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13.2: Fates of Pyruvate under Anaerobic Conditions- Fermentation

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    Search Fundamentals of Biochemistry

    Introduction to Fermentation

    Fermentation is a process in which a fuel molecule is broken down anaerobically (or without oxygen) to meet ATP demands. One of the most notable pathways that can utilize fermentation is glycolysis, which we have just described. During glycolysis, glucose is converted to pyruvate, yielding a total of 2 ATP energy-rich molecules. This occurs in anoxic conditions (or without the requirement of oxygen). However, the process of glycolysis cannot be sustained if the end product is pyruvate. This is because the production of pyruvate also yields 2 molecules of reduced NADH in addition to the production of the two ATP molecules. The pool of available NAD+/NADH is limited within the body, and thus, this electron acceptor/donor molecule must continually be recycled for metabolic pathways to remain functional. For example, if NADH is not oxidized back into NAD+ in a timely manner, the glycolytic pathway can slow. Therefore, to be able to maintain the process of energy generation through glycolysis, the pool of NADH needs to be converted from the reduced form, back into the oxidized form (NAD+) form where it can then accept electrons from intermediates in the glycolytic pathway. This process of recycling NADH to NAD+ can occur aerobically through the Citric Acid Cycle, which is described in more detail in Chapter 16, or it can be processed via anaerobic fermentation (Figure \(\PageIndex{1}\)).

    clipboard_eaacc93a7d985a943b77e1411a43a848d.png

    Figure \(\PageIndex{1}\): Fates of Pyruvate via Aerobic or Anaerobic Pathways.

    In the 1860s, Louis Pasteur first described fermentation very narrowly, as the process that yeast use to convert glucose into ethanol when they are grown in the absence of air. Subsequently, it was discovered that other microorganisms could convert pyruvate to lactate (or lactic acid), instead of ethanol, during the process of anaerobic respiration. These microorganisms became known as lactic acid bacteria and are currently utilized heavily within the food industry to produce a wide array of fermented food products including cheese, yogurt, and sauerkraut, to name a few. As the enzymes within the glycolytic pathway were discovered, it became apparent that muscle tissue could also engage in anaerobic respiration-producing lactate. Thus while fermentative processes are most often described in microbial organisms, the definition of fermentation has since been broadened to include any enzymatic, energy-yielding pathways that occur in the absence of oxygen, including the production of lactate in muscle tissue of animals during the glycolytic process.

    Thus, NADH recycling to NAD+ typically occurs in anaerobic systems by two different routes: ethanolic fermentation or lactate fermentation (Figure \(\PageIndex{1}\)). We will focus on the details of these two systems here.

    Lactate Fermentation

    In lactate fermentation, pyruvate is converted to lactate by the enzyme lactate dehydrogenase. In the process, NAD+ is regenerated. The reaction catalyzed by lactate dehydrogenase (LDH) is shown in Figure \(\PageIndex{2}\).

    Glycolysis_LactDehydro.svg

    Figure \(\PageIndex{2}\): Reaction catalyzed by lactate dehydrogenase

    It is named for the reverse reaction which, as with other dehydrogenases, uses NAD+ as an oxidizing agent. The reaction is reversible with the ΔG0 for conversion of pyruvate to lactate of -3.76 kcal/mol (-15.7 kJ/mol). Under anaerobic conditions when glycolysis is the major source of ATP, pyruvate levels increase, further driving the reaction towards lactate formation and NAD+ regeneration so glycolysis can continue.

    The enzyme is found in the cytoplasm but a mitochondrial form also exists. It is most abundant in muscle, liver, kidney, and also in erythrocytes. Interestingly, mature red blood cells are enucleated and do not contain any mitochondria. Their lifespan is limited to approximately two weeks. During this time, their primary energy resources are generated through the process of anaerobic fermentation via the glycolysis-lactate pathway.

    The active enzyme is a tetramer of various compositions of two different subunits, the heart (H) and muscle (M) forms. The quaternary structures consist of 5 different isozyme forms containing the H and M subunits. LDH-1, found most abundantly in the heart, is a tetramer of 4H subunits (H4). The other forms are as follows: LDH-2 (H3M, prevalent in RBCs), LDL-3 (H2M2, prevalent in lungs), LDH-4 (HM3, prevalent in the kidney), and LDH-5 (M4, prevalent in muscle).

