Lipid and Fatty Acid Metabolism Notes

Lipids: Fatty Acid Metabolism

Triglycerides

• Triglycerides are stored in adipocytes, both white and brown adipose tissue.
• Brown adipose tissue is present in newborn human babies, allowing them to generate heat without shivering.
• Diagram of a 3T3-L1 Adipocyte Cell shows:
  • Membrane
  • Mitochondria
  • Nucleus
  • Cytoplasm
  • Lipid Droplet

Historical Context: Whale Oil

• The book "Moby Dick" is mentioned in the context of whale oil.
• Graph of US Whale Oil and Sperm Oil Imports (1805-1905) shows:
  • Sperm oil and whale oil import trends over time.
  • Peak import levels around 1850s.
  • Decline in later years.
• Sperm whale's spermaceti organ contains oil that changes density with temperature and pressure, aiding in deep dives (up to 1-3 km) for giant squid.

Energy Storage

• Energy is stored through reduction, and released through oxidation.
• $CH<em>2 \rightarrow CHOH \leftrightarrow CHO \leftrightarrow COOH \rightarrow CO</em>2$
• Energy stores in the body:
  • Glycogen in liver and muscle.
  • Fat.
• Sugars dissolve easily in water due to partial charges on –OH groups.
• Images of glycogen granules in mitochondria and chloroplast.

Energy from Fat

• Energy scale for food:
  • Carbohydrates: 4 Calories per gram
  • Protein: 4 Calories per gram
  • Fats: 9 Calories per gram
• Energy distribution in a typical 70 kg human (adapted from Biochemistry by Garrett and Grisham 5th Edition):
  • Fat: $37 kJ/g$ dry weight, $15000 g$, $555000 kJ$ (84.1%)
  • Protein (muscle): $17 kJ/g$ dry weight, $6000 g$, $102000 kJ$ (15.5%)
  • Glycogen (muscle): $16 kJ/g$ dry weight, $120 g$, $1920 kJ$ (0.3%)
  • Glycogen (liver): $16 kJ/g$ dry weight, $70 g$, $1120 kJ$ (0.2%)
  • Glucose: $16 kJ/g$ dry weight, $20 g$, $320 kJ$ (0.05%)
  • Total: $660360 kJ$
• A man weighing 155 pounds with normal fat storage would weigh ~200 pounds if the same energy were stored as glycogen.
• Overall equation for complete oxidation of palmitoyl-CoA (including oxidative phosphorylation):
  • $CH<em>3(CH</em>2)<em>{14}-CoA + 108 P</em>i + 108 ADP + 23 O<em>2 \rightarrow 108 ATP + 16 CO</em>2 + 123 H_2O + CoA$

Efficiency of Fat Storage

• Storing energy as fat is more efficient than storing it as carbohydrate.
  • Oxidation of FAs yields more energy per gram compared to carbohydrate.
  • FAs are hydrophobic and unhydrated, unlike carbohydrates (which carry 2g $H_2O$ / g carbohydrate).
  • Humans can only store ~200 g of glucose (as glycogen), but moderately obese adults can store 10s of kg of triacylglycerides.

Fatty Acids

• Fatty acids consist of a long hydrocarbon chain (mostly saturated carbons) with a terminal carboxylate (carboxylic acid) group.
• Release more than twice the amount of energy per gram compared to carbohydrates.
• FAs are between C4-C36 (C12-C24 most common) in length.
• Most FAs have an even number of C atoms.
• Chains can be saturated (no double bonds) or unsaturated (one or more double bonds).
• Most FAs are linear (no branches).

