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Theory Notes

Sources of cholesterol

Cholesterol is derived from

  • Diet
  • De novo synthesis and
  • Hydrolysis of cholesteryl esters
  • A little more than half the cholesterol of the body arises by synthesis (about 700 mg/d), and the remainder is provided by the average diet.

Site of synthesis

  • The liver and intestine account for approximately 10% each of total synthesis in humans.
  • Virtually all tissues containing nucleated cells are capable of cholesterol synthesis, which occurs in the endoplasmic reticulum and the cytosol.

Steps of synthesis of cholesterol

Remember the formula for cholesterol synthesis

2+2 =4

4+2 = 6

6-1 =5

5+5 = 10

10+5= 15

15 +15 = 30

30-3 = 27

The representation of the numbers (Compounds with given carbon numbers) is as follows-

2 – Acetyl co A

4- Acetoacetyl co A

6- Mevalonate

5- Isopentenyl pyrophosphate (IPP)

5+5 – Isopentenyl pyrophosphate +Dimethyl allyl pyrophosphate

10- Geranyl pyrophosphate

15- Farnesyl pyrophosphate

30 –Squalene and lanosterol

27- Cholesterol

Details of steps

  • Acetyl co A acts as a precursor of cholesterol.
  • All 27 carbon atoms of cholesterol are derived from acetyl CoA in a three-stage synthetic process

Stages of cholesterol synthesis

Stage-1- Synthesis of Isopentenyl pyrophosphate, an activated isoprene unit that is the key building block of cholesterol

Stage-2- Condensation of six molecules of Isopentenyl pyrophosphate to form squalene

Stage-3- Cyclization of squalene to form lanosterol and subsequent conversion to cholesterol

 Stage -1

a) Formation of Mevalonate

  • Initially, two molecules of acetyl-CoA (2+2) condense to form Acetoacetyl-CoA (4) catalyzed by cytosolic thiolase.
  • Acetoacetyl-CoA condenses with a further molecule of acetyl-CoA (4+2) catalyzed by HMG-CoA synthase to form HMG-CoA that is reduced to mevalonate (6) by NADPH catalyzed by HMG-CoA reductase (figure-1).

 Formation of mevalonate

Figure-1- These reactions take place in the cytosol, formation of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) takes place from acetyl CoA.

Significance

  • The synthesis of mevalonate is the committed step in cholesterol formation.
  • The enzyme catalyzing this irreversible step, 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), is an important control site in cholesterol biosynthesis,
  • It is the site of action of the most effective class of cholesterol-lowering drugs, the HMG-CoA reductase inhibitors (statins).

b) Formation of IPP (Isopentenyl pyrophosphate)

  • Mevalonate is converted into 3-isopentenyl pyrophosphate in three consecutive reactions requiring ATP (figure-2)
  • Decarboxylation (6-1 =5) yields Isopentenyl pyrophosphate (5), an activated isoprene unit that is a key building block for many important biomolecules (figure-2).

 

 Formation of IPP

Figure-2- Mevalonate is phosphorylated first to form Mevalonate- 5-P by Mevalonate kinase, subsequently it is phosphorylated at 5th and 3rd position in two different steps to form 5-Pyrophospho mevalonate and 3-phosho-5-pyrophospho mevalonate respectively. 3-phosho-5-pyrophospho mevalonate is highly unstable and undergoes decarboxylation and loses phosphate also to form Isopentenyl pyrophosphate (IPP).

Stage-2- Formation of Squalene

  • Squalene (C30) is synthesized from six molecules of Isopentenyl Pyrophosphate (C5).
  • This stage starts with the isomerization of Isopentenyl pyrophosphate (C5) to dimethylallyl pyrophosphate (C5).
  • Isopentenyl pyrophosphate is isomerized by a shift of the double bond to form dimethylallyl pyrophosphate that condenses with another molecule of Isopentenyl pyrophosphate (5+5 =10) to form the ten-carbon intermediate geranyl pyrophosphate (10).
  • A further condensation with Isopentenyl pyrophosphate forms Farnesyl pyrophosphate (15). Two molecules of Farnesyl pyrophosphate (15+15) condense at the pyrophosphate end to form squalene (30) –figure-3

 Formation of Squalene

Figure-3- Initially, inorganic pyrophosphate is eliminated, forming presqualene pyrophosphate, which is then reduced by NADPH with elimination of a further inorganic pyrophosphate molecule.

Stage-3 –Formation of cholesterol

a) Formation of Lanosterol

  • Squalene can fold into a structure that closely resembles the steroid nucleus
  • Before ring closure occurs, squalene is converted to squalene 2,3-epoxide by a mixed-function oxidase in the endoplasmic reticulum, squalene epoxidase.
  • The methyl group on C14 is transferred to C13 and that on C8 to C14 as cyclization occurs, catalyzed by oxidosqualene: lanosterol cyclase (figure-4).
  • The newly formed cyclized structure is Lanosterol

formation of cholesterol from lanosterol

Figure-4- Formation of Cholesterol from Lanosterol involves three processes-i) loss of three carbons, ii) Shifting of double bond and iii) Saturation of the double bond in the side chain.

b) Formation of cholesterol from Lanosterol (30 – 3 = 27)

  • The formation of cholesterol from lanosterol takes place in the membranes of the endoplasmic reticulum and involves changes in the steroid nucleus and side chain.
  • The methyl groups on C14 and C4 are removed to form 14-desmethyl lanosterol and then zymosterol.
  • The double bond at C8–C9 is subsequently moved to C5–C6 in two steps, forming desmosterol.
  • Finally, the double bond of the side chain is reduced, producing cholesterol (figure-4).

Regulation of cholesterol biosynthesis

Regulation of cholesterol synthesis is  exerted near the beginning of the pathway, at the HMG-CoA reductase step. Following mechanisms are involved at the regulatory step-

 1) Competitive inhibition

  • Statins (Lovastatin, Mevastatin, Atorvastatin etc.) are the reversible competitive inhibitors of HMG Co A reductase (figure-1)
  • They are used to decrease plasma cholesterol levels in patients of hypercholesterolemia.

 2) Feed back inhibition

  • HMG Co A reductase is inhibited by Mevalonate, bile acids and Cholesterol (Figure-1)
  • Mevalonate is the immediate product of HMG Co A reductase catalyzed reaction whereas Cholesterol is the ultimate product of the reaction pathway.

 3) Covalent modification (Role of hormones)- Figure-5

  • Phosphorylation decreases the activity of the reductase.
  • Glucagon favors formation of the inactive (phosphorylated form) form, hence decreases the rate of cholesterol synthesis
  • In contrast , insulin favors formation of the active (dephosphorylated)form of HMG Co A reductase and results in an increase in the rate of cholesterol synthesis
  • Cholesterol synthesis ceases when the ATP level is low.

 covalent modification of HMG Co A reductase

Figure-5- HMG Co A reductase exists in two forms- Dephosphorylated form is the active while  phosphorylated form is the inactive form. Phosphorylation is brought about by reductase kinase enzyme, that itself is phosphorylated by reductase kinase kinase enzyme to be active to bring about subsequent phosphorylation. The ultimate regulator of phosphorylation cascade is  c-AMP which is released by Glucagon. Insulin brings about dephosphorylation by activating phosphatases.

 4) Sterol mediated regulation of transcription          

  • The synthesis of cholesterol is also regulated by the amount of cholesterol taken up by the cells during lipoprotein metabolism.
  • Chylomicron remnants internalized by liver cells, and low density lipoproteins internalized by liver cells and peripheral tissues provide cholesterol which causes a decrease in the transcription of HMG CoA reductase gene, leading to a decrease in cholesterol synthesis.
  • Excess dietary intake thus suppresses endogenous cholesterol synthesis, whereas low cholesterol diet increases transcription of HMG Co A reductase gene causing more availability of enzyme to increase the synthesis of cholesterol.
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Regulation of Acetyl-co A carboxylase- Acetyl co A carboxylase is the rate limiting enzyme that catalyzes the conversion of Acetyl co A to Malonyl co A.

The mammalian enzyme is regulated, by two main mechanisms-

A) Short term control

B) Long term control

A) Short term control of fatty acid synthesis includes

a) Allosteric control by local metabolites

Palmitoyl-CoA (or acyl co A) acts as a feedback inhibitor of the enzyme, and citrate is an activator (figure-1).

  • When there is an increase in mitochondrial acetyl-CoA and ATP, citrate is transported out of mitochondria
  • Citrate becomes both the precursor of cytosolic acetyl-CoA and a signal for the activation of acetyl-CoA carboxylase.
  • Conversely , if acyl-CoA accumulates because it is not esterified quickly enough or because of increased lipolysis or an influx of free fatty acids into the tissue, it will automatically reduce the synthesis of new fatty acid.
  •  Acyl-CoA also inhibits the mitochondrial tricarboxylate transporter, thus preventing activation of the enzyme by egress of citrate from the mitochondria into the cytosol.

 Regulation of acetyl co A carboxylase

Figure-1- Citrate is a positive allosteric modifier, which increases in concentration in the well-fed state and is an indicator of a plentiful supply of acetyl-CoA. Palmitoyl co A or the long chain fatty acids are the negative modifiers of acetyl co A carboxylase (negative feedback inhibition by a product of a reaction).

b) Covalent modification (Phosphorylation and dephosphorylation)

Acetyl-CoA carboxylase is also regulated by hormones such as glucagon, epinephrine, and insulin via changes in its phosphorylation state (figure-2)-

 Covalent modification of acetyl co A carboxylase

Figure-2- Regulation of acetyl co A carboxylase by covalent modification

Acetyl co A carboxylase exists in two forms-

i) Active (dephosphorylated form)

ii) Inactive (phosphorylated form)

Phosphorylation is brought about by c- AMP mediated phosphorylation cascade. Glucagon (and epinephrine) increase cAMP concentration and inactivate the enzyme by bringing about its phosphorylation.

Dephosphorylation, is brought about by protein phosphatase which is stimulated by insulin. In other words, insulin stimulates this enzyme to promote fatty acid synthesis, while glucagon and catecholamines inactivate this enzyme to inhibit fatty acid synthesis.

c) Conformational changes associated with regulation:

  • In the active conformation, Acetyl-CoA Carboxylase associates to form multimeric filamentous complexes. Citrate converts the enzyme from an inactive dimer to an active polymeric form, with a molecular mass of several million.
  • Transition to the inactive conformation is associated with dissociation to yield the monomeric form of the enzyme (protomer). Inactivation is promoted by phosphorylation of the enzyme and by long-chain acyl-CoA molecules.

B) Long term control- Additionally, fatty acid synthesis is regulated at the level of gene expression by induction and repression of gene of acetyl co A carboxylase enzyme

Prolonged consumption of high calorie or high carbohydrate diets causes an increase in acetyl co A carboxylase concentration by increasing the gene expression (induction). Conversely, a low-calorie diet or fasting causes a reduction in fatty acid synthesis by decreasing the synthesis of acetyl co A carboxylase(repression) .

Nutritional state regulates lipogenesis- Excess carbohydrates are stored as fat in many animals in anticipation of periods of caloric deficiency such as starvation, hibernation, etc, and to provide energy for use between meals in animals, including humans, that take their food at spaced intervals. The nutritional state of the organism is the main factor regulating the rate of lipogenesis.

Fatty acid synthesis during Fed state

  • The rate is higher in the well-fed state if the diet contains a high proportion of carbohydrate
  • Lipogenesis converts surplus glucose and intermediates such as pyruvate, lactate, and acetyl-CoA to fat, assisting the anabolic phase of this feeding cycle
  • Lipogenesis is increased when sucrose is fed instead of glucose because fructose bypasses the phosphofructokinase control point in glycolysis and floods the lipogenic pathway

Fatty acid synthesis during Fasting

  • It is depressed by restricted caloric intake, high fat diet, or a deficiency of insulin, as in diabetes mellitus
  • These conditions are associated with increased concentrations of plasma free fatty acids
  • An inverse relationship has been demonstrated between hepatic lipogenesis and the concentration of serum-free fatty acids.

Role of Insulin in fatty acid synthesis

  • Insulin stimulates lipogenesis by several other mechanisms as well as by increasing acetyl-CoA carboxylase activity.
  • It increases the transport of glucose into the cell (e.g., in adipose tissue),
  • Increases the availability of both pyruvate for fatty acid synthesis and glycerol 3-phosphate for esterification of the newly formed fatty acids,
  • Insulin converts the inactive form of pyruvate dehydrogenase to the active form thus provides more of Acetyl co A
  • Insulin also acts by inhibiting c AMP mediated lipolysis in adipose tissue and thereby reduces the concentration of plasma free fatty acids (long-chain fatty acids are inhibitors of lipogenesis.

Note- The fatty acid synthase complex is also similarly regulated.

 

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Step-1- Formation of Malonyl co A 

  • Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA (figure-1 and 4)
  • This irreversible reaction is the committed step in fatty acid synthesis.
  • The synthesis of malonyl CoA is catalyzed by acetyl CoA carboxylase, which contains a biotin prosthetic group.
  • The carboxyl group of biotin is covalently attached to the € amino group of a lysine residue, as in pyruvate carboxylase and propionyl CoA carboxylase.
  • As with these other enzymes, a carboxy biotin intermediate is formed at the expense of the hydrolysis of a molecule of ATP. The activated CO2 group in this intermediate is then transferred to acetyl CoA to form malonyl CoA (figure-1)

 Acetyl co A carboxylase

Figure-1- Conversion of Acetyl co A to Malonyl co A

2) Elongation cycle in fatty acid synthesis

  • Elongation takes place in a cyclic manner, where four processes (Condensation, reduction, dehydration  and  reduction) are repeated till a fatty acid with a required chain length is synthesized.
  • Acyl carrier Protein (ACP) –The intermediates in fatty acid synthesis are linked to an acyl carrier protein. Specifically, they are linked to the sulfhydryl  terminus of a phosphopantetheine group, which is, in turn, attached to a serine residue of the acyl carrier protein.
  • Initially, a priming molecule of acetyl-CoA combines with a cysteine —SH group catalyzed by acetyl transacylase (Figure-2 and 4).
  • Malonyl-CoA combines with the adjacent —SH on the 4′-phosphopantetheine of ACP of the other monomer, catalyzed by malonyl transacylase (reaction-2), to form acetyl (acyl)-malonyl enzyme  (figure-4)
  • Fatty acids with an odd number of carbon atoms are synthesized starting with propionyl ACP, which is formed from propionyl CoA by acetyl transacylase.

