BIOCHEMISTRYSFA 2073

Lipid Metabolism

NIK NORMA NIK MAHMOOD-Ph.D FACULTY OF SCIENCE AND

TECHNOLOGYISLAMIC SCIENCE UNIVERSITY OF

MALAYSIA

Digestion & Absorption• Lipids that gets into the digestive system are

dietary lipids, normally free fatty acids, cholesterols and triglycerides (TAG) and many minor components.

• Digestion starts in the duodenum portion of small intestine:

- is mix with bile that contains HCO3ˉ ions and bile

salts to solubilize fat. This process is called emulsification. The big lipids droplets are broken down into smaller droplets.

(bile is made in liver and stored in gall bladder between meals. When there is food, bile is delivered to the intestine from gall bladder via bile duct)

- TAG is acted by lipase secreted by pancreas liberating monoglyceride and two fatty acids. Monoglyceride, cholesterol and f.fas and bile salts

form amphipathic micelles. These micelles keep the insoluble lipid components in soluble aggregates from which small amounts are released and absorbed by epithelial cells via diffusion.

- Free fatty acids and monoglycerides then recombine into triacylglycerols at the smooth ER and together with cholesterols moves on to Golgi to be converted to chylymicrons. It enters interstitial fluid, then taken up by the lacteals in the intestinal wall and delivered to liver via hepatic portal vein for processing.

• exposure to a large aggregate of triglyceride, the hydrophobic portions of bile acids intercalate into the lipid, with the hydrophilic domains remaining at the surface. Such coating with bile acids aids in breakdown of large aggregates or droplets into smaller and smaller droplets.

• Pancreatic lipases hydrolyse triglyceride into monoglyceride and free fatty acids. The activity of this enzyme is clipping the fatty acids at positions 1 and 3 of the triglyceride, leaving two free fatty acids and a 2-monoglyceride.

**Lipase is a water-soluble enzyme

• Lipids, and products of their digestion are transported through aqueous compartments within the cell as well as in the blood and tissue spaces in the forms LIPOPROTEINS

Why in the form of LIPOPROTEINS?

• Large portion of the lipids’ structures comprise of C-C &C-H rendering lipids hydrophobic in nature i.e lipids are insoluble in aqueous environment thus creates problem to its transport within body-medium which is aqueous in nature.

Dietary triacylglycerols (Tag) & cholesterol and in-vivo Tag and cholesterol (synthesized in liver), must be converted to the soluble form to overcome the problem. This is achieved by forming LIPOPROTEINS

Lipolysis

• Is the breakdown of fat (Tag) stored in fat cells into free fatty acids + glycerol + mono & diglycerides which is catalysed by enzyme lipase

• Induced by hormone epinephrine , norepinephrine, glucagon and adreno corticotropic hormone

• the lipolytic products are then released into the blood

• The free fatty acids bind to serum albumin and transport to tissues that require energy. The energy is generated by catabolic β-oxidation pathway (a 4 steps pathway/cycle)

• How the hormones induce lipolysis ? The hormones trigger 7TM receptors, which activate adenylate cyclase. This results in increased production of cAMP, which activates protein kinase A, which subsequently activate lipases found in adipose tissue.

• β–oxidation of free fatty acid Fatty acid degradation and synthesis are

relatively simple processes and essentially the reverse of each other.

• f.f.a first are activated to acyl-CoA catalyse by Acyl-CoA synthase (ACosyn)prior to transport into mitochondria.

• acylCoA not permeable to inner mitochondria membrane, hence carried across by carnitine carrier system into mitochondrion matrix. It is by conjugation to carnitine.