Naming Fatty Acids

• Lipid Numbers: Numbered from the COOH group (position 1) to give the chain length and number of double bonds.
  • Palmitic Acid: 16:0
  • Oleic Acid: 18:1
• Dx (or delta-x) nomenclature: Indicates the positions of the double bonds.
  • Linoleic Acid: C18:2(D9,12) - Poly Unsaturated Fatty Acid (PUFA)
  • Eicosatetraenoic acid: C20:4(D5,8,11,14)
• cis/trans notation: Indicates the configuration of double bonds.
  • Linoleic Acid: C18:2(cis-, cis-D9, 12) or C18:2(cis-D9, cis-D12)

Common Fatty Acids and Their Names

• 14:0 - myristic acid
• 16:0 - palmitic acid
• 18:0 - stearic acid
• 18:1 cisD9 - oleic acid
• 18:2 cisD9,12 - linoleic acid
• 18:3 cisD9,12,15 - a-linolenic acid
• 20:4 cisD5,8,11,14 - arachidonic acid
• 20:5 cisD5,8,11,14,17 - eicosapentaenoic acid

IUPAC Nomenclature

• Saturated fatty acids are named using the number of carbon atoms (in Greek) followed by “–anoic acid,” sometimes preceded by “n-” for unbranched structures.
  • n-Decanoic acid (10 C)
  • n-Dodecanoic acid (12 C)
  • n-Tetradecanoic acid (14 C)
  • n-Hexadecanoic acid (16 C)
  • n-Octadecanoic acid (18 C)
  • n-Icosadecanoic acid (20 C)
  • n-Docosanoic acid (22 C)
• For unsaturated fatty acids:
  • Number root preceded by cis or trans and positions of double bonds.
  • Number root followed by “-enoic acid” (one double bond), “ -dienoic acid” (two double bonds), “-trienoic acid” (three double bonds), etc.
  • Linoleic acid → cis-,cis-9,12-octadecadienoic acid
  • cis- can be replaced with Z, and trans- with E: (Z)-,(Z)- 9,12-octadecadienoic acid

Omega Notation

• n-x, w-x, omega-x notation: Counting towards double bonds from the carboxylic acid end (a) or from the methyl end (w).
• Useful because the position of double bonds relative to the methyl group is a better indication of their behavior in vivo.
• Linoleic acid: w-6, omega-6 or n-6 fatty acid; 18:2 w-6; 18:2 (n-6); 18:2 omega-6 (or w-6 18:2)

Isomerism

• Most unsaturated FAs (and pretty much all natural FAs) are in the cis configuration.

Esterification

• FAs can be esterified to the –OH groups at position C1, C2 & C3 of glycerol.
• Ester formation: alcohol (-OH) + carboxylic acid (COOH) → ester + H2O

Triglycerides (triacylglycerols/fats)

• Triglycerides are formed by esterifying glycerol with three fatty acids.
• Reaction: Glycerol + Three fatty acids → Triglyceride + 3 $H_2O$

Metabolism of Fats

• The first step is the reversal of esterification (hydrolysis) via a lipase.
• Activation of Triacylglycerol lipase by hormone activity:
  • 7TM receptor → Adenylate cyclase → ATP → cAMP → Protein kinase → Triacylglycerol lipase.
• Breakdown of Triacylglycerols in adipose tissue is regulated by:
  • Glucagon (+)
  • Adrenaline (+)
  • Noradrenaline (+)
  • Insulin (-)
• This connects fatty acid metabolism with diabetes. Hibernating animals have increased levels of adrenaline & noradrenaline to depend on fat stores for energy.

Glycerol and Fatty Acid Metabolism

• Metabolism of glycerol from triacylglycerol occurs via glycolysis/gluconeogenesis pathway (mainly in the liver).
• Free fatty acids (hydrophobic) are carried to target tissues bound to serum albumin where they undergo b-oxidation.