Fatty acid synthesis- step 2

Figure-2- Transfer of acetyl and malonyl co A to the fatty acid synthase complex

a) Condensation- The acetyl group attacks the methylene group of the malonyl residue, catalyzed by 3-ketoacyl synthase, forming 3-ketoacyl enzyme (acetoacetyl enzyme) , freeing the cysteine —SH group (figure-3 and 4) .

  • In the condensation reaction, a four-carbon unit is formed from a two carbon unit and a three-carbon unit, and CO2 is released (figure-3) 

 Condensation reaction

Figure-3- Condensation reaction is catalyzed by keto acyl synthase enzyme.

Why is the four-carbon unit not formed from 2 two-carbon units? In other words, why are the reactants acetyl ACP and malonyl ACP rather than two molecules of acetyl ACP?

  • The answer is that the equilibrium for the synthesis of acetoacetyl ACP from two molecules of acetyl ACP is highly unfavorable. In contrast, the equilibrium is favorable if malonyl ACP is a reactant because its decarboxylation contributes a substantial decrease in free energy.
  •  In effect, ATP drives the condensation reaction, though ATP does not directly participate in the condensation reaction. Rather, ATP is used to carboxylate acetyl CoA to malonyl CoA. The free energy thus stored in malonyl CoA is released in the decarboxylation accompanying the formation of acetoacetyl ACP.
  • Although HCO3 – is required for fatty acid synthesis, its carbon atom does not appear in the product. Rather, all the carbon atoms of fatty acids containing an even number of carbon atoms are derived from acetyl CoA.
  • The acetoacetyl group is then delivered to three active sites in domain 2 of the opposite chain to reduce it to a butyryl unit.

 b) Reduction-The next three steps in fatty acid synthesis reduce the keto group at C-3 to a methylene group (figure-4)

  • First, acetoacetyl ACP is reduced to d-3-hydroxybutyryl ACP. This reaction differs from the corresponding one in fatty acid degradation in two respects:
  • the d rather than the l isomer is formed; and
  • NADPH is the reducing agent, whereas NAD+ is the oxidizing agent in β oxidation.
  • This difference exemplifies the general principle that NADPH is consumed in biosynthetic reactions, whereas NADH is generated in energy-yielding reactions.

c) Dehydration-d-3-hydroxybutyryl ACP is dehydrated to form trans-Δ 2-enoyl ACP (α-β unsaturated acyl ACP)-figure-4

 d) Reduction

  • The final step in the cycle reduces α-β unsaturated acyl ACP to butyryl ACP.
  • NADPH is again the reductant, whereas FAD is the oxidant in the corresponding reaction in β-oxidation.
  • These last three reactions a reduction, a dehydration, and a second reduction convert acetoacetyl ACP into butyryl ACP, which completes the first elongation cycle.
  •  This saturated C4 unit then migrates from the phosphopantetheinyl sulfur atom on ACP to the cysteine sulfur atom on the condensing enzyme (figure-4)
  • The synthase is now ready for another round of elongation.

Second round of Elongation

In the second round of fatty acid synthesis, butyryl ACP condenses with malonyl ACP to form a C6-β-ketoacyl ACP. This reaction is like the one in the first round, in which acetyl ACP condenses with malonyl ACP to form a C4-β- ketoacyl ACP. Reduction, dehydration, and a second reduction convert the C6-β-ketoacyl ACP into a C6-acyl ACP, which is ready for a third round of elongation.

 

 steps of fatty acid synthesis

Figure-4- Biosynthesis of long-chain fatty acids. Details of how addition of a malonyl residue causes the acyl chain to grow by two carbon atoms. (Cys, cysteine residue; Pan, 4′-phosphopantetheine) .

3) Termination of fatty acid synthesis

  • The elongation cycles continue until C16-acyl ACP is formed.
  • Five more rounds of condensation and reduction produce a palmitoyl (C16) chain on the condensing enzyme, which is hydrolyzed to palmitate by the thioesterase on domain 3 of the opposite chain (figure-4).
  • This intermediate is a good substrate for a thioesterase that hydrolyzes C16-acyl ACP to yield palmitate and ACP. 
  • The thioesterase acts as a ruler to determine fatty acid chain length. In mammary gland, there is a separate thioesterase specific for acyl residues of C8, C10, or C12, which are subsequently found in milk lipids.
  • The free palmitate must be activated to acyl-Co A before it can proceed via any other metabolic pathway.
  • Its usual fate is esterification into acylglycerols, chain elongation or desaturation, or esterification to cholesteryl ester.

 The equation  for the overall synthesis of palmitate from acetyl-Co A and malonyl-Co A is-


Overall reaction

For further details-

Follow the links

http://www.namrata.co/fatty-acid-synthesis-power-point-presentation/

http://www.namrata.co/animations-links-fatty-acid-synthesis/

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Details of enzymes

1) Acetyl co A carboxylase catalyzes the initial and rate controlling step in Fatty acid synthesis . Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA. Acetyl CoA carboxylase contains a biotin prosthetic group. The carboxyl group of biotin is covalently attached to the € amino group of a lysine residue. This multienzyme complex contains

  • Biotin
  • Biotin carboxylase
  • Biotin carboxyl carrier protein
  • Transcarboxylase
  • A regulatory allosteric site

Reaction catalyzed by Acetyl co A carboxylase               

The input to fatty acid synthesis is acetyl-CoA, which is carboxylated to malonyl-CoA. The reaction is catalyzed by Acetyl co A carboxylase (Figure-1).

Acetyl co A carboxylase
Figure-1- The reaction takes place in two steps . It is an energy requiring  carboxylation reaction, Biotin is required as a coenzyme.

As with these other enzymes, a carboxy biotin intermediate is formed at the expense of the hydrolysis of a molecule of ATP. The activated CO2 group in this intermediate is then transferred to acetyl CoA to form malonyl CoA (Figure-2)

 Acetyl co A carboxylase

Figure-2-This ATP-dependent carboxylation provides energy input. The CO2 is lost later during condensation with the growing fatty acid.The spontaneous decarboxylation drives the condensation reaction.

2) Fatty acid Synthase complex

  • The Fatty Acid Synthase complex is a polypeptide containing seven enzyme activities
  • In bacteria and plants, the individual enzymes of the fatty acid synthase system are separate, and the acyl radicals are found in combination with a protein called the acyl carrier protein (ACP).
  • In yeast, mammals, and birds, the synthase system is a multienzyme polypeptide complex that incorporates ACP, which takes over the role of CoA.
  • Each chain is folded into three domains joined by flexible regions (Figure-3).
  • Domain-1-Condensation unit- The substrate entry and condensation unit, contains acetyl transferase, malonyl transferase, and β-ketoacyl synthase (condensing enzyme).
  • Domain-2-Reduction unit- The reduction unit, contains the acyl carrier protein, β-ketoacyl reductase, dehydratase, and enoyl reductase.
  • Domain-3-Releasing unit- the palmitate release unit, contains the thioesterase.

Fatty acid synthase complex-1

Figure-3- i ) Domain 1- condensation unit (blue); ii ) Reduction unit ( yellow);  iii) Releasing unit (pink).

AT- Acetyl transacylase ; MT- Malonyl transacylase ; CE- Condensing enzyme (Keto acyl synthase) ; DH- Dehydratase ; ER- Enoyl reductase ; KR- Keto acyl reductase ; ACP- Acyl carrier protein ; TE- Thioesterase

The —SH of the 4′-phosphopantetheine of one monomer is in close proximity to the —SH of the cysteine residue of the ketoacyl synthase of the other monomer, suggesting a “head-to-tail” arrangement of the two monomers (figure-2 and 3).Though each monomer contains all the partial activities of the reaction sequence, the actual functional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously.

Biological Advantage of having Multienzyme complex-

1)    An advantage of this arrangement is that the synthetic activity of different enzymes is coordinated since it is encoded by a single gene.

2)    A multienzyme complex consisting of covalently joined enzymes is more stable than one formed by non covalent attractions.

3)     Furthermore, intermediates can be efficiently handed from one active site to another without leaving the assembly.

Simple representation of Fatty acid synthase complex (Figure-4)-

Fatty acid synthase complex

Figure-4-  Fatty acid synthase complex. There are two polypeptide chains, each containing 7 enzyme activities and ACP. The long flexible arm  of phosphopantetheine helps its thiol to move from one active site to another within the complex. 

Overview of fatty acid synthesis and role of enzymes

Fatty acid synthesis is a cyclic process. The initial step of carboxylation of acetyl co A is catalyzed by Acetyl co A carboxylase, the remaining steps are catalyzed by fatty acid synthase complex. In each cycle, the general processes involved are-

a) Condensation

b) Reduction

c) Dehydration and

d) Reduction

The  process of condensation (addition of 2 carbons to the existing chain in each cycle) is carried out by keto acyl synthase enzyme of condensation unit, while the remaining steps-(reduction, dehydration and reduction) are catalyzed by the enzymes of reduction unit. Once a fatty acid of required chain length is synthesized the releasing enzyme (thioesterase)  catalyzes the release of newly synthesized fatty acid from the enzyme complex.

Details of steps in next post….

 

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Sources of Fatty acids

  • Diet
  • Adipolysis
  • De novo synthesis(from precursors)- Carbohydrates, protein, and other molecules obtained from diet in excess of the body’s need can be converted to fatty acids, which are stored as triglycerides

De novo fatty Acid Synthesis

Characteristics

  • Fatty acids are synthesized by an extra mitochondrial system
  • This system is present in many tissues, including liver, kidney, brain, lung, mammary gland, and adipose tissue.
  • Acetyl-CoA is the immediate substrate, and free palmitate is the end product.
  • Its cofactor requirements include NADPH, ATP, Mn2+, biotin, and HCO3 (as a source of CO2).

Sources of NADPH

  • NADPH is involved as donor of reducing equivalents
  • The oxidative reactions of the pentose phosphate pathway are the chief source of the hydrogen required for the reductive synthesis of fatty acids.
  • Tissues specializing in active lipogenesis—i.e., liver, adipose tissue, and the lactating mammary gland—possess an active pentose phosphate pathway (Figure-1).

 HMP pathway- major source of NADPH

Figure-1- The reaction 1 and 2 are catalyzed by Glucose-6-P dehydrogenase and 6-phospho gluconate dehydrogenase respectively.

  • Other sources of NADPH include the reaction that converts malate to pyruvate catalyzed by the “Malic enzyme” (NADP malate dehydrogenase) – figure-2 and the extra mitochondrial Isocitrate dehydrogenase reaction (probably not a substantial source, except in ruminants) figure-3.

 Malic enzyme- alternative source of NADPH

Figure-2- It is a reversible reaction, pyruvate produced in the reaction reenters the mitochondrion for further utilization

 Cytosolic dehydrogenase- Alternative source of NADPH

Figure-3-There are three isoenzymes of Isocitrate dehydrogenase. One, which uses NAD+, is found only in mitochondria. The other two use NADP+ and are found in mitochondria and the cytosol. Respiratory chain-linked oxidation of Isocitrate proceeds almost completely through the NAD+-dependent enzyme.

Sources of Acetyl co A

  • Acetyl co A is produced primarily from pyruvate, ketogenic amino acids, fatty acid oxidation and by alcohol metabolism
  • It is a substrate of TCA cycle and a precursor for fatty acids, ketone bodies and sterols.

Enzymes and cofactors involved in the process of Fatty acid synthesis

Two main enzymes-

  • Acetyl co A carboxylase
  • Fatty acid Synthase

Both the enzymes are multienzyme complexes

Coenzymes and cofactors are-

  • Biotin
  • NADPH
  • Mn++
  • Mg++

Transportation of Acetyl co A (Figure-4)

  • Fatty acid synthesis requires considerable amounts of acetyl-CoA
  • Nearly all acetyl-CoA used in fatty acid synthesis is formed in mitochondria
  •  Acetyl co A has to move out from the mitochondria to the cytosol
  • Acetate is shuttled out of mitochondria as citrate
  • The mitochondrial inner membrane is impermeable to acetyl-CoA
  • Intra-mitochondrial acetyl-CoA first reacts with oxaloacetate to form citrate, in the TCA cycle catalyzed by citrate synthase
  • Citrate then passes into the cytosol through the mitochondrial inner membrane on the citrate transporter.
  • In the cytosol, citrate is cleaved by citrate lyase regenerating acetyl-CoA.

 Export of Acetyl co A

Figure-4- Transportation of acetyl co A out of the mitochondria through citrate transporter

Fate of Oxalo acetate (Figure-5)

The other product of Citrate cleavage, oxaloacetate can be-

  • Channeled towards glucose production
  • Converted to malate by malate dehydrogenase
  • Converted to Pyruvate by Malic enzyme, producing more NADPH, that can be used for fatty acid synthesis
  • Pyruvate and Malate pass through special transporters present in the inner mitochondrial membrane

 Fate of oxalo acetate

Figure-5- Fate of Oxalo acetate

To be continued …..

 

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Ketone bodies are utilized by extra hepatic tissues via a series of cytosolic reactions that are essentially a reversal of ketone body synthesis; the ketones must be reconverted to acetyl Co A in the mitochondria (figure-1)

Steps

1) Utilization of β-Hydroxy Butyrate

Beta-hydroxybutyrate is first oxidized to acetoacetate with the production of one NADH (Figure-1, step-1). In tissues actively utilizing ketones for energy production, NAD+/NADH ratio is always higher so as to drive the β-hydroxybutyrate dehydrogenase catalyzed reaction in the direction of acetoacetate synthesis. 

Biological significance

D (-)-3-Hydroxybutyrate is oxidized to produce acetoacetate as well as NADH for use in oxidative phosphorylation. D (-)-3-Hydroxybutyrate is the main ketone body excreted in urine.