• carnitine carrier system consisted of 2 enzyme-units CAT I & CAT II

AcylCoA + Carnitine → Acyl-carnitine + Co-ASHAcylCoA + Carnitine → Acyl-carnitine + Co-ASH

CCarnitine arnitine AAcyl cyl TTransferaseransferase

Carnitine (a quaternary ammonium compound) is hydrophilic amino acid derivative, produced endogenously in the kidneys and liver from lysine and methionine of diet’s meat and dairy products. Carnitine binds acyl residues conjugated with coenzyme A.

matrixmatrix

Intermembrane spaceIntermembrane space

cytosolcytosol

Outer membraneOuter membrane

Inner membraneInner membrane

Simplified mitochondrial layoutSimplified mitochondrial layout

ACosyn ACosyn

Fatty acidFatty acid

Acyl-CoAAcyl-CoA

Acyl-carnitineAcyl-carnitine

CAT IICAT II

Carnitine + Acyl-CoACarnitine + Acyl-CoA

CAT ICAT I

CAT: carnitine Acyl CAT: carnitine Acyl transferasetransferase

ACosyn: acyl-CoA ACosyn: acyl-CoA synthetasesynthetase

+ CoASH+ CoASH

+ CoASH+ CoASH

• Followed by (4 steps )

• - oxidation by FAD

• - hydration

• - oxidation by NAD+

• - thiolysis

• The cycle then repeats on the larger fragment while acetyl-CoA fragment channeled to Krebs Cycle

Step 1Step 1

• oxidation by FAD/Acyl-CoA DH : The activated fatty acid is oxidized to introduce a double bond.

• Hydration/Enoyl-CoA Hydratase: to introduce an oxygen via formation of alcohol

Step 2Step 2

Step 3Step 3

• oxidation by NAD+/Hydroxy-CoA- DH: the alcohol is oxidized to a ketone.

• Thiolysis- Thiolase/CoA-SH : cleaving of the acylCoA into two fragments, acetyl CoA and an acylCoA of fatty acid chain two carbons shorter.

Step 4Step 4

Steps: 1 and 2 3 and 4Steps: 1 and 2 3 and 4

• β-oxidation of unsaturated fatty acids poses a problem. Unsaturated f.a are the cis type. This prevents the formation of the required bond orientation, trans-δ2 bond, in the enoyl intermediate. These situations are handled by an additional of two enzymes.

• Process occurs in cytoplasm of liver (major) and adipose tissue cells.

• Fatty acids are formed by the following 3 rxn-stages:

i- acetyl-CoA Carboxylase rxn

ii- fatty acid synthase rxn

iii- desaturase rxn

• This process is the de novo synthesis of F.A

• The initiator substrate acetyl-CoA is the product of β-oxidation catabolic pathway

Anabolism Of Fatty Acidin Human

citratecitrate

malate

malate

oxaloacetate

oxaloacetate

Acetyl CoA

pyruvatepyruvate

Acetyl CoA

Fatty Acid

Fatty Acid

MitochondriaCytoplasm

β-oxidation

synthesis

Malate-oxaloacetate shuttle: Transfer OF Acetyl CoA from Mitochondria to

cytoplasm

NADH

NAD+NADH

NAD+

NADP+

NADPH

NADP+

NADPH

ATP-citrate lyaseCitrate

synthase

Malate DHMalic enzyme

transporter

ACETYL-CoA

Glucose

Pyruvate

Fatty Acids

Ketogenic

Amino Acids

Oxidation

Fatty Acid &

Cholesterol

Steroid hormones

Ketone bodies

SOURCES AND UTILIZATION OF ACETYL-CoA

Acetyl-CoA Carboxylase rxn

• Initiator to fatty acid synthesis is acetyl-CoA• Acetyl-CoA carboxylase catalyses carboxylation of acetyl-CoA to

malonyl-CoA via 2-steps reaction. • The enzyme is biotin bound. In mammals acetyl-CoA carboxylase

is a large enzyme controlled by conversion inactive ══> active (inactive: protomers (4 subunits; one biotin); active: 1 unit)

conversion promoted by citrate, but inhibited by fatty acyl CoA. Also

by hormonal controlled: in liver by glucagon – PO4rylation to inactive form; in adipose tissue by adrenalin (epinephrin) – PO4rylation to inactive form