β-Oxidation

• The main process of fatty acid breakdown.
• Energy is stored in fats as fixed carbon and metabolic energy.
• β-Oxidation involves four repeated steps until the fatty acid is completely metabolized.
• In eukaryotes, primarily takes place in the mitochondrial matrix.
• In eukaryotes, oxidation of fatty acids mostly occurs in the mitochondrion.
• Fatty acids are first “activated” in the cytoplasm:
  • $DG’0 = -15 kJ mol^{-1}$
  • Reaction is driven in the forward direction by the hydrolysis of $PP<em>i$ to $2 P</em>i$ ($DG’0 = -19 kJ mol^{-1}$).
  • Overall: $DG’0 = -34 kJ mol^{-1}$ (equivalent to 2 molecules of ATP under standard conditions).
• Catalyzed by Acyl-CoA synthase (located on the outer mitochondrial membrane). Different enzymes for different chain lengths.
  • Very long chain (C12-C24)
  • Long chain (C8-C20)
  • Medium chain (C4-C12)
  • Short-chain (C4-C6)

Transport into Mitochondria

• Long chain FA (> C12) cannot diffuse through the inner mitochondrial membrane and require a transport mechanism.
  • Fatty acids are first conjugated to Carnitine: Carnitine + Acyl CoA → Acyl-Carnitine + CoA (Carnitine Acyltransferase I)
  • Carried into the matrix by Acyl Carnitine Translocase
  • Acyl-Carnitine + CoA → Carnitine + Acyl CoA (Carnitine Acyltransferase II)

Oxidation Process

• Once fatty acids enter the matrix, they are committed to oxidation.
• Fatty acid is oxidized via 2-C units, released as acetyl-CoA.
• Repetitive four-step processes:
  • Step 1: Oxidation introduces a trans double bond into the Fatty Acyl CoA and generates a molecule of FADH2 (Acyl-CoA dehydrogenase).
• There are several Acyl Co A dehydrogenases present:
  • long-chain Acyl Co A dehydrogenase (> C12)
  • medium-chain Acyl Co A dehydrogenase (C4-C14)
  • short-chain Acyl Co A dehydrogenase (C4-C6)
• $FADH_2$ ® ETF ® ETF:ubiquinone reductase ® Ubiquinone Complex III: Generates ~ 1.5 ATP
• $FADH_2$ of Acyl CoA Dehydrogenase is re-oxidized by transfer of 2 electrons to an Electron Transfer Flavoprotein (ETF), which in turn passes the electrons to coenzyme Q of the respiratory chain.
• Half Cell Reaction:
  Note the correct notation the half cell reaction gives the reduction. The oxidation will go in the opposite direction.
• Water is added in a reaction catalyzed by Enoyl-CoA hydratase (crotonase).
  • This reaction is addition to a double bond.
• The hydroxyl is oxidized to a ketone by L-Hydroxyacyl-CoA dehydrogenase (or 3-Hydroxyacyl-CoA dehydrogenase).
  • More reducing potential is released for oxidative phosphorylation at this step.
• The B-carbon is now oxidized from Acyl-CoA to B-Ketoacyl-CoA, hence, B-oxidation.

Thiolase Reaction

• Each reduction in chain length of a saturated fatty acid by two carbon atoms generates:
  • One molecule of $FADH_2$
  • One molecule of NADH
  • One molecule of Acetyl CoA
• Acetyl-CoA is fed into the TCA cycle.
  • Oxidation of acetyl-CoA generates more NADH and $FADH_2$ as well as one molecule of GTP.

ATP Production

• Each reduction in chain length of a saturated fatty acid by two carbon atoms generates:
  • One molecule of $FADH_2$ → 1.5 ATP
  • One molecule of NADH → 2.5 ATP
  • One molecule of Acetyl CoA → 3 NADH plus 1 $FADH_2$ plus 1 GTP → 9 ATP plus 1 GTP
  • Giving a total of about 13 ATP molecules and 1 GTP molecule.
• Each two-carbon unit of carbohydrate comes with an extra two oxygen atoms.
  • A mole of $C<em>2H</em>4O<em>2$ weighs 60 g, but a mole of $C</em>2H_4$ only weighs 28 g, and yet metabolism yields more ATP molecules.