2) Utilization of Acetoacetate

a) Coenzyme A must be added to the acetoacetate. The thioester bond is a high energy bond, so ATP equivalents must be used. In this case the energy comes from a trans esterification of the CoASH from succinyl CoA to acetoacetate by Coenzyme A transferase (Figure-1, step-2), also called Succinyl co A: Acetoacetate co A transferase, also known as Thiophorase.

The Succinyl CoA comes from the TCA cycle. This reaction bypasses the Succinyl-CoA synthetase step of the TCA cycle; hence there is no GTP formation at this step although it does not alter the amount of carbon in the cycle.

Biological significance

The liver has acetoacetate available to supply to other organs because it lacks this particular CoA transferase and that is the reason “Ketone bodies are synthesized in the liver but utilized in the peripheral tissues”. The latter enzyme is present at high levels in most tissues except the liver. Importantly, very low-level of enzyme expression in the liver allows the liver to produce ketone bodies but not to utilize them. This ensures that extra hepatic tissues have access to ketone bodies as a fuel source during prolonged fasting and starvation, and also, lack of this enzyme in the liver prevents the futile cycle of synthesis and breakdown of acetoacetate.

b) The Acetoacetyl CoA is now cleaved by thiolase to produce two acetyl CoA molecules (figure-1-step-3).

This implies that the TCA cycle must be running to allow ketone body utilization; a fact  which  is  necessarily true,  because  the TCA cycle  is  necessary to allow generation of energy from acetyl-Co A.

If the blood level is raised, oxidation of ketone bodies increases until, at a concentration of approximately 12 mmol/L, they saturate the oxidative machinery. When this occurs, a large proportion of the oxygen consumption may be accounted for by the oxidation of ketone bodies.

Utilization of ketone bodies 

Figure-1- Utilization of ketone bodies, Acetoacetate can  also be converted to Acetoacetyl co A by direct attachment of Co A  from Co ASH, same as  in activation of fatty acid, but this is a minor pathway .The major pathway proceeds through transfer of  CoA from succinyl co A.

Regulation of ketosis

Ketogenesis is regulated at three steps (Figure-2)

1) Lipolysis in Adipose tissue

  • Ketosis does not occur unless there is an increase in the level of circulating free fatty acids that arise from lipolysis of triacylglycerol in adipose tissue.
  • When glucose levels fall, lipolysis induced by glucagon secretion causes increased hepatic ketogenesis due to increased substrate (free fatty acids) delivery from adipose tissue.
  • Conversely, insulin, released in the well-fed state, inhibits ketogenesis via the triggering dephosphorylation and inactivation of adipose tissue hormone sensitive lipase (HSL).

2) Fate of fatty acid-free fatty acids are either oxidized to CO2 or ketone bodies or esterified to triacylglycerol and phospholipids. 

  •  There is regulation of entry of fatty acids into the oxidative pathway by carnitine Acyl transferase-I (CAT-I)
  •  Malonyl-CoA, the initial intermediate in fatty acid biosynthesis formed by acetyl-CoA carboxylase in the fed state, is a potent inhibitor of CAT-I.
  •  Under these conditions, free fatty acids enter the liver cell in low concentrations and are nearly all esterified to Acylglycerols and transported out of the liver in very low density lipoproteins (VLDL).

3) Fate of Acetyl co A

  • The acetyl-CoA formed in beta-oxidation is oxidized in the citric acid cycle, or it enters the pathway of ketogenesis to form ketone bodies.
  • As the level of serum free fatty acids is raised, proportionately more free fatty acids are converted to ketone bodies and less are oxidized via the citric acid cycle to CO2.
  • Entry of acetyl CoA into the citric acid cycle depends on the availability of Oxaloacetate for the formation of citrate, but the concentration of Oxaloacetate is lowered if carbohydrate is unavailable or improperly utilized. 

 Regulation of ketosis

Figure-2- Regulation of ketosis takes place at 3 steps. 1) Lipolysis in adipose tissue, 2) Entry of free fatty acid in to the mitochondrion 3) Entry of acetyl co A in to TCA cycle

Clinical significance

Ketonemia – increased concentration of ketone bodies in blood

  • It is due to increased production of ketone bodies by the liver rather than to a deficiency in their utilization by extra hepatic tissues.
  • The production of ketone bodies occurs at a relatively low rate during normal feeding and under conditions of normal physiological status. 
  • Normal physiological responses to carbohydrate shortages cause the liver to increase the production of ketone bodies from the acetyl-CoA generated from fatty acid oxidation.
  • Ketonemia progresses to ketonuria (excessive excretion of ketone bodies in urine), that can be detected by Nitroprusside test.

Ketoacidosis– Both β-hydroxybutyrate and acetoacetate are organic acids. These compounds are released in the protonated form, which means that their release tends to lower the pH of the blood. In normal individuals, other mechanisms compensate for the increased proton release, but their excessive production leads to lowering of pH causing ketoacidosis.

a) Diabetic ketoacidosis

DKA results from relative or absolute insulin deficiency combined with counter regulatory hormone excess (glucagon, catecholamines, cortisol, and growth hormone).

The decreased ratio of insulin to glucagon promotes gluconeogenesis, glycogenolysis, and ketone body formation in the liver, as well as increases in substrate delivery from fat and muscle (free fatty acids, amino acids) to the liver

In most cases, the increase in ketone body concentration in blood is due to increased synthesis in liver; in severe ketoacidosis, cells begin to lose ability to use ketone bodies also.

b) Starvation induced ketosis

Prolonged fasting may result

  • From an inability to obtain food
  • From the desire to lose weight rapidly, or
  • In clinical situations in which an individual cannot eat because of trauma, surgery, neoplasms, burns etc.

Biochemical basis of starvation induced ketogenesis

  • In the absence of food the plasma levels of glucose, amino acids and triacylglycerols fall, triggering a decline in insulin secretion and an increase in glucagon release. The decreased insulin to glucagon ratio makes this period of nutritional deprivation a catabolic state, characterized by degradation of glycogen, triacylglycerol and protein (Figure-3)
  • This sets in to motion an exchange of substrates between liver, adipose tissue, muscle and brain that is guided by two priorities-

 (i) the need to maintain glucose level to sustain the energy metabolism of brain ,red blood cells and other glucose requiring cells and

(ii) to supply energy to other tissues by mobilizing fatty acids from adipose tissues and converting them to ketone bodies to supply energy to other cells of the body.

  • In early stages of starvation, heart and skeletal muscle consume primarily ketone bodies to preserve glucose for use by the brain. 
  •  After several weeks of starvation, ketone bodies become the major fuel of the brain. 

 starvation induced ketosis

Figure-3- During starvation, glucagon induced adipolysis increases the flow of free fatty acids to liver for oxidation. Excess of Acetyl co A are channeled towards the pathway of ketogenesis. Acetoacetate and beta hydroxy butyrate are poured in blood to be transported to peripheral cells for utilization. Liver cells lack the enzyme for their utilization.

Conditions causing ketosis

  • Uncontrolled diabetes mellitus
  • Starvation
  • Chronic alcoholism
  • Von- Gierke’s disease
  • Heavy exercise
  • Low carbohydrate diet- For weight loss
  • Glycogen storage disease type 6 (due to phosphorylase kinase deficiency)
  • Pyruvate carboxylase deficiency
  • Prolonged ether anesthesia
  • Toxemia of pregnancy
  • Certain conditions of alkalosis 
  • Nonpathologic forms of ketosis are found under conditions of high-fat feeding
  • After severe exercise in the post absorptive state.

 

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Acetoacetate, D(-3) -hydroxybutyrate (Beta hydroxy butyrate), and acetone are often referred to as ketone bodies (figure-1).

 Ketone bodies

Figure-1- Acetoacetate is the primary ketone body , the other ketone bodies are derived from it.

The term “ketones” is a misnomer because 3-hydroxybutyrate is not a ketone and there are ketones in blood that are not ketone bodies, eg, pyruvate, fructose.

Ketogenesis

Ketogenesis takes place in liver using Acetyl co A as a substrate or a precursor molecule. Enzymes responsible for ketone body formation are associated mainly with the mitochondria.

Steps of synthesis– Acetoacetate (First ketone body) is formed from Acetyl CoA in three steps (Figure-2 ).

1) Two molecules of acetyl CoA condense to form acetoacetyl CoA .This reaction, which is catalyzed by thiolase, is the reverse of the thiolysis step in the oxidation of fatty acids.

2) Acetoacetyl CoA then reacts with acetyl CoA and water to give 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) and CoA .The reaction is catalyzed by HMG co A synthase .This enzyme is exclusively present in liver mitochondria. There are two isoforms of this enzyme-cytosolic and mitochondrial. The mitochondrial enzyme is needed for ketogenesis while the cytosolic form is associated with cholesterol biosynthesis.

3) 3-Hydroxy-3-methylglutaryl CoA is then cleaved to acetyl CoA and acetoacetate in the presence of HMG Co A  lyase (Figure-2)

The carbon atoms split off in the acetyl-CoA molecule are derived from the original acetoacetyl-CoA molecule. Both enzymes must be present in mitochondria for ketogenesis to take place. This occurs solely in liver and rumen epithelium.

The other two ketone bodies-Acetone and D(-)- 3-Hydroxybutyrate are formed from Acetoacetate, the primary ketone body.

4) Acetone is formed by decarboxylation in the presence of decarboxylase enzyme and, because it is a beta-keto acid, acetoacetate also undergoes a slow, spontaneous decarboxylation to acetone (figure-2). The odor of acetone may be detected in the breath of a person who has a high level of acetoacetate in the blood.  “Acetone-breath” has been used as  a  crude  method  of  diagnosing  individuals  with  untreated Type  I  diabetes mellitus.

 5) D(-)-3-Hydroxybutyrate is formed by the reduction of acetoacetate in the mitochondrial matrix by D(-)3-hydroxybutyrate dehydrogenase (figure-2).

  • D(-)-3-Hydroxybutyrate is quantitatively the predominant ketone body present in the blood and urine in ketosis.
  • The β-hydroxybutyrate dehydrogenase reaction has two functions: 1) it stores energy equivalent to an NADH in the ketone body  for  export  to the tissues, and  2)  it  produces  a  more  stable molecule. Acetoacetate and β-hydroxybutyrate, in particular, also serve as major substrates for the biosynthesis of neonatal cerebral lipids.
  • The ratio of β hydroxybutyrate to acetoacetate depends on the NADH/NAD+ ratio inside mitochondria , if NADH concentration is high, the liver releases a higher proportion of β-hydroxybutyrate.

In vivo, the liver appears to be the only organ in nonruminants to add significant quantities of ketone bodies to the blood. Extrahepatic tissues utilize them as respiratory substrates .The net flow of ketone bodies from the liver to the extrahepatic tissues results from active hepatic synthesis coupled with very low utilization.The reverse situation occurs in extra hepatic tissues.

While an active enzymatic mechanism produces acetoacetate from acetoacetyl-CoA in the liver, acetoacetate once formed cannot be reactivated directly except in the cytosol, where it is used in a much less active pathway as a precursor in cholesterol synthesis. This accounts for the net production of ketone bodies by the liver.

 Steps of ketogenesis

Figure-2- Steps of ketogenesis

Biological Significance

Ketone bodies serve as a fuel for extra hepatic tissues

The  brain  is  an  important organ. It  is  metabolically  active  and  metabolically privileged. The brain generally uses 60-70% of total body glucose requirements, and always  requires  some  glucose  for  normal  functioning. Under  most  conditions, glucose is essentially the sole energy source of the brain. The brain cannot use fatty acids, which  cannot  cross  the  blood-brain  barrier.  Because  animals  cannot synthesize significant amounts of glucose from fatty acids, as glucose availability decreases, the brain is forced to use either amino acids or ketone bodies for fuel.

Individuals eating diets extremely high in fat and low in carbohydrates, or starving, or  suffering  from  a severe  lack  of  insulin  (Type I  diabetes  mellitus) therefore increase the synthesis and utilization of ketone bodies

During high rates of fatty acid oxidation, primarily in the liver, large amounts of acetyl-Co A are generated.These exceed the capacity of the TCA cycle, and one result is the synthesis of ketone bodies.The synthesis of the ketone bodies (ketogenesis) occurs in the liver mitochondria allowing this process to be intimately coupled to rate of hepatic fatty acid oxidation.Conversely, the utilization of the ketones (ketolysis) occurs in the peripheral cells, in the cytosol.

The acetyl CoA formed in fatty acid oxidation enters the citric acid cycle only if fat and carbohydrate degradation are appropriately balanced. The reason is that the entry of acetyl CoA into the citric acid cycle depends on the availability of oxaloacetate for the formation of citrate, but the concentration of oxaloacetate is lowered if carbohydrate is unavailable or improperly utilized. Oxaloacetate is normally formed from pyruvate, the product of glycolysis, by pyruvate carboxylase (Figure-3). This is the molecular basis of the adage that fats burn in the flame of carbohydrates.

 Conditions causing ketosis

Figure-3- the pathway of ketogenesis in conditions of non availability of Oxaloacetate

In fasting or diabetes, oxaloacetate is consumed to form glucose by the gluconeogenic pathway (figure-3) and hence is unavailable for condensation with acetyl CoA. Under these conditions, acetyl CoA is diverted to the formation of acetoacetate and β-hydroxybutyrate.

These substances diffuse from the liver mitochondria into the blood and are transported to peripheral tissues. These ketone bodies were initially regarded as degradation products of little physiological value. However, Acetoacetate and β-hydroxybutyrate are normal fuels of respiration and are quantitatively important as sources of energy. Indeed, heart muscle and the renal cortex use acetoacetate in preference to glucose. In contrast, the brain adapts to the utilization of acetoacetate during starvation and diabetes. In prolonged starvation, 75% of the fuel needs of the brain are met by ketone bodies.

To be continued …

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The β oxidation accounts for the bulk of the energy production from fatty acids in human. These reactions must be supplemented by other mechanisms, so that all types of ingested fatty acids can be oxidized.

 Over view of minor pathways of biological importance of fatty acid oxidation

1) α- Oxidation- Oxidation occurs at C-2 instead of C-3, as in β oxidation

2) ω- Oxidation – Oxidation occurs at the methyl end of the fatty acid molecule.