    During anaerobic metabolism, lactate is produced by muscle tissue and released into the bloodstream where it can travel back to the liver. Once in the liver, lactate is converted back into pyruvate and can be utilized to produce glucose through a pathway called gluconeogenesis. The liver can then export the glucose into the blood from where it can be taken up by the muscle for ATP production. This cycle is called the Cori cycle and is illustrated in Figure \(\PageIndex{3}\).

    Cori Cycle.png

    Coricycle.svg

    Figure \(\PageIndex{3}\): Cori cycle Upper diagram is a cartoon image of the Cori cycle and the lower diagram demonstrates the recycling of NAD+/NADH in different locations within the body. Figure modified from Servier Medical Art

    Notably, anaerobic metabolism can only be sustained for short periods in animals due to its high energy demand. Aerobic respiration is required to maintain an adequate ATP supply. However, the production of lactate by certain tissues, such as white, or fast-twitch muscle cells occurs regularly and releases lactate into the bloodstream, where it can be taken up and used as an energy source by other neighboring tissues such as red, slow-twitch muscle. Brain tissue can also effectively use lactate as an energy source as well. Within these 'consumer' tissues, lactate is converted back into pyruvate using the mitochondrial LDH enzyme where it can then be processed by aerobic respiration producing high levels of ATP.

    Thus, the production of lactate can be thought of as a strain response that occurs during times of metabolic stress, such as intense cardiovascular exercise. It has also been noted that after an injury or head trauma, the activation of epinephrine will cause an increase in lactate production and blood levels of lactate will increase. Thus, it has been hypothesized that lactate may play a role in the repair of damaged tissue. Clinical trials and experiments are currently underway to determine if lactate can help in the healing and recovery process for conditions such as traumatic brain injury, myocardial infarction, and sepsis. Early studies have shown that lactate can increase the production of Brain-Derived Neurotropic Factor (BDNF) which supports neuronal growth, providing further support for the role of lactate in recovery and repair.

    Spectroscopy analyses show the geometry and electrostatics of the active sites of LDHs from all organisms are essentially the same. The mechanism for the reverse oxidation of lactate by NAD+ by spiny dogfish lactate dehydrogenase (LDH) is shown in Figure \(\PageIndex{4}\)

    lactatedehyrdorogenaseMech.svg

    Figure \(\PageIndex{4}\): Mechanism for the reduction of lactate by NAD+ by spiny dogfish lactate dehydrogenase (LDH)

    Lactate is shown with a deuterium (D), which moves as a deuteride to NAD+ to form NADH, simply to illustrate the stereochemistry of the reaction. In the reverse reaction, the reduction of pyruvate to lactate (to regenerate more NAD+ so anaerobic glycolysis can continue), the deuterium (or the proR H of non-deuterated NADH) is removed.

    Figure \(\PageIndex{5}\) shows an interactive iCn3D model of dogfish M4 apo-lactate dehydrogenase (1LDM).

    Dogfish M4 apo-lactate dehydrogenase (1LDM).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{5}\): Dogfish M4 apo-lactate dehydrogenase (1LDM).(Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...FBzuqJ2gEPUHM8

    Only the monomer of the active tetramer is shown. NAD and oxamic acid, structurally similar to lactate/pyruvate and a competitive inhibitor of the enzyme, are labeled. His193, the active site general base/acid, is near Arg 169 and to Asp 166 and Asn 138, to which it is hydrogen bonded.

    LDH exists in two major conformational states, T (inactive) and R (active) state, as we have seen with other allosteric proteins. Figure \(\PageIndex{6}\) shows an interactive iCn3D model of the T state (2ZQY) and R state (2ZQZ) of Lacticaseibacillus casei L-lactate dehydrogenase.