Additional noteAdditional note• Acetyl-CoA originated from pyruvate in mitochondria and transported

to cytosol as citrate by condensing with oxaloacetate• In cytosol citrate is broken down to yield acetyl-CoA and oxaloacetate

by ATP-citrate lyase.• Acetyl-CoA undergoes carboxylation by Acetyl-CoA carboxylase to

malonyl-CoA

Figure at right is expansion of reaction in figure on leftFigure at right is expansion of reaction in figure on left

reaction at site 2reaction at site 2

• ATP-dependent carboxylation of the biotin, carried out at one active site (1)

• transfer of the carboxyl group to acetyl-CoA at a second active site (2).

• Reaction is spontaneous,

HCO3

- + ATP + acetyl-CoA → ADP + Pi + malonyl-CoA

FFatty atty aacid cid ssynthase rxnynthase rxn• The reaction is a multi-steps .• The enzyme(in mammal) is a very large

polypeptide of many domains that includes an acyl carrier protein domain.

• Has a number of prosthetic grps.• Individual domain catalyses a single step .

• the precursor of fatty acid synthesis is malonyl-CoA

• the initial action is binding of acetyl-CoA (2C) and malonyl-CoA (3C) to specific domain of FAS leading to formation of acyl-ACP intermediates -5C (steps 1&2)

•Followed by formation of β-ketoacyl-ACP (4C) with evolution of CO2 (step 3)

•β-ketoacyl is reduced to an alcohol, by electron transfer from NADPH (step 4).

• Dehydration yields a trans double bond (step 5).

•Reduction at the double bond by NADPH yields a saturated Acyl-ACP chain- 4C (step 6). This is 1 cycle.

• Acyl-ACP and malonyl ACP then repeat step 3 and reaction proceeds to step 7.

• Acyl chain lengthens by 2C / cycle.

• Elongation process stops when acyl 16C is formed. Hydrolysis of the ester bond takes place with liberation of palmitate.

All of the reactions of fatty acid synthesis are carried out by the All of the reactions of fatty acid synthesis are carried out by the multiple enzymatic activities of multiple enzymatic activities of fatty acid synthasefatty acid synthase FASFAS

**The active enzyme is a **The active enzyme is a dimerdimer of identical subunits of identical subunits. .

FASFAS

REGULATION of f.f a synthesis

• The major site of fatty acid synthesis regulation is at reaction catalysed by acetyl-CoA carboxylase (ACC). ACC requires a biotin co-factor

Activity of ACC is associated Activity of ACC is associated with conformational change with conformational change of the enzyme, and conc. of of the enzyme, and conc. of citrate and palmitoyl-CoA. citrate and palmitoyl-CoA. When [citrate] is high, When [citrate] is high, monomeric form associates monomeric form associates to the multimeric form. Active to the multimeric form. Active conformation is the conformation is the multimeric form. When multimeric form. When [palmitoyl] is high, multimeric [palmitoyl] is high, multimeric form dissociates into form dissociates into monomeric,it becomes monomeric,it becomes inactive.inactive.

inactiveinactiveactiveactive

nn monomeric –PO4 monomeric –PO4 (multimeric)(multimeric)nn + P + Pii

citratecitrate

palmitoylpalmitoyl

Catabolism Of Triglycerides (TG)

• Initial rxn is in small intestine where TG is mixed with bile salt.

• Bile salt are steroids with detergent properties. 2 most abundant componants are cholate and deoxycholate, and they are normally conjugated with either glycine or taurine

Taurocholic acid. In mammal it Taurocholic acid. In mammal it

exists as Naexists as Na++ salt. salt. In medical In medical

use, it is administered as use, it is administered as

cholagogue and choleretic. and choleretic.