3) Peroxisomal fatty acid oxidation- Occurs for the chain shortening of very long chain fatty acids.

Details of α- Oxidation

  • Takes place in the microsomes of brain and liver,
  •  Involves decarboxylation process for the removal of single carbon atom at one time
  • An odd chain fatty acid is produced by decarboxylation
  •  Subsequent process involves  beta oxidation for energy production
  • Strictly an aerobic process.
  • No prior activation of the fatty acid is required.
  • The process involves hydroxylation of the alpha carbon with a specific α-hydroxylase enzyme that requires Fe++ and vitamin C/FH4 as cofactors.

A) Biological significance of alpha oxidation

1) Oxidation of methylated fatty acids- Although the use of the α- Oxidation scheme is relatively less in terms of total energy production, but it is significant in the metabolism of dietary fatty acids that are methylated. A principal example of these is Phytanic acid (Figure-1) α- Oxidation is most suited for the oxidation of phytanic acid, produced from dietary phytol, a constituent of chlorophyll of plants. Phytanic acid is a significant constituent of milk lipids and animal fats and normally it is metabolized by an initial α- hydroxylation followed by dehydrogenation and decarboxylation. Beta oxidation can not occur initially because of the presence of 3- methyl groups, but it can proceed after decarboxylation. The whole reaction produces three molecules of propionyl co A, three molecules of Acetyl co A, and one molecule of iso butyryl co A (Figure-2).

 Phytanic acid

 Figure-1-Phytanic acid -3, 7, 11, 15-tetramethylhexadecanoic acid

 Phytanic acid oxidation

Figure-2- Phytanic acid is oxidized by Phytanic acid α oxidase to yield CO2 and odd chain fatty acid Pristanic acid that can be subsequently oxidised by beta oxidation. This process involves hydroxylation of the alpha carbon, removal of the terminal carboxyl group and concomitant conversion of the alpha hydroxyl group to a terminal carboxyl group, and linkage of CoA to the terminal carboxyl group. This branched substrate will function in the beta-oxidation process, ultimately yielding propionyl-CoA, acetyl Co As and, in the case of phytanic acid, 2-methyl propionyl CoA (Iso butyryl Co A)

2) The hydroxy fatty acids produced as intermediates of this pathway like Cerebronic acid can be used for the synthesis of cerebrosides and sulfatides

3) Odd chain fatty acid produced upon decarboxylation in this pathway, can be used for the synthesis of sphingolipids and also undergo beta oxidation  to form propionyl co A and Acetyl co A .The number of acetyl co A depend upon the chain length. Propionyl co A is converted to Succinyl co A to gain entry in to TCA cycle for further oxidation.

B) Clinical significance of alpha oxidation of fatty acids

Refsum disease (RD)

Refsum disease (RD) is a neurocutaneous syndrome that is characterized biochemically by the accumulation of phytanic acid in plasma and tissues. Refsum first described this disease.

Biochemical defect

Refsum disease is an autosomal recessive disorder. Patients with Refsum disease are unable to degrade phytanic acid because of a deficient activity of Phytanic acid oxidase enzyme catalyzing the first step of phytanic acid alpha-oxidation (Figure-2)

Consequently, this unusual, exogenous C20-branched-chain (3, 7, 11, 15-tetramethylhexadecanoic acid) fatty acid accumulates in brain, blood and other tissues. It is almost exclusively of exogenous origin and is delivered mainly from dietary plant chlorophyll and, to a lesser extent, from animal sources. Blood levels of phytanic acid are increased in patients with Refsum disease. These levels are 10-50 mg/dL, whereas normal values are less than or equal to 0.2 mg/dL, and account for 5-30% of serum lipids. Phytanic acid replaces other fatty acids, including such essential ones as Linoleic and Arachidonic acids, in lipid moieties of various tissues. This situation leads to an essential fatty acid deficiency, which is associated with the development of ichthyosis.

Refsum disease is rare, with just 60 cases observed so far.

Clinical manifestations

Classic Refsum disease manifests in children aged 2-7 years; however, diagnosis usually is delayed until early adulthood. Infantile Refsum disease makes its appearance in early infancy. Peripheral polyneuropathy, cerebellar ataxia, retinitis pigmentosa, and Ichthyosis (rough, dry and scaly skin) are the major clinical components. The symptoms evolve slowly and insidiously from childhood through adolescence and early adulthood.

The disease is characterized by

  • Night blindness due to degeneration of the retina (retinitis pigmentosum)
  • Loss of the sense of smell (anosmia)
  • Deafness
  • Concentric constriction of the visual fields
  • Cataract
  • Signs resulting from cerebellar ataxia –Cardiac arrhythmias
    • Progressive weakness
    • Foot drop
    • Loss of balance
  • Some individuals will have shortened bones in their fingers or toes.
  • The children usually have moderately dysmorphic features that may include epicanthal folds, a flat bridge of the nose, and low-set ears.

Laboratory Diagnosis

  • Levels of plasma cholesterol and high- and low-density lipoprotein are often moderately reduced.
  • Blood phytanic acid levels are elevated.
  • Cerebrospinal fluid (CSF) shows a protein level of 100-600 mg/dL.
  • Routine laboratory investigations of blood and urine do not reveal any consistent diagnostic abnormalities.
  • Phytanic oxidase activity estimation in skin fibroblast cultures is important

Imaging

Skeletal radiography is required to estimate bone changes.

Treatment

  • Eliminate all sources of chlorophyll from the diet.
  • Plasmapheresis – Patients may also require plasma exchange (Plasmapheresis) in which blood is drawn, filtered, and reinfused back into the body, to control the buildup of phytanic acid.
  • The major dietary exclusions are green vegetables (source of phytanic acid) and animal fat (phytol).
  • The aim of such dietary treatment is to reduce daily intake of phytanic acid from the usual level of 50 mg/d to less than 5 mg/d.

Prognosis – in untreated patients generally is poor.

2) Omega oxidation of fatty acids

  • Another minor pathway for the fatty acid oxidation
  • Involves hydroxylation  of omega carbon (the methyl carbon at the other end of the molecule from the carboxyl group or on the carbon next to the methyl end).
  • Occurs in the endoplasmic reticulum of many tissues.
  • Hydroxylation involves the “mixed function oxidase” type of reaction requiring cytochrome P450, O2 and NADPH, as well as the necessary enzymes (Figure-3)

Omega oxidation -1

 

Figure-3- The methyl end of the fatty acid is hydroxylated by a specialized hydroxylase, called Mixed function oxidase, that uses molecular oxygen and NADPH for hydroxylation.

  • Hydroxy fatty acids can be further oxidised to a dicarboxylic acid via sequential reactions of Alcohol dehydrogenase and aldehyde dehydrogenases (Figure-4 and 5)

 Omega oxidation-2

Figure-4- Omega hydroxy fatty acid is oxidized by alcohol dehydrogenase to form Omega aldo acid.

  • The process occurs primarily for medium chain fatty acids.

Omega oxidation-3

Figure-5- Omega aldo acid is further oxidized by aldehyde dehydrogenase to form Dicarboxylic acid

  • The dicarboxylic acids so formed can be activated at either end of molecule to form a Co A ester, which can undergo beta oxidation to produce shorter chain dicarboxylic acids such as Adipic acids(C6) and succinic acid (C4).

Clinical significance of Omega oxidation

The microsomal (endoplasmic reticulum, ER) pathway of fatty acid ω-oxidation represents a minor pathway of overall fatty acid oxidation.

However, in certain pathophysiological states, such as diabetes, chronic alcohol consumption, and starvation, the ω-oxidation pathway may provide an effective means for the elimination of toxic levels of free fatty acids. 

3) Peroxisomal oxidation of very long chain fatty acids

  • Although most fatty acid oxidation takes place in mitochondria, some oxidation takes place in cellular organelles called peroxisomes.
  •  Peroxisomes are a class of sub cellular organelles with distinctive morphological and chemical characteristics.
  • These organelles are characterized by high concentrations of the enzyme catalase, which catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen
  • Fatty acid oxidation in these organelles, which halts at octanoyl CoA, may serve to shorten long chains to make them better substrates of beta-oxidation in mitochondria.
  • Peroxisomal oxidation differs from beta oxidation in the initial dehydrogenation reaction (Figure-6).

 Peroxisomal oxidation

 

Figure-6- The first reaction is catalyzed by acyl co A dehydrogenase same as Beta oxidation but the reduced FADH2 is not oxidized in the electron transport chain instead it is oxidized  using direct molecular oxygen to form H2O2 which is subsequently decomposed by catalase to form water and oxygen.

  • In peroxisomes, a flavoprotein dehydrogenase transfers electrons to O2 to yield H2O2 instead of capturing the high-energy electrons as FADH2, as occurs in mitochondrial beta oxidation.
  • Catalase is needed to convert the hydrogen peroxide produced in the initial reaction into water and oxygen.
  • Subsequent steps are identical with their mitochondrial counterparts, although they are carried out by different isoforms of the enzymes.
  • The specificity of the peroxisomal enzymes is for somewhat longer chain fatty acids. Thus peroxisomal enzymes function to shorten the chain length of relatively long chain fatty acids to a point at which beta oxidation can be completed in mitochondria.
  • Other peroxisomal reactions include chain shortening of dicarboxylic acids, conversion of cholesterol to bile acids and formation of ether lipids.
  • It has been suggested that peroxisomes may function in a protective role against oxygen toxicity.
  • Several lines of evidence suggest that they are also involved in the lipid catabolism. A number of drugs used clinically to decrease triglyceride levels in patients cause a marked increase in peroxisomes.

Clinical significance

Zellweger syndrome

Zellweger syndrome, also called cerebrohepatorenal syndrome is a rare, congenital disorder (present at birth), characterized by the reduction or absence of Peroxisomes in the cells of the liver, kidneys, and brain.

Biochemical defect

Zellweger syndrome is one of a group of four related diseases called peroxisome biogenesis disorders (PBD), which are part of a larger group of diseases known as the leukodystrophies.  It is characterized by an individual’s inability to beta-oxidize very-long chain fatty acids in the Peroxisomes of the cell, due to a genetic disorder in one of the several genes involved with peroxisome biogenesis.

Clinical Manifestations

The most common features of Zellweger syndrome include-

  • Enlarged liver
  • High levels of iron and copper in the blood stream, and
  • Vision disturbances.
  • Some affected infants may show prenatal growth failure.
  • Symptoms at birth may include a lack of muscle tone, an inability to move and glaucoma.
  • Other symptoms may include unusual facial characteristics, mental retardation, seizures, and an inability to suck and/or swallow.
  • Jaundice and gastrointestinal bleeding may also occur.
  • More than 90% show postnatal growth failure.

Laboratory diagnosis

There are several noninvasive laboratory tests that permit precise and early diagnosis of peroxisomal disorders.  The abnormally high levels of VLCFA ( Very long chain fatty acids ), are most diagnostic .

Treatment

 There is no cure for Zellweger syndrome, nor is there a standard course of treatment.  Since the metabolic and neurological abnormalities that cause the symptoms of Zellweger syndrome are caused during fetal development, treatments to correct these abnormalities after birth are limited. Most treatments are symptomatic and supportive.

Prognosis

The prognosis for infants with Zellweger syndrome is poor.  Most infants do not survive past the first 6 months, and usually succumb to respiratory distress, gastrointestinal bleeding, or liver failure.

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Beta oxidation of odd chain fatty acids

Fatty acids with an odd number of carbon atoms are oxidized by the pathway of β-oxidation, producing acetyl-CoA, until a three-carbon (propionyl-CoA) residue remains.

Fate of propionyl co A

This compound is converted to Succinyl-CoA, a constituent of the citric acid cycle (figure-1).

 Fate of propionyl co A

Figure-1- Propionyl co A is carboxylated to produce D-methyl malonyl co A, that is converted to its L-isomer by Racemase enzyme. L- Methyl malonyl co A is finally converted to Succinyl co A , an intermediate of TCA cycle.

Biological Significance

The propionyl residue from an odd-chain fatty acid is the only part of a fatty acid that is glucogenic. Acetyl CoA cannot be converted into pyruvate or Oxaloacetate in animals.

Clinical significance

Vitamin B12 deficiency leads to impaired conversion of L-methyl malonyl co A to Succinyl co A, due to reduced activity of the enzyme, causing methyl malonic aciduria.

Beta oxidation of unsaturated fatty acids

  • In the oxidation of unsaturated fatty acids, most of the reactions are the same as those for saturated fatty acids, only two additional enzymes an isomerase and a reductase are needed to degrade a wide range of unsaturated fatty acids.
  •  Energy yield is less by the oxidation of unsaturated fatty acids since they are less reduced.
  •  Per double bonds 2 ATP are less formed, since the first step of dehydrogenation to introduce double bond is not required, as the double already exists.

a) Beta oxidation of mono unsaturated fatty acids

  • Palmitoleoyl Co A undergoes three cycles of degradation, which are carried out by the same enzymes as in the oxidation of saturated fatty acids (Figure-2)
  • The cis Δ 3-enoyl CoA formed in the third round is not a substrate for acyl CoA dehydrogenase.
  •  An isomerase converts this double bond into a trans- Δ 2 double bond.
  • The subsequent reactions are those of the saturated fatty acid oxidation pathway, in which the trans- Δ 2-enoyl CoA is a regular substrate.

 Beta oxidation of Palmitoleic acid

Figure-2- Beta oxidation of Palmitoleic acid (Mono unsaturated fatty acid)

b) Beta oxidation of polyunsaturated fatty acids (figure-3)

  • A different set of enzymes is required for the oxidation of Linoleic acid, a C18 polyunsaturated fatty acid with cis-Δ 9 and cis-Δ12 double bonds  (figure-3) .
  • The cis- Δ 3 double bond formed after three rounds of  β oxidation is converted into a trans- Δ 2 double bond by isomerase.
  • The acyl CoA produced by another round of β oxidation contains a cis- Δ 4 double bond. Dehydrogenation of this species by acyl CoA dehydrogenase yields a 2,4-dienoyl intermediate, which is not a substrate for the next enzyme in the β -oxidation pathway.
  • This impasse is circumvented by 2,4-dienoyl CoA reductase, an enzyme that uses NADPH to reduce the 2,4-dienoyl intermediate to trans-D 3-enoyl CoA. 
  •  cis-Δ 3-Enoyl CoA isomerase then converts trans– Δ 3-enoyl CoA into the trans- Δ 2 form, a customary intermediate in the beta-oxidation pathway.