    T and R states in the crystals of bacterial L-lactate dehydrogenase reveal the mechanism for allosteric control (1LTH).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{6}\): T state (2ZQY) and R state (2ZQZ) of Lacticaseibacillus casei L-lactate dehydrogenase. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...zjrtVYjLXdhDp8

    Toggle between the magenta T state and cyan R state using the "a" key. Orient the proteins to see the best view of the allosteric changes caused by binding regulators. The spheres show Arg 171 whose disposition changes significantly in the T and R states. Pyruvate (a reactant/product) activates the enzyme (conversion of T to R state) and is considered a homotropic regulator since it is a substrate. This is similar to the allosteric "activation" of hemoglobin by its ligand O2, which preferentially binds to the R state and promotes the T-to-R state transition. Fructose 1,6-bisphosphate, a glycolytic intermediate, also activates the Lacticaseibacillus casei L-lactate dehydrogenase and since it is not a reactant/product of the enzyme, it is considered a heterotropic allosteric regulator. The enzyme hence is effectively regulated by the concentration of substrate (pyruvate) and by NADH levels. High concentrations of ethanol consumption lead to high levels of NADH through the activity of alcohol dehydrogenase. High levels of NADH would lead to increased lactate production as well.

    Ethanol Fermentation in Yeast

    In this process, pyruvate is decarboxylated first to acetaldehyde by the thiamine pyrophosphate (TPP)-requiring enzyme pyruvate decarboxylase. The resulting product, acetaldehyde, is then reduced by NADH to form ethanol by the enzyme ethanol dehydrogenase in a process that reforms NAD+. Yeast are facultative (not obligate) anaerobes in that they can produce energy by glycolysis and ethanol fermentation in the absence of oxygen Figure \(\PageIndex{7}\):. Of course, in the presence of oxygen, the pyruvate produced from glycolysis in yeast is preferentially converted to acetyl-CoA which enters the citric acid cycle and oxidative phosphorylation pathways to maximize ATP production.

    clipboard_e5df1e8fb7af941e5fe4b4ad30470b66b.png

    Figure \(\PageIndex{7}\): Summary of Ethanol Fermentation in Yeast

    Figure from Thomas Baldwin

    Pyruvate decarboxylase

    Pyruvate decarboxylase catalyzes the first step in the alcoholic fermentation pathway converting pyruvate into acetaldehyde and carbon dioxide. Interestingly, in addition to yeast, some species of fish, such as goldfish and carp, have a homologous enzyme that allows for the production of ethanol when oxygen is scarce. Pyruvate decarboxylase is tetrameric occurring as a dimer of dimers with two active sites shared between the monomer subunits of each dimer. The decarboxylation reaction requires two cofactors, thiamine pyrophosphate (TPP) and magnesium (Figure \(\PageIndex{8}\)). Within the active site, the acidic Glu-477 and Glu-51 residues and the Mg2+ cofactor interact with and stabilize the TPP cofactor. The aminopyrimidine ring on TPP acts as a base and enables the formation of the TPP nucleophile with the removal of the C2 proton. This reaction is stabilized by the protonation of Glu-51. The nucleophilic attack of pyruvate causes the release of carbon dioxide. The acetaldehyde intermediate is still covalently attached to the TPP cofactor following the release of carbon dioxide.

    pyruvatedecarboxylaseYeastMech.svg

    Figure \(\PageIndex{8}\): Part 1 Pyruvate Decarboxylase mechanism - decarboxylation


    Once carbon dioxide diffuses away from the active site, the double bond of the enol-intermediate abstracts a proton from Asp-28, which is stabilized by a neighboring His-115 residue. TPP can then serve as a good leaving group during the formation of the carbonyl functional group and causes the release of acetaldehyde (Figure \(\PageIndex{9}\):).

    pyruvatedecarboxylaseYeastMechPart2.svg

    Figure \(\PageIndex{9}\): Part 2 Pyruvate Decarboxylase mechanism - acetaldehyde generation


    Figure \(\PageIndex{10}\) shows an interactive iCn3D model of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae (1PVD).

    thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae (1PVD).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{10}\): Thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae (1PVD).(Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...DMM4gjodX4q1G9 TTP is shown in spacefill. The enzyme is a homotetramer.

    Alcohol dehydrogenase (ADH)

    Alcohol dehydrogenase enzymes catalyze the interconversion of aldehydes or ketones with alcohol functional groups. In humans and other animals, they are utilized to break down alcohols that are ingested, as well as participate in the biosynthesis of numerous metabolites. There are at least 5 classes of alcohol dehydrogenases that use NAD+ as an oxidizing agent to convert alcohols to aldehydes or ketones. We are mainly concerned with the yeast ADH, a member of Family I, that is involved in ethanol fermentation. Figure \(\PageIndex{11}\) shows the reaction catalyzed by alcohol dehydrogenase.