Problem and Solution

• Assume we get:
  • 10 ATP per acetyl CoA
  • 2.5 ATP per NADH
  • 1.5 ATP per $FADH_2$
• Oxidizing one mole of Palmitic Acid (C-16) by b-oxidation gives how much energy?
  • $ΔG°$ for hydrolysis of ATP to ADP= -32 kJ mol-1
• Each cycle (spiral) of the b-oxidation spiral generates:
  • $FADH_2$
  • NADH
  • acetyl CoA
  • Fatty Acyl CoA (2 carbons shorter)
• The balanced equation for oxidizing one molecule of Palmitic Acid (C-16) by seven cycles of b-oxidation is:
  • Palmitoyl-CoA + 7 HS-CoA + 7 FAD + 7 NAD+ + 7 $H<em>2O$ ® 8 acetyl CoA + 7 $FADH</em>2$ + 7 NADH + 7 H+
• Assume we get:
  • 10 ATP per acetyl CoA
  • 2.5 ATP per NADH
  • 1.5 ATP per $FADH_2$
• 8 acetyl CoA gives:
  • 80 ATP
• 7 NADH gives:
  • 17.5 ATP
• 7 $FADH_2$ gives:
  • 10.5 ATP
• Total: 108 ATP
• ATP expended to activate Fatty Acid: 2 ATP
• Net total ATP generated: 106 ATP
• 106 ATP × -32 kJ mol-1 = -3,392 kJ (under standard conditions)
  • $ΔG°$ for hydrolysis of ATP to ADP = -32 kJ mol-1

Lipids: Fatty Acid Synthesis

Fatty Acid Synthesis

• In eukaryotes, fatty acid synthesis is largely carried out in the cytoplasm, in animals by a multi-enzyme complex called a fatty acyl synthase.
• The formation of Acetyl ACP was described as “first” because this is the starting point for the elongating chain
• Acetyl transferase can use propionyl-CoA, which will give a chain with an odd number of carbon atoms (malonyl transferase cannot). A thioesterase removes the elongating fatty acid from ACP and thus ends chain elongation.
• The first step is the irreversible carboxylation of acetyl–CoA to form malonyl-CoA.
• This is catalysed by a highly regulated enzyme called acetyl-CoA carboxylase.
• It is upregulated by citrate and downregulated by palmitoyl-CoA

Comparison with β-Oxidation

• Fatty acyl synthesis is close to being the reverse of β-oxidation, but there are interesting differences:
  • Location: In eukaryotes β-oxidation is largely mitochondrial and fatty acyl synthesis is cytoplasmic.
  • NAD+/NADH and FAD/FADH2 are used in β-oxidation and NADP+/NADPH in fatty acyl synthesis.
    • NAD+/NADH is involved in catabolic reactions and NADP+/NADPH in anabolic reactions.
  • CoA is used as the acyl group carrier in β-oxidation and the acyl carrier protein (ACP) in fatty acyl synthesis (although ACP has a prosthetic group chemically similar to CoA).

Formation of Double Bonds

• Formation of double bonds in fatty acids (to form unsaturated or polyunsaturated fatty acids) requires desaturase enzymes.
• Eukaryotic desaturases work on “complete” fatty acids (although the chain can be lengthened by additional C 2 units after desaturation).
• Different desaturases incorporate double bonds in specific positions.
• In bacteria, double bonds are formed by a modified step in fatty acid chain elongation by a thioester dehydrase.
  • The reaction is peculiar and complex involving oxidation of both the fatty acid and one molecule of NADPH or NADH.
• Pathway of electron transfer in the desaturation of fatty acids by stearoyl-CoA desaturase (SCD).
• Mammals do not have Fatty Acyl-CoA Desaturases capable of introducing double bonds “below” the D9 position.
• Human cells need unsaturated fatty acids with double bonds below this position.
  • prostaglandins are derived from arachidonic acid (20:4 D5,8,11,14)
  • arachidonic acid and docosahexaenoic acid (22:6 D4,7,10,13,16,19) are the main fatty acids in membranes of nerves in the eye and central nervous system, and therefore depend on dietary sources of unsaturated fatty acids with double bonds nearer to the methyl end of the chain.
  • Linoleic acid (18:2 D9,12) a-Linolenic acid (18:3 D9,12,15)
• Linoleic acid (nuts, cheeses, safflower or sunflower oil) and a-Linolenic acid (flax seeds, walnuts, tofu, flax or rapeseed oil) are essential dietary fatty acids in humans.
• Other necessary fatty acids (such as arachidonic and docosahexaenoic acid) can be synthesised from Linoleic acid and a-Linolenic acid