 

 Beta oxidation of Linoleic acid

Figure-3 -Beta oxidation of Linoleic acid (Dienoic acid- containing two double bonds)

Regulation of fatty acid oxidation(figure-4)

  • There is regulation at the level of entry of fatty acids into the oxidative pathway by carnitine palmitoyl transferase-I (CPT-I), CPT-I activity is low in the fed state, leading to depression of fatty acid oxidation, and high in starvation, allowing fatty acid oxidation to increase.
  • Malonyl-CoA, the initial intermediate in fatty acid biosynthesis (Figure-4), formed by acetyl-CoA carboxylase in the fed state, is a potent inhibitor of CPT-I. Under these conditions, free fatty acids enter the liver cell in low concentrations and are nearly all esterified to acylglycerols and transported out of the liver in very low density lipoproteins (VLDL).
  • However, as the concentration of free fatty acids increases with the onset of starvation, acetyl-CoA carboxylase is inhibited directly by acyl-CoA, and [malonyl-CoA] decreases, releasing the inhibition of CPT-I and allowing more acyl-CoA to be -oxidized.
  • These events are reinforced in starvation by decrease in the [insulin]/[glucagon] ratio.
  • Thus, β -oxidation from free fatty acids is controlled by the CPT-I gateway into the mitochondria, and the balance of the free fatty acid uptake not oxidized is esterified.

 Regulation of fatty acid oxidation

Figure-4- CPT-1 (Carnitine palmitoyl Transferase -1) is inhibited by malonyl co A, the product of first step of fatty acid synthesis. Active fatty acid synthesis takes place in the well fed state under the effect of insulin, thus when fatty acid synthesis is active, fatty acid oxidation is inhibited. Both the processes do not occur simultaneously.

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Steps of beta oxidation

Step-1 Dehydrogenation-The first step is the removal of two hydrogen atoms from the 2(α)- and 3(β)-carbon atoms, catalyzed by acyl-CoA dehydrogenase and requiring FAD. This results in the formation of Δ2-trans-enoyl-CoA and FADH2.

 Acyl co A dehydrogenase

Figure-1- Acyl co A is dehydrogenated to form α-β Unsaturated Acyl Co A (Δ2trans- Enoyl Co A)

  • Electrons from the FADH2 prosthetic group of the reduced acyl CoA dehydrogenase are transferred to electron-transferring flavoprotein (ETF).
  • ETF donates electrons to ETF: ubiquinone reductase, an iron-sulfur protein of the electron transport chain, Consequently,  2 (1.5) molecules of ATP are generated per molecule of FADH2 formed in this dehydrogenation step.

 Biological Significance

There are chain specific acyl co A dehydrogenases- Short chain, medium and long chain acyl co A dehydrogenases.

Clinical Significance

1) Jamaican Sickness- Jamaican vomiting sickness is caused by eating the unripe fruit of akee tree, which contains the toxin hypoglycin, that inactivates medium and short-chain acyl-CoA dehydrogenases, inhibiting β oxidation and thereby causing hypoglycemia.

2) Nonketotic hypoglycemia which is caused by lack of mitochondrial medium chain acyl-CoA dehydrogenases. Impaired fatty acid oxidation results in energy imbalance producing hypoglycemia.

3) Dicarboxylic aciduria is characterized by excretion of C6–C10 -dicarboxylic acids and by nonketotic hypoglycemia, and is caused by a lack of mitochondrial medium-chain acyl-CoA dehydrogenase.

4) Acute fatty liver of pregnancy

  • Manifests in the second half of pregnancy, usually close to term, but may also develop in the postpartum period.
  • The patient developed symptoms of hepatic dysfunction at 36 weeks of gestation.
  • Short history of illness, hypoglycemia, liver failure, renal failure, and coagulopathy are observed.
  • Diagnosis is made based on an incidental finding of abnormal liver enzyme levels.
  • Affected patients may become jaundiced or develop  encephalopathy from liver failure, usually reflected by an elevated ammonia level.
  • Profound hypoglycemia is common.

 Step-2- Hydration

Water is added to saturate the double bond and form 3-hydroxyacyl-CoA, catalyzed by Δ 2-enoyl-CoA hydratase (figure-2).

 Hydratase

Figure-2- Reaction  catalyzed by hydratase enzyme

 Step-3- dehydrogenation-

The 3-hydroxy derivative undergoes further dehydrogenation on the 3-carbon catalyzed by L(+)-3-hydroxyacyl-CoA dehydrogenase to form the corresponding 3-ketoacyl-CoA compound. In this case, NAD+ is the coenzyme involved (Figure-3).

 

Beta hydroxy acyl co A dehydrogenase

Figure-3- Reaction catalyzed by Beta hydroxy acyl co A dehydrogenase

Biological Significance– NADH and H+ thus produced enter the electron transport chain through Complex-I to yield  3 ATP molecules.

Step-4- Thiolysis-

3-ketoacyl-CoA is split at the 2,3- position by thiolase (3-ketoacyl-CoA-thiolase), forming acetyl-CoA and a new acyl-CoA two carbons shorter than the original acyl-CoA molecule (figure-4). The acyl-CoA formed in the cleavage reaction reenters the oxidative pathway at reaction 2.

 Since acetyl-CoA can be oxidized to CO2 and water via the citric acid cycle the complete oxidation of fatty acids is achieved

 Thiolase

Figure-4- Reaction catalyzed by Thiolase enzyme

Summary (Figure-5)

Fatty acid oxidation

 

Figure-5- Long-chain acyl-CoA is cycled through reactions 2–5, each time  acetyl-is  split off  through each cycle, by thiolase (reaction 5). When the acyl radical is only four carbon atoms in length, two acetyl-CoA molecules are formed in reaction 5.

The overall reaction can be represented as follows-

 Over all reaction of beta oxidation

Energy yield by the complete oxidation of one mol of Palmitic acid-

The degradation of palmitoyl CoA (C16-acyl Co A) requires seven reaction cycles. In the seventh cycle, the C4-ketoacyl CoA is thiolyzed to two molecules of acetyl CoA.

Energetics of palmitic acid 

106 (129 As per old concept) ATP are produced by the complete oxidation of one mol of Palmitic acid.

Details

Per cycle energy output

1NADH +1FADH2 = 3 + 2 = 5 ATP

In 7 cycles of beta oxidation

7×5 = 35

Energy output from complete oxidation of Acetyl co A

Per Acetyl co A = 12 ATP

8 Acetyl co A    = 8X12 = 96

Total output = 35 +96 = 131

Energy consumed during activation of palmitate to Palmitoyl CoA

2 ATP equivalents         (ATP ————> AMP + PPi)

                                          (PPi ————->  2 Pi)

Net Energy output- 131-2 = 129 ATP

                                     131-2 = 129  

 To be continued …….

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A fatty acid contains a long hydrocarbon chain and a terminal carboxylate group. The hydrocarbon chain may be saturated (with no double bond) or may be unsaturated (containing double bond).

 Types of fatty acids

Fatty acids can be obtained from-

  • Diet
  • Adipolysis
  • De novo synthesis

Functions of fatty acids – Fatty acids have four major physiological roles.

1) Fatty acids are building blocks of phospholipids and glycolipids.

2) Many proteins are modified by the covalent attachment of fatty acids, which target them to membrane locations

3) Fatty acids are fuel molecules. They are stored as triacylglycerols. Fatty acids mobilized from triacylglycerols are oxidized to meet the energy needs of a cell or organism.

4) Fatty acid derivatives serve as hormones and intracellular messengers e.g. steroids, sex hormones and prostaglandins.

Triglycerides

  • Triglycerides are the  highly concentrated stores of energy because they are reduced and anhydrous.
  • —The yield from the complete oxidation of fatty acids is about 9 kcal g-1 (38 kJ g-1)
  • Triacylglycerols are nonpolar, and are stored in a nearly anhydrous form, whereas much more polar proteins and carbohydrates are more highly hydrated.

Triglycerides V/S Glycogen

  • — A gram of nearly anhydrous fat stores more than six times as much energy as a gram of hydrated glycogen, which is likely the reason that triacylglycerols rather than glycogen were selected in evolution as the major energy reservoir.
  • The glycogen and glucose stores provide enough energy to sustain biological function for about 24 hours, whereas the Triacylglycerol stores allow survival for several weeks.

Provision of dietary fatty acids

Most lipids are ingested in the form of triacylglycerols, that must be degraded to fatty acids for absorption across the intestinal epithelium.

 dietary fattya cids

Figure-1- Free fatty acids and monoacylglycerols obtained by digestion of dietary triglycerides are absorbed by intestinal epithelial cells. Triacylglycerols are resynthesized and packaged with other lipids and apoprotein B-48 to form chylomicrons, which are then released into the lymph system.

Provision of fatty acids from adipose tissue

The triacylglycerols are degraded to fatty acids and glycerol by hormone sensitive lipase. The released fatty are transported to the energy-requiring tissues.

 

 Adipolysis

Figure-2- The enzyme hormone sensitive lipase (triacylglycerol lipase)is activated by glucagon and catecholamines through cAMP mediated cascade. This enzyme is inhibited by methyl xanthines such as caffeine and theophylline. Insulin antagonizes the effect of the lipolytic hormones.

Transportation of free fatty acids

  • Free fatty acids—also called unesterified (UFA) or nonesterified (NEFA) fatty acids—are fatty acids that are in the unesterified state.
  • In plasma, longer-chain FFA are combined with albumin, and in the cell they are attached to a fatty acid-binding protein.
  • Shorter-chain fatty acids are more water-soluble and exist as the un-ionized acid or as a fatty acid anion.
  • By these means, free fatty acids are made accessible as a fuel in other tissues.

Types of fatty acid oxidation

Fatty acids can be oxidized by-

1) Beta oxidation- Major mechanism, occurs in the mitochondria  matrix. 2-C units are released as acetyl CoA per cycle.

2) Alpha oxidation- Predominantly takes place in brain and liver, one carbon is lost in the form of CO2 per cycle.

3) Omega oxidation- Minor mechanism, but becomes important in conditions of impaired beta oxidation

4) Peroxisomal oxidation- Mainly for the trimming of very long chain fatty acids.

Beta oxidation

A saturated acyl Co A is degraded by a recurring sequence of four reactions:

1) Oxidation by flavin adenine dinucleotide (FAD)

2) Hydration,

3) Oxidation by NAD+, and

4) Thiolysis by Co A

The fatty acyl chain is shortened by two carbon atoms as a result of these reactions, and FADH2, NADH, and acetyl Co A are generated. Because oxidation is on the β carbon and the chain is broken between the α (2)- and β (3)-carbon atoms—hence the name – β oxidation.

Activation of fatty acid

Fatty acids must first be converted to an active intermediate before they can be catabolized. This is the only step in the complete degradation of a fatty acid that requires energy from ATP.  The activation of a fatty acid is accomplished in two steps-

 Activation of fatty acid

Figure-3- In the presence of ATP and coenzyme A, the enzyme acyl-CoA synthetase (thiokinase) catalyzes the conversion of a fatty acid (or free fatty acid) to an “active fatty acid” or acyl-CoA, which uses one high-energy phosphate with the formation of AMP and PPi .The PPi is hydrolyzed by inorganic pyrophosphatase with the loss of a further high-energy phosphate, ensuring that the overall reaction goes to completion. Acyl-CoA synthetases are found in the endoplasmic reticulum, peroxisomes, and inside and on the outer membrane of mitochondria.

Transport of fatty acid in to the mitochondrial matrix (Figure-4)

  • Fatty acids are activated on the outer mitochondrial membrane, whereas they are oxidized in the mitochondrial matrix.
  • Activated long-chain fatty acids are transported across the membrane by conjugating them to carnitine, a zwitterionic alcohol.

Carnitine (ß-hydroxy-Υ-trimethyl ammonium butyrate), (CH3)3N+—CH2—CH(OH)—CH2—COO, is widely distributed and is particularly abundant in muscle. Carnitine is obtained from foods, particularly animal-based foods, and via endogenous synthesis.

Role of carnitine

The acyl group is transferred to the hydroxyl group of carnitine to form acyl carnitine. This reaction is catalyzed by carnitine acyl transferase I

 Chemistry of carnitine

2) Acyl carnitine is then shuttled across the inner mitochondrial membrane by a translocase.

3) The acyl group is transferred back to CoA on the matrix side of the membrane. This reaction, which is catalyzed by carnitine acyl transferase II.

Finally, the translocase returns carnitine to the cytosolic side in exchange for an incoming acyl carnitine.

 

 Role of carnitine

Figure-4- Role of carnitine in the transport of long-chain fatty acids through the inner mitochondrial membrane. Long-chain acyl-CoA cannot pass through the inner mitochondrial membrane, but its metabolic product, acylcarnitine, can cross through with the help of carnitine shuttle.

Significance of fatty acid transport through carnitine shuttle

1) Biological significance- This counter-transport system provides regulation of the uptake of fatty acids into the mitochondrion for oxidation. As long as there is free CoA available in the mitochondrial matrix, fatty acids can be taken up and the carnitine returned to the outer membrane for uptake of more fatty acids. However, if most of the CoA in the mitochondrion is acylated, then the fatty acid uptake is inhibited.

This carnitine shuttle also serves to prevent uptake into the mitochondrion (and hence oxidation) of fatty acids synthesized in the cytosol in the fed state; malonyl CoA (the precursor for fatty acid synthesis) is a potent inhibitor of carnitine palmitoyl transferase I in the outer mitochondrial membrane.

Short and medium chain fatty acids do not require carnitine for their transportation across the inner mitochondrial membrane.