    EtOHDehydorgenasesSummary.svg

    Figure \(\PageIndex{11}\): Reaction catalyzed by alcohol dehydrogenase

    Figure \(\PageIndex{12A}\) below shows a mechanism for yeast alcohol dehydrogenase (ADH1). ADH1 requires zinc as a metal cofactor and utilizes NADH as the reducing agent in the conversion of acetaldehyde into ethanol.

    AlcoholDehydrogenase_ForRevRxMechFig2.svg

    Figure \(\PageIndex{12A}\): Simplified catalytic mechanism of ADH reactions. Upon binding of both substrates (e.g., NAD(P)+ and alcohol; 0->1->2) a hydride transfer occurs from the alcohol-carbon atom to the oxidized nicotinamide moiety yielding the Zn-coordinated carbonyl product and NAD(P)H (3). Both can dissociate from the active site yielding apo-ADH (0). Alternatively, only NAD(P)H stays bound and the reduced ADH can undergo a reductive conversion (1’->2’-> 3’-> 1). Amanda Silva de Miranda, et al. Front. Catal., 10 May 2022. Sec. Biocatalysis. https://doi.org/10.3389/fctls.2022.900554. Creative Commons Attribution License (CC BY).

    Figure \(\PageIndex{12B}\) shows key amino acids in the active site of yeast ADH1. Note that in this figure, two cysteines are the coordinating ligands.

    ADH1_MECH.svg

    Figure \(\PageIndex{12B}\): Abbreviated mechanism for yeast alcohol dehydrogenase (ADH1)

    If you look at the reverse reduction reaction, the acetaldehyde (or a ketone for 20 alcohols) carbonyl is sp2 hybridized and planar, so the hydride could be added to either of the two faces, re or si, of the plane (using the same rules used to define R or S enantiomers). Likewise, the methylene carbon atom with two hydrogens, one of which is transferred as a hydride, is prochiral. Hence four different stereochemical hydride transfer pathways are possible, as shown in Figure \(\PageIndex{13A}\) below. Hydride transfers from the si-face of the prochiral aldehyde/ketone result in (R)-configured alcohols whereas hydride attacks from the re-face yield (S)-alcohols. In both cases, the hydride transferred can stem from either the re- or si-face of the nicotinamide ring.

    AlcoholDehydrogenase_ForRevRxMechFig3.svg

    Figure \(\PageIndex{13A}\): Possible stereochemical courses of the hydride transfer from NAD(P)H to the ketone. Attacks from the si-face of the ketone result in (R)-alcohols (E1 and E2) whereas hydride attacks from the re-face (E3 and E4) result in (S)-alcohols.

    ADHs catalyzing hydride addition from the re-face of the ketone (or abstraction of a hydride from (S)-alcohols) are termed Prelog-selective ADHs whereas those ADHs attacking from the si-face (or abstracting a hydride from the (R)-alcohol) are termed anti-Prelog ADHs. Different ADHs are available for the different reaction stereochemistries.

    The stereochemistry for the reaction of ADH1 with ethanol is shown in Figure \(\PageIndex{13B}\) below. Deuterium (D) is shown in the figure to better illustrate the stereochemistry of the reaction. With the deuterium label, the carbon is now chiral and the enantiomer shown is the R isomer. The corresponding H in the undeuterated and prochiral form of ethanol is removed by the enzyme, as shown in Figure \(\PageIndex{13B}\) below.

    NAD_Re_Si_ProR_ADH.svg

    Figure \(\PageIndex{13B}\): Stereochemistry of human alcohol dehydrogenase reaction.


    Figure \(\PageIndex{14}\) shows an interactive iCn3D model of the Yeast alcohol dehydrogenase (ADH I) with bound substrate analogs- ADH1 (4W6Z).

    Yeast alcohol dehydrogenase structure with bound substrate analogs (4W6Z).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{14}\): Yeast alcohol dehydrogenase structure with bound substrate analogs (4W6Z). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...feE7toF6wU6MB9

    All four monomers of the homotetramer are shown. The light gray subunit has the key catalytic and binding residues shown in sticks and labeled. The numbering for the key residues is a bit different than in the mechanism shown above. The cofactor analog, nicotinamide-8-iodo-adenine-dinucleotide, is shown in sticks and the substrate analog, trifluoroethanol is shown in spacefill.