Glycocholic acid (Cholic Glycocholic acid (Cholic acid+ glycin). It occurs as acid+ glycin). It occurs as a sodium salt in the bile a sodium salt in the bile of mammalsof mammals

• Starts by break-up of the glyceride (TG) into fatty acids and monoacylglycerol by pancreatic lipases. This step takes place because TG cannot be transported across the plasma membrane of the intestinal wall cells (enterocytes) due to its size.

• The 2 products are transported into the cell. Once in the cell, recombination occurs and triacylglyerols TG1 are reformed.

• TG1 is combined with dietary cholesterol, newly synthesized phospholipids and protein into compound chylomicrons (a large, low-density lipoproteins).

• Lipoprotein lipase (synthesize by a number of sources) acts on TG1 portion in the chylomicron liberating F.F.A and glycerol.

F.F.A is metabolised by either: - converted to new TG - catabolic pathway(β-oxidation) - used in membrane synthesis

• The glycerol is transported to and absorbed by the liver or kidney where it is converted to glycerol-3-phosphate by the enzyme glycerol kinase,GK. Glycerol 3-phosphate (especially from hepatic) converted into dihydroxyacetonephosphate (DHAP) then glyceraldehyde-3-phosephate(G3P) to join glycolysis and gluconeogenesis pathway.

TG1 in chylomicronTG1 in chylomicron

F.F.AF.F.A GlycerolGlycerolGlycerol kinaseGlycerol kinase

Glycerol-3-POGlycerol-3-PO44TGTG

ββ-oxidation-oxidationMembrane Membrane synthesissynthesis

GlucoseGlucose

TGTG

phospholipidsphospholipids

Lipoprotein lipaseLipoprotein lipase

IN INTESTINEIN INTESTINE

CatabolismCatabolism

Anabolism Of Triglyceride

• Precursor is L-glycero-3-phosphate

• Proceed by condensation with acyl-CoA to form lysophospha- tidic acid (l.p.a), catalyse by enzyme E1

PEPPEP L-glycerol-3-POL-glycerol-3-PO44GlycerolGlycerol

11 22

*1 is glycerol-3-PO*1 is glycerol-3-PO4 4 DH ; 2 is glycerol kinaseDH ; 2 is glycerol kinase

**E1E1 is is glycerol-3-POglycerol-3-PO4 4 acyltranferaseacyltranferase

Further reactions on l.p.a till formation of TGFurther reactions on l.p.a till formation of TG

Cholesterols

CHOLESTEROL

• Is a soft, fat-like, waxy substance found in the bloodstream and membrane of cells (especially of the liver, spinal cord), and myelin sheaths and some hormones.

• require by cells as a precursor to bile

acids. • it is transported in the circulatory system

within lipoproteins.

• The most abundant of the steroids

**Steroids are complex derivatives of triterpenes They are characterized by a carbon skeleton consisting of four fused rings. 

• normal adult utilized ~1 gram of cholesterol daily. Approximately 70% of the amount produces by the liver. The other 30% comes from dietary intake

• Cholesterol is the precursor for all steroids. It is a common component of animal cell membranes and functions to help stabilize the membrane. Thus it is a crucial molecule

*high levels of it in the blood may contribute to atherosclerosis.

• Is not the usual mode i.e broken- down to smaller molecules

• Instead it is converted to the more soluble derivatives to facilitate its degradation and excretion.

• Most important mechanism is the formation of bile acids, in liver.

Catabolism Of Cholestrols

• Bile Acids (BA) are mixtures of compounds and possess digestive function as agent for emulsification and absorption of dietary fats. BA are important component of bile.