2) Clinical significance

Carnitine deficiency

Causes of carnitine deficiency include the following:

  • Inadequate intake (e.g., due to fad diets, lack of access, or long-term TPN)
  • Inability to use carnitine due to enzyme deficiencies (e.g., carnitine palmitoyl Transferase deficiency)
  • Decreased endogenous synthesis of carnitine due to a severe liver disorder
  • Excess loss of carnitine due to diarrhea, diuresis, or hemodialysis
  • A hereditary disorder in which carnitine leaks from renal tubules (Primary carnitine deficiency)
  • Increased requirements for carnitine when ketosis is present or demand for fat oxidation is high (eg, during a critical illness such as sepsis or major burns; after major surgery of the GI tract)

Clinical manifestations

Symptoms and the age at which symptoms appear depend on the cause.

Carnitine deficiency may cause-

  • Muscle aches and fatigue
  • Muscle necrosis, myoglobinuria
  • Fasting hypoketotic hypoglycemia 
  • Fatty liver and hyperammonemia,.
  • Cardiomyopathy, heart failure
  • Coma, and sudden unexpected death 

Hypoglycemia in carnitine deficiency, is a consequence of impaired fatty acid oxidation with the resultant imbalance between demand and supply of glucose which is the sole of source of energy in such individuals.

Deficiencies in the carnitine acyl Transferase enzymes I and II can cause similar symptoms. Inherited CAT-I deficiency affects only the liver, resulting in reduced fatty acid oxidation and ketogenesis, with hypoglycemia. CAT-II deficiency affects primarily skeletal muscle and, when severe, the liver.

Diagnosis

  • Extremely reduced carnitine levels in plasma and muscle (1–2% of normal).
  • Fasting ketogenesis may be normal  if liver carnitine transport is normal, but it may be impaired if dietary carnitine intake is interrupted and there is associated liver disorder.
  • The fasting urinary organic acid profile may show a hypoketotic dicarboxylicaciduria pattern if hepatic fatty acid oxidation is impaired, but it is otherwise unremarkable.

Treatment

  • Treatment of this disorder with pharmacologic doses of oral carnitine is highly effective in correcting the cardiomyopathy and muscle weakness as well as any impairment in fasting ketogenesis.
  • All patients must avoid fasting and strenuous exercise.
  • Some patients require supplementation with medium-chain triglycerides and essential fatty acids (eg, Linoleic acid, Linolenic acid).
  • Patients with a fatty acid oxidation disorder require a high-carbohydrate, low-fat diet.

To be continued……

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  • Glycerol, short  and medium chain fatty acids (chain length less than 14 carbons) are directly absorbed from the intestinal lumen in to the portal vein and taken to liver for further utilization.
  • Long chain fatty acids, free cholesterol  and β- acyl glycerol together with bile salts form mixed micelles.

Micelles

  • Micelles are disk-shaped clusters of amphipathic lipids that coalesce with their hydrophobic groups on the inside  and their hydrophilic groups on the outside of clusters (Figure-1).
  • Mixed micelles are soluble in the aqueous environment of the intestinal lumen
  • They are about 200 times smaller than emulsion droplets (4-7 nm versus 1µm for emulsion droplets).
  • Micelles are necessary because they transport the poorly soluble monoglycerides and fatty acids to the surface of the enterocyte where they can be absorbed.
  • The micelles approach the brush border membrane of the enterocytes .This membrane is  separated from the liquid contents of the intestinal lumen by an unstirred water layer that mixes poorly with the bulk fluid (Figure-2).
  • The hydrophilic surface of the micelles facilitate the transport of the hydrophobic lipids through the unstirred water layer to the brush border where they are absorbed.

 Micelle structure

Figure-1- Micelles are disk-shaped clusters of amphipathic lipids that coalesce with their hydrophobic groups on the inside and hydrophilic groups on the outside of the clusters.

  • Micelles constantly break  down and re-form
  • It is the monoglycerides and fatty acids that are free in solution that are absorbed, NOT the micelles.
  • Because of their nonpolar nature, monoglycerides and fatty acids can just diffuse across the plasma membrane of the enterocyte.
  • Some absorption may be facilitated by specific transport proteins

 Absorption of dietray fat

Figure-2- Absorption of lipids contained in mixed micelles by intestinal mucosal cell

 Cholesterol absorption

Some of the cholesterol in the small intestine is dietary cholesterol, and some is poured  there by the liver, arriving via the bile (Figure-3). Of the total cholesterol that passes through the small intestine, only half is typically absorbed, and the rest is eliminated in the feces. Thus, cholesterol in the bile is an example of a substance that is targeted for excretion via the digestive tract.

 Cholesterol absorption

Figure-3- Absorption and excretion of cholesterol

 Clinical Significance

  • The drug ezetimibe blocks a protein that specifically mediates cholesterol transport across the apical plasma membrane of enterocytes.
  • Ezetimibe has been shown to be effective at reducing levels of LDL cholesterol, particularly when combined with a statin, a drug that inhibits cholesterol synthesis in the liver.

 Lipid Malabsorption (Steatorrhea)

  • Lipid malabsorption results in increased lipids including fat soluble vitamins A,D E and K in the feces.
  • Cause may be pancreatic insufficiency, including cystic fibrosis , chronic diseases of pancreas or surgical removal of pancreas
  • Shortened bowel, Celiac diseases, sprue or crohn’s disease
  • May be bile duct obstruction due to gall stones, tumor of head of pancreas, enlarged lymph nodes etc.
  • Milk  and coconut oil are used therapeutically since they  contain medium chain fatty acids.

 Secretion of lipids from enterocytes

  • Once inside the enterocyte, monoglycerides and fatty acids are re-synthesized into TAG.
  • Lysophospholipids are recycled to form phospholipids.
  • Cholesterol is re acylated to form Cholesteryl esters
  • Long chain fatty acids are used for esterification to form TGs, phospholipids and cholesteyl esters.
  • Short and medium chain fatty acid are released in to the portal circulation and are carried by serum albumin to liver.
  • The TGs are packaged, along with cholesterol and fat soluble vitamins, into chylomicrons  (Figure-4)
  • Chylomicrons are lipoproteins, special particles that are designed for the transport of lipids in the circulation.
  • Chylomicrons are released by exocytosis at the basolateral surface of the enterocytes. Because they are particles, they are too large to enter typical capillaries.
  • Instead they enter lacteals, lymphatic capillaries that poke up into the center of each villus.
  • Chylomicrons then flow into the circulation via lymphatic vessels.

 Lipid absorption and transport

Figure-4- Absorption and transport of lipids

 Structure of Chylomicron

  • Size: 0.1–1 µm
  • Average composition (figure-5)
  • TG (84%)
  • Cholesterol(2%)
  • Ester Cholesterol (4%)
  • Phospholipid (8%)
  • Apo lipoproteins (2%)

 Structure of chylomicron

Figure-5- The nonpolar lipid core consists of mainly triacylglycerol and cholesteryl ester and is surrounded by a single surface layer of amphipathic phospholipid and cholesterol molecules . These are oriented so that their polar groups face outward to the aqueous medium, as in the cell membrane.The protein moiety of a lipoprotein is known as an apolipoprotein or apoprotein, (B48).

Transport and Utilization of chylomicrons

The clearance of chylomicrons from the blood is rapid, the half-time of disappearance being under 1 hour in humans. Fatty acids originating from chylomicron triacylglycerol are delivered mainly to adipose tissue, heart, and muscle (80%), while about 20% goes to the liver. Triacylglycerol is hydrolyzed progressively through a diacylglycerol to a monoacylglycerol and finally to free fatty acids plus glycerol. Some of the released free fatty acids return to the circulation, attached to albumin, but the bulk is transported into the tissues. The resulting  chylomicron remnants, rich in cholesterol  are taken up by the liver by receptor-mediated endocytosis.

 Clinical significance of Chylomicron synthesis and utilization

  • Defective synthesis- Due to deficiency of apo-B 48 protein. The triglyceride may accumulate in intestinal cells.
  • Chyluria-  Due to an abnormal connection between urinary tract and lymphatic drainage system of the intestines, forming Chylous fistula. Characterized by passage of Milky urine.
  • Chylothorax- There is an abnormal connection between pleural space and the lymphatic drainage of small intestine resulting in accumulation of lymph in pleural cavity giving Milky pleural effusion

Summary of lipid digestion and Absorption (Figure-6)

 Summary of digestion and absorption of lipids

Figure-6- 1) Triglycerides are broken down by lipases 2) absorption is mediated by micelles,3)  inside the intestinal cells chylomicrons are synthesized for transportation of absorbed lipids 4) Chylomicrons deliver absorbed TAG to the body’s cells. TAG in chylomicrons and other lipoproteins are hydrolyzed by lipoprotein lipase, an enzyme that is found in capillary endothelial cells. Monoglycerides and fatty acids released from digestion of TAG then diffuse into cells.

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Lipids are a heterogeneous group of water insoluble organic molecules that can be extracted from tissues by non polar solvents.They are the major source of energy for the body.

Dietary fat Composition

More than 95% are triglycerides, the other are – Cholesteryl esters, phospholipids  and unesterified fatty acids.

Dietary sources of Lipids

a)  Animal Sources

  • Dairy products -Milk , butter, ghee
  • Meat , fish, pork, and eggs

b) Vegetable Sources

  • Cooking oils -Sun flower oil, Mustard oil, Ground nut oil
  • Fats from other vegetable sources.

 Enzymes of lipid digestion

  • Lipases- For the digestion of triglycerides
  • Phospholipase A2- for the digestion of Phospholipids
  • Cholesterol esterase-For the digestion of Cholesteryl esters

Lipases- Three main lipases

  • Lingual
  • Gastric and
  • Pancreatic

 General reaction catalyzed by lipase

Triglycerides are degraded by lipases to form free fatty acids and glycerol. The reaction proceeds in a step wise manner (Figure-1).

 Action of lipase

Figure-1-Triglycerides are degraded by lipases to form free fatty acids and glycerol

A) Digestion in Mouth

Hydrolysis of triacylglycerols is initiated by lingual and gastric lipases, which attack the sn-3 ester bond forming 1,2-diacylglycerols and free fatty acids, aiding emulsification.

Lingual lipase:

Characteristics

  • Secreted by dorsal surface of tongue
  • Active at low pH (pH 2.0 – 7-5)
  • Optimum pH 4.0-4.5
  • Ideal substrate-Short chain TGS
  • Milk fat contains short chain fatty acids which are esterified at -3 position, thus it is the best substrate for lingual lipase
  • Enzymatic action continues in stomach
  • Short chain fatty acids, released are absorbed directly from the stomach wall and enter the portal vein.

B) Digestion in Stomach

Characteristics

  • Gastric Lipase is secreted in small quantities
  • More effective at alkaline p H (Average p H 7.8)
  • Requires the presence of Ca++
  • Less effective  in stomach due to acidic pH except when intestinal contents are regurgitated in to the gastric lumen
  • Not effective for long chain fatty acids, most effective for short and medium chain fatty acids
  • Milk, egg yolk and fats containing short chain fatty acids are suitable substrates for its action

 Role of fats in gastric emptying

  • Fats delay the  rate of emptying of stomach
  • Action is brought about by secretion of Enterogastrone
  • Enterogastrone inhibits gastric motility and retards the discharge  of bolus of food from the stomach.
  • Thus fats have a  high satiety value.

Significance of Lingual and Gastric Lipases

  • Play important role in lipid digestion in neonates since milk is the main source of energy
  • Important digestive enzymes in pancreatic insufficiency such as Cystic fibrosis or other pancreatic disorders
  • Lingual and gastric lipases can degrade triglycerides with short and medium chain fatty acids in patients with pancreatic disorders despite a near or complete absence of pancreatic lipase

Emulsification and digestion

  • Lipids are hydrophobic, and thus are poorly soluble in the aqueous environment of the digestive tract. 
  • The digestive enzyme, lipase, is water soluble and can only work at the surface of fat globules. 
  • Digestion is greatly aided by emulsification, the breaking up of fat globules into much smaller emulsion droplets.
  • Triacylglycerol digestion occurs at lipid-water interfaces
  • Rate of TAG digestion depends on surface area of this interface which is increased by churning peristaltic movements of the intestine ,
  • combined with the emulsifying action of bile salts
  • The critical process of emulsification takes place in the duodenum.

C) Digestion in small intestine

  • Major site of fat digestion
  • Effective digestion due to the presence of Pancreatic lipase and bile salts.
  • Bile salts act as effective emulsifying agents for fats (figure-4)
  • Secretion of pancreatic juice is stimulated by-
  • Passage of acid gastric contents in to the duodenum
  • By secretion of Secretin, Cholecystokinin and Pancreozymin, the gastro intestinal hormones

Gastro Intestinal hormones

  • Secretin- Increases the secretion of electrolytes and fluid components of pancreatic juice
  • Pancreozymin of CCK -PZ stimulates the secretion of the pancreatic enzymes
  • Cholecystokinin of CCK-PZ- causes the contraction of the gall bladder and discharges the bile in to the duodenum.
  • Hepatocrinin- Released by intestinal mucosa, stimulates more bile formation which is relatively poor in bile acid content .

Contents of Pancreatic Juice

  • Pancreatic Lipase- For the digestion of triglycerides (figure-5)
  • Cholesterol esterase-For the digestion of Cholesteryl esters (figure-7)
  • Phospholipase A2- for the digestion of Phospholipids (figure-8)

 Role of Bile Salts

  • Bile salts are required for the proper functioning of the pancreatic lipase enzyme
  • They help in combination of  lipase with two molecules of a small protein called as Colipase. This combination enhances the lipase activity.
  • Bile salts also help in the emulsification of fats (figure-4)
  • They are synthesized in the liver and are stored in the gall bladder
  • They are derivatives of cholesterol
  • They consist of a sterol ring structure  with a side chain to which a molecule of glycine or taurine is covalently attached by an amide linkage (figure-2).

 Structure of bile salts

Figure-2- Bile salts are derivatives of cholesterol containing a sterol ring structure  with a side chain to which a molecule of glycine or taurine is covalently attached by an amide linkage.