    Only two subunits (a homodimer) have both bound cofactor and substrate. The substrate trifluoroethanol in these subunits is ligated to the catalytic Zn2+ through its oxygen, with the other ligands provided by the side chains of Cys 43, Cys 153, and His 66. The other two monomers (the other homodimer) have a different conformation and use Glu 67 to coordinate the Zn2+ instead of His 66 and no substrate is bound. This may be an intermediate in the process that displaces water bound to Zn2+ with the substrate.

    Alcohol Metabolism in Humans:

    When ethanol is consumed, it is oxidatively metabolized primarily in the liver. Cytosolic Alcohol Dehydrogenase (ADH) and mitochondrial Acetaldehyde Dehydrogenase 2 (ALDH2) are the main enzymes involved in this metabolic pathway, first converting ethanol to acetaldehyde and then acetaldehyde to acetate (Figure \(\PageIndex{15}\):). Liver mitochondria have a limited capacity to use the acetate in the Kreb cycle because the enzyme needed to convert acetate to acetyl-CoA (acetyl-CoA synthase 2) is almost absent in the liver, but is abundant in the heart and skeletal muscles. Thus, most of the acetate resulting from ethanol metabolism escapes the liver into the blood circulation and is eventually metabolized to CO2 by way of the Kreb cycle in cells with mitochondria that contain enzymes to convert acetate to acetyl CoA, such as heart, skeletal muscle, and brain.

    clipboard_ef33c320587711613df26de82173ac2cb.png

    Figure \(\PageIndex{15}\): Summary of Ethanol Metabolism in Humans

    Figure modified from Zakhari, S. (2013) Alcohol Research: Current Reviews 35(1):6-16


    Like many substances, the consumption of alcohol can be both beneficial and detrimental, depending on the quantity and frequency of consumption. This is known as a hormetic response (Figure \(\PageIndex{16}\):). At low doses (up to 2 daily drinks for men and 1 daily drink for women) the consumption of alcohol can be cardioprotective. At higher consumption rates this protective effect is lost and the detrimental effects of alcohol consumption become apparent and include cardiotoxicity, liver damage, and increased cancer risk. Not to mention the debilitation that can accompany addiction.

    Alcohol and agingFig1.svg

    Figure \(\PageIndex{16}\): Alcohol Consumption and Hormesis

    Figure from Adamson, S.S., et al (2017) Translational Medicine of Aging 1:18-23.

    The graph presented in Figure \(\PageIndex{17}\) exemplifies the hormetic nature of alcohol consumption. Maintaining ethanol consumption between one (for women) and two (for men) daily drinks will significantly reduce the overall risk of mortality. However, mortality risk skyrockets at high ethanol consumption.

    clipboard_eff913bc46c99f24136c9649cab5564e3.png

    Figure \(\PageIndex{17}\): Risk of Mortality and Alcohol Consumption

    Figure from Adamson, S.S., et al (2017) Translational Medicine of Aging 1:18-23.

    Moderate alcohol use of no more than 2 drinks per day for men, and no more than 1 drink per day for women can reduce the risk of coronary artery disease by 29%, and reduce the risk of dying from any cardiovascular disease by 25%. These statistics are significant. Do note that at these levels that there is an increased risk of dying from hemorrhagic stroke. Increased alcohol consumption, however, can have dire effects on health and can lead to unwanted addiction. The Centers for Disease Control estimates that alcohol abuse leads to approximately 80,000 deaths annually in the United States and that up to 40% of deaths related to liver disease are caused by alcohol abuse.

    Harvard Medical School has evaluated thousands of studies and has tried to come up with a method for alcohol risk assessment. They categorize low-risk drinking behavior for men as no more than four drinks in a single day or 14 drinks in a week and no more than 3 drinks in a single day or a total of 7 drinks in a single week for women. Women have lower drinking tolerance due to their smaller sizes, but also due to metabolic differences. Women have less ADH and typically higher fat levels which disperse and retain ethanol longer. Addiction and damage to the liver tend to occur more quickly in women as well. Harvard ranks people at increased risk if their drinking is above either the single day or the weekly limit (which is estimated at 29% of drinkers). High-risk drinkers break both the daily and weekly limits (estimated at 14% of drinkers).