Coenzymes

• A few coenzymes have been involved in these reactions:
  • NAD+
  • Biotin
  • FAD
  • Coenzyme A
• Co-enzymes are generally essential but required in very small quantities.
• Most are derived from dietary vitamins.
  • Vitamin B3 - Niacin NAD+
  • Vitamin B2 - Riboflavin
  • Vitamin B5 - Pantothenic Acid
  • Biotin is Vitamin B7

Supplementary Material: More complicated situations in fatty acid metabolism

Unsaturated fatty acids

• This is relatively simple if the closest to the carboxylic acid of the carbon atoms joined by a double bond is odd numbered (this may not be what you expect).
• There is already a double bond in the chain (but in the wrong place) and so this step is skipped.
• However, Enoyl-CoA hydratase (which catalyses the next step) only acts on trans double bonds and so cis double bonds must be converted to trans double bonds before the next step.
• $D<em>3$,$D</em>2$-enoyl-CoA-isomerase catalyses “movement” of the double bond “one step up” the chain and reforms it as a trans bond.
• Less ATP is produced because one fewer molecule of $FADH_2$ is produced per double bond.
• If the closest to the carboxylic acid of the carbon atoms joined by a double bond is cis and even numbered then enoyl-CoA hydratase cannot process it without modification. This takes place in several steps.
• The first happens after a trans bond has been formed two carbons further “up” the chain (this happens as part of normal b-oxidation. At this stage there will be a trans bond between carbons 2 and 3 and a cis bond between carbons 4 and 5
• 2,4-dienoyl-CoA reductase breaks both existing double bonds and instead forms a new double bond between carbons 3 and 4 (some isozymes produce cis and some trans bonds.
• One molecule of NADPH is oxidised in this reaction, indirectly reducing the ATP yield.
• Finally the $D<em>3$ bond is converted to a trans- $D</em>2$ bond by enoyl-CoA isomerase.

Branched Fatty Acids

• A fatty acid with a branch on its a-carbon can be metabolised by b- oxidation in the peroxisome.
  • Pristanic acid is present in human milk fat and butter fat (also krill, whales and freshwater sponges).
  • Also from a-oxidation of phytanic acid.
• A fatty acid with a branch on its b-carbon cannot be metabolised by b- oxidation and is instead metabolised by a-oxidation, again in the peroxisome.
  • This shortens the fatty acid chain by one carbon atom allowing b-oxidation to occur.
  • Phytanic acid is produced in ruminants by metabolism of chlorophyll and therefore enters the human diet in dairy products and ruminant fats (also some fish).

Fatty Acids with Odd Numbers of Carbon Atoms

• Most fatty acids have an even number of carbon atoms but there are exceptions and propionate can enter the body through the diet in some cases (for example, it is sometimes added to bread to inhibit mold growth)
• Both propionate and odd numbered “ends” of fatty acids are metabolised from propionyl-CoA.
• Propionyl-CoA is converted to D -methylmalonyl-CoA by addition of $HCO_3^{-}$.
• This is then first converted to L- methylmalonyl-CoA (by methylmalonyl-CoA epimerase) And then to succinyl-CoA (by methylmalonyl-CoA mutase).
• Succinyl-CoA enters the Citric Acid Cycle (about half way around).

ω-Oxidation

• There is also a process by which fatty acids can be metabolised from the w end in the endoplasmic reticulum. For obvious reasons this is called w-oxidation.