  • Bile salts are formed from bile acids
  • The primary bile acids are cholic acid (found in the largest amount) and chenodeoxycholic acid .
  • The primary bile acids enter the bile as glycine or taurine conjugates.
  • In the alkaline bile, the bile acids and their conjugates are assumed to be in a salt form—hence the term “bile salts.“

Enterohepatic circulation of Bile salts

The bile salts present in the body are not sufficient to fully process the fats in a typical meal, thus they need to be recycled. This is achieved by the enterohepatic circulation. Specific transporters in the terminal ileum move bile salts from the lumen of the digestive tract to the intestinal capillaries. They are then transported directly to the liver via the hepatic portal vein. Hepatocytes take up bile salts from the blood, and increase the secretion of bile salts into the bile canaliculi, small passage ways that convey bile into the larger bile ducts. 95% of the bile that is released to the small intestine is recycled via the enterohepatic circulation, while 5% of the bile salts are lost in the feces. (Figure-3).

Emulsification by bile salts

Bile salts as emulsifying agents interact with the dietary lipid particles and the aqueous duodenal contents, thereby stabilizing the lipid particles as they become smaller, and preventing them from coalescing (Figure-4) .

Triacyl glycerol degradation by pancreatic lipase (Figure-5)

  • Pancreatic lipase is specific for the hydrolysis of primary ester linkages (Fatty acids present at position 1 and 3)
  • It cannot hydrolyze the ester linkages of position -2
  • Digestion of triglycerides  proceeds by removal of a terminal fatty acid to produce  an α,β diglyceride.
  • The other terminal fatty acid is then removed to produce β mono glyceride.
  • The last fatty acid is linked by secondary ester group, hence cannot be hydrolyzed by pancreatic lipase.
  • β- Mono acyl glycerol can be converted to α- Mono acyl glycerol  by isomerase enzyme and then hydrolyzed by Pancreatic lipase.
  • The primary product of hydrolysis are β- Mono acyl glycerol (78%), α- Mono acyl glycerol (6%) with free fatty acids and glycerol (14%).

 Enterohepatic circulation of bile salts

Figure-3-Enterohepatic circulation of bile salts   

Role of bile salts in emulsification

Figure-4- Role of bile salts in emulsification

 

 pancreatic lipase

Figure-5- Triglyceride digestion by pancreatic lipase.

Summary  of Emulsification and Digestion of Triglycerides (Figure-6)

 Summary of pancreatic digestion

Figure-6- steps of emulsification and digestion of dietary triglycerides.

Significance of Pancreatic lipase

  • The enzyme is present in high concentration in pancreas. Only very severe pancreatic deficiency such as cystic fibrosis  results in malabsorption of fats due to impaired digestion.
  • Orlistat, an antiobesity drug inhibits , gastric and pancreatic lipases, thereby decreasing fat digestion and absorption resulting in weight loss.

Cholesteryl ester degradation

  • Dietary cholesterol is mainly present in the free (Non esterified) form
  • Only 10-15% is present in the esterified form
  • Cholesteryl esters are hydrolyzed by pancreatic Cholesteryl esterase (Cholesterol ester hydrolase) to produce cholesterol and free fatty acid (figure-7)
  • The enzymatic activity is greatly increased in the presence of bile salts.

 

 Cholesterol esterase

Figure-7- degradation of cholesteryl esters by cholestrol esterase (cholesterol ester hydrolase)

Phospholipid degradation

  • The enzyme – Phospholipase A 2requires bile salts for optimum activity.
  • Removes one fatty acid from carbon 2 of Phospholipid to form lysophospholipid (figure-8)
  • The remaining fatty acid at position 1  can be removed by lysophospholipase , leaving a glycerylphosphoryl base that may be excreted in the feces, further degraded or absorbed.

 Reaction catalyzed by phospholipase A2

Figure-8- Reaction catalyzed by phospholipase A2.

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Enzyme

Pathway

Effect of substrate concentration

Allosteric Modification/ Fed back Inhibition

Induction/ Repression

Clinical Significance

Acetyl co A Carboxylase

Fatty acid synthesis

Activity increases during well fed state

Activity decrease during fasting

Activator-

Citrate,

ATP

Acetyl co A

Insulin-by causing de phosphorylation by stimulating protein phosphatase

Inhibitors

Long chain fatty acids, Epinephrine, Glucagon- via changes in phosphorylation state through c AMP mediated phosphorylation cascade

Induced by Insulin

 

Repressed by Glucagon

Activity decreases in diabetes Mellitus

Carnitine Acyl Transferase

Carnitine shuttle

Activity low in fed state, high during fasting

Activated by Glucagon through lipolysis and provision of fatty acids for oxidation

 

Inhibited by insulin and malonyl co A

 

Inherited CAT-I deficiency affects only the liver, resulting in reduced fatty acid oxidation and ketogenesis, with hypoglycemia.

HMG co A Reductase

Cholesterol synthesis

Activity low in fasting state,

Activated by Insulin, Thyroid hormone

 

Inhibited by – Glucagon, Glucocorticoids,(By reversible phosphorylation)

Dietary cholesterol (Hepatic synthesis)

Mevalonate and cholesterol ,the products of pathway

 

Expression of HMG COA reductase is regulated by sterol regulatory element binding protein

Also induced by Insulin

Activity high in Diabetes mellitus die to availability of excess Acetyl co A.

 

Activity inhibited by Statins used as cholesterol lowering drugs.

 

 


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Hyperlipoproteinemia

 A) Primary Hyperlipoproteinemia

TYPE CLASSIFICATION BIOCHEMICAL DEFECT INHERITANCE PLASMA LIPIDS AND LIPOPROTEINS CLINICAL FEATURES
I. Familial lipoprotein lipase deficiency(Hyperchylomicronemia) Deficiency of lipoprotein lipase. A variant of this diseases can be produced by deficiency of apo CII. Autosomal recessive TG↑, cholesterol may also be increased, Chylomicrons ++, VLDL↑, HDL and LDL ↓.Refrigeration test confirms the presence of chylomicrons in serum Present in early childhood, Eruptive xanthomas, Recurrent abdominal pain, no premature cardiovascular disease
II. Familial Hypercholesterolemia(FHC) No enzyme deficiency but there is increased synthesis of apo- B protein and there is impaired degradation of LDL Autosomal dominant LDL ↑, total cholesterol ↑, VLDL  and TG are also raised Xanthomas, corneal arcus, increased risk of premature Cardiovascular disease.
III. Familial Dysbeta lipoproteinemia Also called- Broad beta disease , ‘Remnant removal disease” Increase concentration of apo-E, increased synthesis of Apo B, conversion of normal VLDL to IDL and its degradation without conversion to LDL. Defect in Remnant removal. Autosomal dominant LDL↑, VLDL↑, actual rise in IDL which appears as Broad beta band on electrophoresis. TG↑, cholesterol may also be increased. Tuberous and Palmar xanthomas, increased risk of premature Cardiovascular disease and peripheral vascular disease.
IV. Familial Hypertriglyceridemia(FHTG) Increased synthesis  and decreased catabolism of endogenous TGs Autosomal dominant Hyper pre beta lipoproteinemia VLDL↑, TG and cholesterol also↑, α and β-lipoproteins are subnormal (HDL↓ and LDL↓). There is associated impaired Glucose Tolerance Presents in early childhood, and is commonly associated with CHD, Diabetes Mellitus, Alcoholism and  obesity
V. Combined Hyperlipidemia Usually secondary to other disorders like obesity, excessive alcohol intake, renal failure and pancreatitis etc. Exact biochemical defect is not known Autosomal dominant Complex lipoprotein pattern, Increase in both chylomicrons and pre β-lipoproteins (VLDL), TG and cholesterol also↑↑, α and β-lipoproteins are subnormal (HDL↓ and LDL↓). Xanthomas are frequently present impaired glucose tolerance, frequently found associated with CHD, Diabetes Mellitus, Alcoholism and  obesity

B) Secondary Hyperlipoproteinemia

S. .No Disease Serum total Cholesterol Serum triglycerides
1. Diabetes Mellitus Increased Increased
2. Nephrotic syndrome Increased Increased
3. Hypothyroidism Increased Increased
4. Biliary obstruction Increased Normal
5. Pregnancy Increased Normal
6. Alcoholism Normal Increased
7. Oral contraceptives Normal

Hypolipoproteinemia

Disease Biochemical defect Inheritance Laboratory findings Clinical manifestations
Familial Hypobetalipoproteinemia Inability to synthesize ApoB100 and Apo-B48 Autosomal dominant LDL between 10 to 50 % of normal, Cholesterol low but Triglycerides and Chylomicrons normal. Mostly asymptomatic, but there is decreased risk of CHD
Abetalipoproteinemia No synthesis or secretion  of Apo B containing lipoproteins Rare inherited disease VLDL↓, LDL↓↓, B-100↓, B-48↓, decrease in TG and a marked ↓in Serum total cholesterol, prolonged prothrombin time. Malabsorption, mental and physical retardation, acanthocytosis, Atypical retinitis pigmentosa, fatty infiltration of intestinal mucosal cells and liver.
Hypoalphalipoproteinemia Not known Autosomal dominant HDL↓, Cholesterol and TGs normal Increased risk of CHD
Familial Alpha lipoprotein deficiency(Tangier’s disease) Deficiency of alpha lipoprotein. Cholesteryl esters are deposited in the reticulo endothelial system, but functions of major organs are not affected Autosomal recessive Low or absent HDL, Lack of Alpha band on electrophoresis. Orange yellow tonsils and adenoids, muscle weakness, atrophy, recurrent peripheral neuropathies and depressed tendon reflexes. The pharyngeal and rectal mucosa is also orange colored.(The orange color is due to deposition of cholesteryl esters.) There is increased predisposition to Atherosclerosis.

 

 

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The sphingolipidosis (lipid storage diseases) are a group of inherited diseases that are caused by a genetic defect in the catabolism of lipids containing sphingosine. They are part of a larger group of lysosomal disorders and exhibit several constant features:

(1) Complex lipids containing ceramide accumulate in cells, particularly neurons, causing neuro degeneration and shortening the life span.

(2) The rate of synthesis of the stored lipid is normal.

(3) The enzymatic defect is in the lysosomal degradation pathway of sphingolipids.

(4) The extent to which the activity of the affected enzyme is decreased is similar in all tissues.

There is no effective treatment for many of the diseases, although some success has been achieved with enzyme replacement therapy and bone marrow transplantation in the treatment of Gaucher’s and Fabry’s diseases. Other promising approaches are substrate deprivation therapy to inhibit the synthesis of sphingolipids and chemical chaperone therapy. Gene therapy for lysosomal disorders is also currently under investigation.

 

Disease Enzyme Deficiency Lipid Accumulating
Clinical Symptoms
Tay Sach’s Disease Hexosaminidase A GM2 Ganglioside Mental retardation, blindness, muscular weakness
Fabry’s disease α-Galactosidase Globotriaosylceramide Skin rash, kidney failure (full symptoms only in males; X-linked recessive).
Metachromatic leukodystrophy Arylsulfatase A Sulfogalactosylceramide Mental retardation and Psychologic disturbances in adults; demyelination.
Krabbe’s disease β-Galactosidase Galactosylceramide Mental retardation; myelin almost absent.
Gaucher’s disease β -Glycosidase Glucosyl ceramide Enlarged liver and spleen, erosion of long bones, mental retardation in infants.
Niemann-Pick disease Sphingomyelinase  Sphigomyelin Enlarged liver and spleen, mental retardation; fatal in early life.
Farber’s disease Ceramidase Ceramide Hoarseness, dermatitis, skeletal deformation, mental retardation; fatal in early life

 

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Sr.no. Disease Biochemical Defect Inheritance Clinical Manifestations Lab. Diagnosis Treatment
1. Refsum disease There is deficiency of phytanic acid oxidase enzyme.Characterized biochemically by the accumulation of phytanic acid in plasma and tissues. Autosomal recessive The disease is characterized by night blindness, loss of smell, deafness, muscle weakness and development of dysmorphic features in children. -Serum total cholesterol, HDL and LDL are moderately reduced. -Blood phytanic acid levels are elevated. -Phytanic oxidase activity estimation in skin fibroblast cultures is diagnostic. Eliminate all sources of chlorophyll from diet.Plasmapheresis is needed to remove Phytanic acid from blood.
2. Zellwegar syndrome Zellwegar syndrome is characterized by an individual’s inability to beta-oxidize very-long chain fatty acids in the Peroxisomes of the cell, due to a genetic disorder in one of the several genes involved with peroxisome biogenesis.

Zellwegar syndrome is the most severe of the PBDs(Peroxisome biogenesis Syndrome 

Autosomal Recessive Symptoms at birth may include a lack of muscle tone, an inability to move and glaucoma.

Other symptoms may include unusual facial characteristics, mental retardation, seizures, and an inability to suck and/or swallow. Jaundice and gastrointestinal bleeding may also occur. More than 90% growth failure.

 

The abnormally high levels of VLCFA ( Very long chain fatty acids ), are most diagnostic. There is no cure for Zellwegar syndrome, nor is there a standard course of treatment. 

Most treatments are symptomatic and supportive.

 

 

 

 

 

 

 

 

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HMG Co A reductase- 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), is an important control site in cholesterol biosynthesis, this enzyme catalyzes the formation of Mevalonate, the committed step in cholesterol biosynthesis. HMG-CoA reductase is an integral membrane protein in the endoplasmic reticulum and spans the membrane. The active site for this enzyme is found on the cytosolic side of the membrane.

The enzyme catalyzes the irreversible step,

 

Regulation of HMG co A reductase/ cholesterol Biosynthesis- HMG CoA reductase is controlled in multiple ways:

This regulation is mediated primarily by changes in the amount and activity of 3-hydroxy-3-methylglutaryl CoA reductase.

A) Regulation of enzyme activity-

1) Feed back inhibition –HMG-CoA reductase in liver is inhibited by Mevalonate, the immediate product of the pathway, and by cholesterol, the main product. The rate of cholesterol formation is highly responsive to the cellular level of cholesterol.