    During ethanol metabolism, when circulating ethanol is in the millimolar range, acetaldehyde is in the micromolar range, and acetate is in the millimolar range (Figure \(\PageIndex{18}\):). When heavy drinking or chronic drinking occurs the Acetaldehyde Dehydrogenase enzyme cannot keep up with the Alcohol Dehydrogenase enzyme and the pool of Acetaldehyde increases. This aldehyde has many toxic effects within biological systems (Figure \(\PageIndex{19}\):).

    clipboard_e857a2f68242179e924af3707f29f2665.png

    Figure \(\PageIndex{18}\): The Effects of Binge or Chronic Drinking on Ethanol Metabolites.

    Figure modified from Zakhari, S. (2013) Alcohol Research: Current Reviews 35(1):6-16

    Acetaldehyde can form adducts with proteins. For example, acetaldehyde adducts on cytoskeletal components such as microtubulin, lead to the swelling of hepatocytes (liver cells). If secreted from the cell, these protein adducts can also be recognized as foreign by the immune system and cause an autoimmune response causing further inflammation and damage to the liver. Acetaldehyde also causes oxidative damage to lipids and DNA and can alter mitochondrial function. Overall, the liver is stressed and unhappy when too much ethanol is consumed.

    clipboard_e03662d4a990d37c617593d043949099c.png

    Figure \(\PageIndex{19}\): Acetaldehyde Toxicity

    Figure modified from Servier Medical Art

    Heavy chronic drinking can also lead to epigenetic modifications that alter protein expression patterns within the cell. Due to the oxidation of alcohol to acetate, the metabolism of alcohol also leads to an increase in the NADH: NAD+ ratio altering carbohydrate metabolism. Heavy ethanol consumption promotes the formation of reactive oxygen species and can also promote apoptosis. Overall, heavy drinking is extremely hard on liver function. It can lead progressively from the formation of a fatty liver to liver cirrhosis and increased risk of liver cancer. The risk for several other types of cancer is also heightened with heavy alcohol use.

    Chronic heavy alcohol use can also alter gene expression, especially in the liver. Figure \(\PageIndex{20}\) is a schematic representation of DNA methylation, which converts cytosine to 5′methyl-cytosine via the actions of DNA methyltransferase (DNMT). DNA methylation typically occurs at cytosines that are followed by a guanine (i.e., CpG motifs). Within the liver, chronic heavy drinking reduces pools of S-adenosylmethionine (SAM) while increasing homocysteine and S-adenosylhomocysteine (SAH). SAH further inhibits DNA methyltransferases (DNMTs) by negative feedback inhibition, ultimately resulting in global hypomethylation of DNA. This hypomethylation leads to the inappropriate expression of many genes, especially within the liver tissue.

    clipboard_ecf2c285942c1050e9ba36066114a8c45.png

    Figure \(\PageIndex{20}\): Effects of Chronic Alcohol Use on DNA Methylation

    Figure modified from Zakhari, S. (2013) Alcohol Research: Current Reviews 35(1):6-16

    Two genes that are upregulated due to this hypomethylation express the Catalase enzyme and the p450 oxidoreductase enzyme, CYP2E1. Both of these enzymes are involved in oxidative pathways of alcohol metabolism that produce the toxic acetaldehyde intermediate (Figure \(\PageIndex{21}\)). Expression of both of these proteins become more prevalent in chronic alcohol consumption or when blood alcohol levels are high, as in cases of binge or heavy drinking. The activity of these enzymes can also lead to the formation of reactive oxygen species that contribute to global cellular damage (lipid peroxidation, DNA damage, protein damage, etc).

    clipboard_eae5c180da4b4c6640664887a92df8384.png

    Figure \(\PageIndex{21}\): Oxidative Pathways of Alcohol Metabolism

    Figure from Zakhari, S. (2013) Alcohol Research: Current Reviews 35(1):6-16

    Gene regulation in other areas of the body is also affected in response to chronic heavy alcohol consumption. This is due to the production of acetate during the metabolic pathway of alcohol that is released from the liver into the bloodstream. In other areas of the body, acetate is converted to acetyl-CoA by the enzyme Acetyl-coenzyme A (acetyl-CoA) synthetase (AceCS). AceCS is activated by Sirtuin 1, also known as NAD-dependent protein deacetylase (SIRT1). Acetyl-CoA is used by histone acetyltransferase (HAT) to acetylate the lysine residues in histone proteins. Histone acetylation causes these proteins to release the bound DNA, allowing regions to be opened up for transcription (Figure \(\PageIndex{22}\)). Thus, higher levels of acetate promote histone acetylation and increased gene expression.