Ketone Bodies

• Acetyl-CoA produced by fatty acid metabolism in the liver can either feed into the Citric Acid Cycle, or it can be converted to ketone bodies (acetoacetate, D- b- hydroxybutyrate and acetone) and transported to other organs, where they are converted back to acetyl-CoA and used to feed the Citric Acid Cycle for ATP production there.
• Ketone bodies increase when glucose availability is limited:
  • In uncontrolled diabetes (because of an absence of glycogen stores and because an insulin response is needed for glucose uptake)
  • In a diet low in carbohydrates (or indeed low in everything).
  • Under these circumstances the available pool of oxaloacetate is required for both gluconeogenesis and to receive acetyl-CoA produced from metabolism of fatty acids and protein. Therefore insufficient oxaloacetate is available to use the acetyl-CoA produced in the liver and the excess is converted to ketone bodies.
• This is particularly notable for the brain, which has a very substantial energy requirement but no carbohydrate or fat reserves, or surplus protein.
• This source of energy is also important for the heart, renal cortex (in the kidney) and skeletal muscle.

Supplementary Material: Dietary Fats and Lipoproteins

Lipoproteins

• Components:
  • apoB-100(Non HDL non-exchangeable)
  • cholesterol
  • triglyceride
  • Exchangeable apolipoprotein
  • cholesterylester
  • phospholipid
• The image presents a chart about lipoproteins.
  • Density, %total lipid, % total protein, Particle diameter.
• Prediction table of the 10-y risk of coronary heart disease and angina.
• Table about
  • Total cholesterol(mmol/L)(mg/dL)
  • Age
  • HDL cholesterol(mmol/L)(mg/dL)
• TABLE 1 shows Prevalence of risk factors in premature coronary heart disease'
  • Cases,
  • Controls,
  • %,Cigarette smoking (past year),
  • Low HDL cholesterol<0.9 mmol/L (35 mg/dL),
  • Hypertension,
  • Elevated LDL cholesterol ≥4.1 mmol/L (160 mg/dL),
  • Elevated homocysteine,
  • Elevated lipoprotein(a),
  • Diabetes
• The image presents a chart about density, total lipid, total protein, Particle diameter in lipoproteins.
  • Chylomicrons, VLDL, IDL, LDL, HDL.
• There is a note about the lipoproteins being Good or Bad.
• Endogenous Pathway (LDL)
• Reverse Transport Pathway (HDL)
• Exogenous Pathway (chylomicrons)
• Recycling and return of cholesterol.
• Transport of fats and lipids from the gut to the liver.
• Transport of lipids to tissues.
• Hyperlipidemia certainly correlates with CHD.
• https://www.flickr.com/photos/euthman/121061911/in/set- 72057594114099781/ Atheroma Plaques formed in arteries, substantially composed of white blood cells that have taken up LDLs.
• Lipoprotein ratios: Physiological significance and clinical usefulness in cardiovascular prevention
• Role of omega-3 fatty acids in obesity, metabolic syndrome, and cardiovascular diseases: a review of the evidence
• Saturated Fats Versus Polyunsaturated Fats Versus Carbohydrates for Cardiovascular Disease Prevention and Treatment
• Use of dietary linoleic acid for secondary prevention of coronary heart disease and death: evaluation of recovered data from the Sydney Diet Heart Study and updated meta-analysis
• Meta-analysis of prospective cohort studies evaluating the association of saturated fat with cardiovascular disease
• Saturated fats, unsaturated fats and carbs
• Consumption of industrial and ruminant trans fatty acids and risk of coronary heart disease: a systematic review and meta-analysis of cohort studies
• The negative effects of hydrogenated trans fats and what to do about them
• Intake of saturated and trans unsaturated fatty acids and risk of all cause mortality, cardiovascular disease, and type 2 diabetes: systematic review and meta-analysis of observational studies
• Long-term coronary heart disease risk associated with very-low-density lipoprotein cholesterol in Chinese
• Very Low Levels of Atherogenic Lipoproteins and the Risk for Cardiovascular Events: A Meta-Analysis of Statin Trials
• Meta-Analysis of Impact of Different Types and Doses of Statins on New- Onset Diabetes Mellitus
• Trans fats, cholesterol and statins (and a bit more on saturated fats)