defense mechanism • binds to free radical defense proteins, inactivating them. • Damages mitochondrial ETC, progressively uncouples ETC from ATP synthase. • Shuts down FA oxidation, so FA accumulate even more. • Damages mitochondrial ALDH à increases level of free acetaldehyde (vicious cycle). Free Radicals: • Large amounts of ethanol / chronic use induces CYP2E1 • FAD / FMN and heme in the CYP2E1 transfer two electrons, one at a time. • Increased likelihood of ROS / RNS generation. • Hydroxyethyl radical ( CH3CH2O• ) may be released as well (BAD). • Increased free radicals cause damage to lipids, membranes, proteins, DNA, etc. • Damage additive to acetaldehyde damage. 18 18 Question Which of the following enzymes is/are most often responsible for the metabolism of ethanol to acetate in the liver following consumption of small amounts of ethanol (i.e., one can of beer or one glass of wine, etc)? Alcohol dehydrogenase Acetaldehyde dehydrogenase Alcohol dehydrogenase and acetaldehyde dehydrogenase MEOS MEOS and acetaldehyde dehydrogenase 19 19 Question A patient has a deficiency in his acetaldehyde dehydrogenase enzyme such that it is completely inactive. Which of the following cellular results would be expected? MEOS will oxidize a greater fraction of acetaldehyde to compensate. Acetaldehyde will be exported to the blood instead of the mitochondria. Increased acetaldehyde-dependent cellular damage. Increased superoxide production by MEOS. 20 20 Question Metabolism of ethanol produces NADH, causing cellular levels of NADH to rise. Which of the following pathways would most likely be inhibited by the NADH produced by ethanol metabolism? Fatty acid oxidation Fatty acid oxidation, glycolysis and the TCA cycle Glycolysis Pentose Phosphate Pathway TCA cycle 21 21 Thank You! 22 22