    clipboard_e615ba6caf91c9b5738361a3a9ba71e47.png

    Figure \(\PageIndex{22}\): The Effects of Chronic Alcohol Consumption on Histone Acetylation. Note in the diagram shown, that SIRT1 also deacetylates histones, resulting in gene silencing. Thus, SIRT1 is a sensor that balances gene activation and silencing in the cell based on the cell’s energy status. Alcohol metabolism results in acetate formation, which is used in extrahepatic tissues to produce acetyl-CoA, upregulating histone acetylation within those tissues. NOTES: AceCS1 = Acetyl-CoA synthase 1; ADH = alcohol dehydrogenase; ALDH = Aldehyde dehydrogenase.  Figure from Zakhari, S. (2013) Alcohol Research: Current Reviews 35(1):6-16

    In addition to these effects, both ADH and ALDH utilize the cofactor nicotinamide adenine dinucleotide (NAD+), which is reduced to NADH; as a consequence, during ethanol oxidation the ratio NADH/NAD+ is significantly increased, altering the cellular redox state and triggering several adverse effects, related to alcohol consumption. Glycolysis and the Kreb Cycle are downregulated due to low NAD+ levels. This results in lower pyruvate levels, and lower conversion of pyruvate to acetyl-CoA and also causes a decrease in gluconeogenesis (ie there is not enough pyruvate to drive glucose production). Thus, the pyruvate that does form favors anaerobic conversion to lactate and can result in lactic acidosis or lowering of the blood pH levels. Low rates of gluconeogenesis can also contribute to hypoglycemia which can be seen during binge drinking.

    Hyperuricemia or an increase in blood levels of uric acid can also occur due, in part, to increased production of ketone bodies and lactic acid. Both the ketone bodies and lactate can compete with uric acid for excretion into the urine within the kidney. The uric acid gets retained and heightens blood levels.

    Ketone bodies typically form during periods of starvation when carbohydrate stores have been depleted. When the liver can no longer efficiently maintain blood glucose levels, it will break down fatty acids into ketone bodies and secrete these into the bloodstream (Figure \(\PageIndex{23}\)). Ketone bodies, such as acetoacetate, acetone, and D-beta-hydroxybutyrate (which isn’t a real ketone, but is still referred to as a ketone body) are released into the bloodstream to compensate for the reduced glucose levels. Brain, heart, and skeletal muscle tissue can utilize ketone bodies as an energy source and this is a good short term solution to starvation. However, the formation of lactate and ketone bodies can severely reduce blood pH levels and induce a life-threatening state known as ketoacidosis. In addition to starvation, heavy alcohol consumption can induce ketogenesis inappropriately.

    clipboard_e10a9fa8bb5991931ce9814b2ceafe8ad.png

    Figure \(\PageIndex{23}\): Formation of Ketone Bodies. Figure from Sav vas

    As noted before, high levels of lactate and ketone bodies within the bloodstream can result in dehydration and reduced excretion of uric acid, leading to hyperuricemia. Over time, high uric acid levels can cause uric acid to precipitate, especially in joints where it causes painful gout flare-ups (Figure \(\PageIndex{24}\)).

    clipboard_e8dcf18c9f996f1b2eb344466295d7d04.png

    Figure \(\PageIndex{24}\): Hyperuricemia and Gout.   Image from www.scientificanimations.com

    According to the American Cancer Society, excessive alcohol use can also increase the risk for several different forms of cancer.

    • Cancers of the mouth, throat, voice box, and esophagus: Alcohol use raises the risk of these cancers. Drinking and smoking together raise the risk of these cancers even more than drinking or smoking alone. This might be because alcohol can help harmful chemicals in tobacco get inside the cells that line the mouth, throat, and esophagus. Alcohol may also limit how these cells can repair damage to their DNA caused by the chemicals in tobacco.
    • Liver cancer: Long-term alcohol use has been linked to an increased risk of liver cancer. Regular, heavy alcohol use can damage the liver, leading to inflammation and scarring. This might raise the risk of liver cancer.
    • Colon and rectal cancer: Alcohol use has been linked with a higher risk of cancers of the colon and rectum. The evidence for this is generally stronger in men than in women, but studies have found the link in both sexes.
    • Breast cancer: Even a few drinks a week are linked with an increased risk of breast cancer in women. This risk may be especially high in women who do not get enough folate (Vitamin B12) in their diet or through supplements. Alcohol can also raise estrogen levels in the body, which may explain some of the increased risks. Cutting back on alcohol may be an important way for many women to lower their risk of breast cancer.