2) Covalent modification Insulin or thyroid hormone increases HMG-CoA reductase activity, whereas glucagon or glucocorticoids decrease it. Activity is reversibly modified by phosphorylation-dephosphorylation mechanisms, some of which may be cAMP-dependent and therefore immediately responsive to glucagon. Phosphorylation decreases the activity of the reductase.  This enzyme, like acetyl CoA carboxylase(which catalyzes the committed step in fatty acid synthesis, is switched off by an AMP-activated protein kinase. Thus, cholesterol synthesis ceases when the ATP level is low. (Insulin causes dephosphorylation, while glucagon causes phosphorylation).

3) Effect of statins-Becausethe enzyme HMG-CoA reductase is the rate-limiting step of cholesterol biosynthesis,this enzyme is the target for many cholesterol lowering drugs. Statins act by inhibiting HMG-CoA reductase and up-regulating LDL receptor activity. Examples currently in use include atorvastatin, simvastatin, fluvastatin, and pravastatin.

B) Regulation of concentration of HMG Co A reductase- The concentration of HMG Co A Reductase is regulated by three main mechanisms-

i)The rate of synthesis of reductase mRNA(Transcription ) – Transcription of  HMG Co A reductase gene is controlled by the sterol regulatory element binding protein (SREBP).

SREBPs are a family of proteins that regulate the transcription of a range of genes involved in the cellular uptake and metabolism of cholesterol and other lipids. This transcription factor binds to a short DNA sequence called the sterol regulatory element(SRE) on the 5’ side of the reductase gene. In its inactive state, the SREBP is anchored to the endoplasmic reticulum or nuclear membrane. When cholesterol levels fall, the protein is released from its association with the membrane by two specific proteolytic cleavages. The released protein migrates to the nucleus and binds the SRE of the HMG-CoA reductase gene, as well as several other genes in the cholesterol biosynthetic pathway, to enhance transcription.Low concentrations of cholesterol increase the level of mRNA for HMG-CoA reductase, whereas high concentrations of cholesterol decrease the mRNA level.

When cholesterol levels rise, the proteolytic release of the SREBP is blocked, and the SREBP in the nucleus is rapidly degraded. These two events halt the transcription of the genes of the cholesterol biosynthetic pathways. It is feed back regulation.Dietary cholesterol also decreases the endogenous cholesterol synthesis. However, it is only hepatic synthesis that is inhibited by dietary cholesterol. .

ii)The rate of translation of reductase mRNA -is inhibited by non sterol metabolites derived from Mevalonate as well as by dietary cholesterol. Reverse occurs when Mevalonate concentration is low, hence translation is enhanced and amount ofHMG Co A reductase is increased.

iii) The degradation of the reductase is stringently controlled. In response to increasing concentrations of sterols such as cholesterol, the enzyme becomes more susceptible to proteolysis. A combination of these three regulatory devices can regulate the amount of enzyme over a 200-fold range.

 

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 a)  Acetyl co A carboxylase-Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonylCo A catalyzed by Acetyl co A carboxylase . This irreversible reaction is the committed step in fatty acidsynthesis.

 Acetyl CoA carboxylase, contains a biotin prosthetic group. The carboxyl group of biotin is covalently attached to the  epsilon amino group of a lysine residue, as in pyruvate carboxylase and propionyl CoA carboxylase .The enzyme is a multienzyme protein containing a variable number of identical subunits, each containing biotin, biotin carboxylase, biotin carboxyl carrier protein, and transcarboxylase, as well as a regulatory allosteric site.

 The reaction takes place in two steps: (1) carboxylation of biotin involving ATP and (2) transfer of the carboxyl to acetyl-CoA to form malonyl-CoA.

 Regulation of Acetyl co A carboxylase- This enzyme is also the essential regulatory enzyme for fatty acid metabolism.

 a) Hormonal control- The carboxylase is controlled by three global signals glucagon, epinephrine, and insulin that correspond to the overall energy status of the organism. Insulin stimulates fatty acid synthesis by activating the carboxylase, whereas glucagon and epinephrine have the reverse effect.

 b) Allosteric modification-The levels of citrate, palmitoyl Co A, and AMP within a cell also exert control. Citrate, a signal that building blocks and energy are abundant,activates the carboxylase. Palmitoyl CoA and AMP, in contrast, lead to the inhibition of the carboxylase. Citrate facilitates the polymerization of the inactive octamers into active filaments. The level of citrate is high when both acetyl CoA and ATP are abundant.

The stimulatory effect of citrate on the carboxylase is antagonized by palmitoyl CoA, which is abundant when there is an excess of fatty acids. Palmitoyl CoA causes the filaments to disassemble into the inactive octamers.

Palmitoyl CoA also inhibits the translocase that transports citrate from mitochondria to the cytosol, as well as glucose 6-phosphate dehydrogenase, which generates NADPH in the pentose phosphate pathway

 c) Covalent Modification- is carried out by means of reversible phosphorylation and dephosphorylation mediated by hormonal action. AcetylCoA carboxylase is switched off by phosphorylation and activated by dephosphorylation. Epinephrine and glucagon activate protein kinase A to bring about phosphorylation. Hence, these catabolic hormones switch off fattyacid synthesis by keeping the carboxylase in the inactive phosphorylated state.

Insulin stimulates the carboxylase by causing its dephosphorylation by stimulating phosphatase enzyme.

 d)Response to Diet

Fatty acid synthesis and degradation are reciprocally regulated so that both are not simultaneouslyactive. In starvation, the level of free fatty acids rises because hormones such as epinephrine and glucagon stimulate adipose-cell lipase.  In well fed state, Insulin, in contrast, inhibits lipolysis. Acetyl CoA carboxylase also plays a role in the regulation of fatty acid degradation. Malonyl CoA, the product of the carboxylase reaction, is present at a high level when fuel molecules are abundant. Malonyl CoA inhibits carnitine acyl transferaseI, preventing access of fatty acyl CoA s to the mitochondrial matrix in times of plenty.

 e) Long-term control is mediated by changes in the rates of synthesis and degradation of the enzymes participating in fatty acid synthesis.Animals that have fasted and are then fed high-carbohydrate, low-fat diets show marked increases in their amounts of acetyl CoA carboxylase and fatty acid synthase (Another multienzyme complex of fatty acid bio synthetic pathway) within a few days. This type of regulation is known as adaptive control.

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Lipids absorbed from the diet and synthesized by the liver and adipose tissue must be transported between various cells and organs for utilization and storage. Since lipids are insoluble in water, the problem of transportation in the aqueous plasma is solved by associating nonpolar lipids (triacylglycerols and cholesteryl esters) with amphipathic lipids(phospholipids and cholesterol) and proteins to make water-miscible Lipoproteins.

General Structure of Lipo protein

 Lipoproteins Consist of a Nonpolar Core & a Single Surface Layer of Amphipathic Lipids

The nonpolar lipid core consists of mainly triacylglycerol and cholesteryl ester and is surrounded by a single surface layer of amphipathic phospholipid and cholesterol molecules (Figure-1). These are oriented so that their polar groups face outward to the aqueous medium. The protein moiety of a lipoprotein is known as an apolipoprotein or apoprotein,constituting nearly 70% of some HDL and as little as 1% of Chylomicons. Some apolipoproteins are integral and cannot be removed, whereas others can be freely transferred to other lipoproteins.

 

Figure-1- showing general structure of lipoprotein

 Classification of Lipoproteins

 Lipoproteins can be classified in three ways

 1) Based on density

Because fat is less dense than water, the density of a lipoprotein decreases as the proportion of lipid to protein increases.  Lipoproteins with high lipid content will have low density and so float on centrifugation. Those with high protein content sediment easily and have a high density. They are separated by Ultracentrifugation. Depending upon the floatation constant (Sf), Five major groups of lipoproteins have been identified that are important physiologically and in clinical diagnosis. These are

 (i) Chylomicons, derived from intestinal absorption of triacylglycerol and other lipids; Density is generally less than0.95 while the mean diameter lies between 100- 500 nm

 (ii) Very low density lipoproteins(VLDL), derived from the liver for the export of triacylglycerol; density lies between 0.95- 1.006 and the mean diameter lies between 30-80 nm.

 (iii) Intermediate density lipoproteins (IDL) are  derived from the catabolism of VLDL,with a density  ranging intermediate between Very low density and Low density lipoproteins i.e. ranging between 1.006-1.019 and the mean diameter ranges between 25-50nm.

 (iv)Low-density lipoproteins (LDL), representing a final stage in the catabolism of VLDL; density lies between 1.019-1.063 and mean diameter lies between 18-28 nm

 (iv) High-density lipoproteins (HDL),involved in cholesterol transport and also in VLDL and chylomicron metabolism. Density ranges between 1.063-1.121 and the mean diameter varies between 5-15 nm. (Table)

 

Figure- 2-showing the relationship of density and mean diameter of lipoproteins

Triacylglycerol is the predominant lipid in chylomicron and VLDL, whereas cholesterol and phospholipid are the predominant lipids in LDL and HDL, respectively. (Table)

 2) Based on electrophoretic mobilities

Lipoproteins may be separated according to their electrophoretic properties into alpha , beta, pre-beta,and broad beta lipoproteins. The mobility of a  lipoprotein is mainly dependent upon protein content. Those with higher protein content will move faster towards the anode and those with minimum protein content will have minimum mobility.

 HDL are alpha , LDLbeta, VLDL pre-beta, and IDL are broad beta lipoproteins. Free fatty acids and albumin complex although not a lipoprotein is an important lipid fraction in serum and is the fastest moving fraction. Chylomicons remain at the origin since they have more lipid content. VLDL with less protein content than LDL move faster than LDL, this is due to nature of apoprotein present.

Table- showing the composition of lipoproteins. As the lipid content increases, density decreases and size increases, that is why Chylomicons are least dense but biggest in size, while HDL are rich in proteins , hence most dense but smallest in size.

 3)Based on nature of Apo- protein content

One or more apolipoproteins (proteins or polypeptides) are present in each lipoprotein. The major apolipoproteins of HDL Alpha Lipoproteins) are designated A.The main apolipoprotein of LDL (beta -lipoprotein) is apolipoprotein B(B-100), which is found also in VLDL. Chylomicons contain a truncated form of apo B (B-48) that is synthesized in the intestine, while B-100 is synthesized in the liver. Apo B-100 is one of the longest single polypeptide chains known,having 4536 amino acids and a molecular mass of 550,000 Da. Apo B-48 (48% ofB-100) is formed from the same mRNA as apo B-100 after the introduction of a stop signal by an RNA editing enzyme. Apo C-I, C-II, and C-III are smaller polypeptides (molecular mass 7000–9000 Da) freely transferable between several different lipoproteins. Apo E is found in VLDL, HDL, Chylomicons, andchylomicron remnants; it accounts for 5–10% of total VLDL apolipoproteins in normal subjects.

 Functions of Apoproteins- Apolipoproteins carry out several roles: 

 (1) They can form part of the structure of the lipoprotein, eg, apo B is a structural component of VLDL and Chylomicons

 (2)They are enzyme cofactors, e.g. C-II for lipoprotein lipase, A-I for lecithin: cholesterolacyl transferase (LCAT), 

 (3) They act as enzyme inhibitors, eg, apo A-II and apo C-III for lipoprotein lipase, apo C-I for cholesteryl ester transfer protein;

 (4)They act as ligands for interaction with lipoprotein receptors in tissues, eg,apo B-100 and apo E for the LDL receptor, apo A-I for the HDL receptor. The functions of apo A-IV and apo D, however, are not yet clearly defined, although apo D is believed to be an important factor in human neuro degenerative disorders and acts as cholesteryl ester transfer protein required for the exchange of triglycerides and cholesteryl esters between VLDL,chylomicron remnants and HDL.

 All apoproteins are synthesized mainly in liver but small amounts can be synthesized in almost all organs.

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“Although chemical processes involved are the same but Fatty acid synthesis is not simply a reversal of fatty acid oxidation”.             

Fatty acid synthesis seems simply a reversal of the degradative pathway, but it consists of a new set of reactions, exemplifying the principle that synthetic and degradative pathways are almost always distinct.Some important differences between the pathways are:
1. Synthesis takes place in the cytosol, in contrast with degradation, which takes place primarily in the mitochondrial matrix.
2. Intermediates in fatty acid synthesis are covalently linked to the sulfhydryl groups of an acyl carrier protein (ACP), whereas intermediates in fatty acid breakdown are covalently attached to the sulfhydryl group of coenzyme A.
3. The enzymes of fatty acid synthesis in higher organisms are joined in a single polypeptide chain called fatty acid synthase. In contrast, the degradative enzymes do not seem to be associated.
4. The growing fatty acid chain is elongated by the sequential addition of two-carbon units derived from acetyl Co A. The activated donor of two carbon units in the elongation step is malonyl ACP. The elongation reaction is driven by the release of CO2.
5. The reductant in fatty acid synthesis is NADPH, whereas the oxidants in fatty acid degradation are NAD + and FAD.
6. Elongation by the fatty acid synthase complex stops on formation of Palmitate(C16). Further elongation and the insertion of double bonds are carried out by other enzyme systems.
Over view of fatty acid oxidation- The process of degradation converts an aliphatic compound into a set of activated acetyl units (acetyl CoA) that can be processed by the citric acid cycle. An activated fatty acid is oxidized to introduce a double bond; the double bond is hydrated to introduce oxygen; the alcohol is oxidized to a ketone; and,finally, the four carbon fragment is cleaved by coenzyme A to yield acetyl CoA and a fatty acid chain two carbons shorter. If the fatty acid has an even number of carbon atoms and is saturated, the process is simply repeated until the fatty acid is completely converted into acetyl CoA units. (Figure)

Overview of fatty acid synthesis- Fatty acid synthesis is essentially the reverse of this process. Because the result is a polymer, the process starts with monomers in this case with activated acyl group (most simply, an acetyl unit) and malonyl units (see Figure). The malonyl unit is condensed with the acetyl unit to form a four-carbon fragment. To produce the required hydrocarbon chain, the carbonyl must be reduced. The fragment is reduced, dehydrated, and reduced again, exactly the opposite of degradation, to bring the carbonyl group to the level of a methylene group with the formation of butyryl CoA. Another activated malonyl group condenses with the butyryl unit and the process is repeated until a C16 fatty acid is synthesized. (See figure)

 
Figure-Showing the chemical processes involved in fatty acid synthesis and oxidation
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