    So how does alcohol consumption contribute to increased risks of cancer? Alcohol may help other harmful chemicals, such as those in tobacco smoke, enter the cells lining the upper digestive tract more easily. This might explain why the combination of smoking and drinking is much more likely to cause cancers in the mouth or throat than smoking or drinking alone. In other cases, alcohol may slow the body’s ability to break down and get rid of some harmful chemicals. Alcohol might affect the body’s ability to absorb some nutrients, such as folate. Folate is a vitamin needed as a cofactor for enzymes involved in amino acid biosynthesis. Absorption of nutrients can be even worse in heavy drinkers, who often have low levels of folate. These low levels may play a role in the risk of some cancers, such as breast and colorectal cancer. Alcohol can raise the levels of estrogen, a hormone important in the growth and development of breast tissue. This could affect a woman’s risk of breast cancer. Too much alcohol can also add extra calories to the diet, which can contribute to weight gain in some people. Being overweight or obese is known to increase the risks of many types of cancer.

    Overall, heavy ethanol consumption produces a wide spectrum of hepatic lesions (Figure \(\PageIndex{25}\)). Fatty liver (i.e., steatosis) is the earliest, most common response that develops in more than 90 percent of problem drinkers who consume 4 to 5 standard drinks per day. With continued drinking, alcoholic liver disease can proceed to liver inflammation (i.e., steatohepatitis), fibrosis, cirrhosis, and even liver cancer (i.e., hepatocellular carcinoma). Heavy drinking can also damage other organs, such as the pancreas and the brain, and can raise blood pressure. It also increases the risk of heart disease and stroke. In pregnant women, alcohol use, especially heavy drinking, may lead to birth defects or other problems with the fetus.

    clipboard_e995745554018f16d42d3075dd8d137a5.png

    Figure \(\PageIndex{25}\): The Effects of Heavy Chronic Drinking on the Liver

    Figure from Osna, N.A. et al (2017) Alcohol Research: Current Reviews 38(2)

    In 1951, the FDA approved disulfiram for the treatment of alcoholism in the US (Figure \(\PageIndex{26}\)). Initially, the drug was prescribed in very high doses, often as high as 3,000 mg per day. The high doses led to reports of severe reactions to alcohol, some of which were fatal. It is an inhibitor of the ALDH-2, which will lead to an even higher increase in acetaldehyde concentration if alcohol is consumed. Thus, alcoholics that are taking this drug to quit drinking need to maintain sobriety. If they drink while taking this drug, they will become very ill due to the accumulation of acetaldehyde. Overall, the use of this drug supports abstinence, which is often hard to realistically achieve.

    clipboard_e006a6bbf5555bf0a830f16d0067d1e25.png

    Figure \(\PageIndex{26}\): Disulfiram

    At one time it was thought that before prescribing disulfiram to people, patients should experience mixing the drug with alcohol in a supervised setting. Researchers felt it was important for the individuals to have full knowledge of what would happen if they mixed disulfiram and alcohol. This practice is no longer used, but every person must be educated on the reactions of combining alcohol with disulfiram before a prescription is written.

    Along with these effects, alcohol may contribute to cancer growth in other, unknown ways.

    and other mammals, various ADHs are used to oxidatively metabolize ethanol to acetaldehyde (also toxic) which is converted to acetate by the enzyme aldehyde dehydrogenase which catalyzes the following reaction:

    CH3CHO + NAD+ + H2O → acetate + NADH + H+

    They can also convert methanol to the very toxic and reactive formaldehyde, which makes methanol poisoning so dangerous if not fatal. Mammalian enzymes are dimers of up to nine different monomers.


    This page titled 13.2: Fates of Pyruvate under Anaerobic Conditions- Fermentation is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski and Patricia Flatt.