Cholic acid and deoxycholate are 2 of the components of BA.

cholesterolcholesterol

7-7-αα-hydroxycholestrl-hydroxycholestrl

Many2 stepsMany2 steps

Cholic acidCholic acid glycocholateglycocholate

Metabolism of CholesterolMetabolism of Cholesterol

(-ve charge)(-ve charge)(-ve charge)(-ve charge)glycineglycine

Cholic acidCholic acid

OxidationOxidation

HydrogenationHydrogenation

HydroxylationHydroxylation

Usually the BA are converted to a more soluble form by conjugation with glycine or taurine

glycine /NH2CH2CO2H

taurine (an a.a)/NH2CH2CH2SO3H

e.g Conjugation:

cholic acid +cholic acid + GlycineGlycine → → glycocholateglycocholate

cholic acid + taurine cholic acid + taurine → taurocholic acid → taurocholic acid

Taurocholic acid. In mammal it Taurocholic acid. In mammal it

exists as Naexists as Na++ salt. salt. In medical In medical

use, it is administered as use, it is administered as

cholagogue and choleretic.cholagogue and choleretic.

Glycocholic acid. It occurs Glycocholic acid. It occurs as a sodium salt in the as a sodium salt in the bile of mammalsbile of mammals

• Regulation of cholesterols level in blood -Absorbed dietary cholesterol increased

linearly with the increase of dietary cholesterol intake.

- The higher the fractional and absolute absorption of dietary cholesterol the lower the rates of biliary secretion, fecal elimination, and cholesterol synthesis (regulate cholesterol elimination and synthesis).

- high serum levels of total, LDL, and HDL cholesterol were associated with high cholesterol absorption

Anabolism Of Cholesterol

• Condensation of precursors, acetyl-CoA and acetoacetyl-CoA catalyse by Hydroxymethyl glutaryl CoA synthase (HMG-CoA synthase).

• Reactions proceed to formation of Mevalonate, catalysed by HMG-CoA Reductase . This rxn is rate-limiting

Reactions occur in cytosolReactions occur in cytosol

• To this structure other rings are added to form the final product

• Enzyme HMG-CoA Reductase is highly regulated and the target of pharmaceutical intervention.

Regulation of Cholesterol Synthesis:

• is not direct on cholesterol but through B.A• B.A is removed from pool by dietary fibers• Depletion of B.A induces synthesis of

cholesterol: activation of HMG-CoA synthase (Hydroxymethyl glutaryl CoA formation step) and HMG-CoA Reductase (mevalonate formation step)

The products of these phospholipases are called The products of these phospholipases are called lysophospholipids and can be substrates for acyl transferases lysophospholipids and can be substrates for acyl transferases utilizing different acyl-CoA groups. Lysophospholipids can also utilizing different acyl-CoA groups. Lysophospholipids can also accept acyl groups from other phospholipids in an exchange accept acyl groups from other phospholipids in an exchange reaction catalyzed by lysolecithin:lecithin acyltransferase reaction catalyzed by lysolecithin:lecithin acyltransferase (LLAT).(LLAT).

Catabolism of PhospholipidsCatabolism of Phospholipids

• phospholipase A2, lysophospholipase, and other enzymes are involved in phospholipid metabolism,

• Phospholipase A2 is an important enzyme, its activity is responsible for the release of arachidonic acid from the C-2 position of membrane phospholipids. The released arachidonate is then a substrate for the synthesis of the prostaglandins and leukotrienes.

• glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) are

competitive inhibitors of lysophospholipase activity, inhibits lysophospholipid hydrolysis

Anabolism of Phospholipids

Choline Acetylcholine                                                                CDP-Choline              

PE PS PC

**Phosphatidylserine (PS) Phosphatidylcholine (PC) CDP: cytidine-5’-diphospho

- CO- CO22

+ CO+ CO22

1,2-diglyceride1,2-diglyceride

** CDP: cytosinediphosphate** CDP: cytosinediphosphate

• DPGs:diphosphatidylglycerols. Also known as cardiolipins are synthesized by the condensation of CDP-diacylglycerol with phosphatidylglycerols (PG).

Clinical Effect• has not yet been fully evaluated, but

scientists have studied the role of choline and phospholipids in age related cognitive decline (ARCD), Alzheimer’s disease, and Parkinson’s disease

• good dietary intake of phospholipids, cholin lead to an improvement in learning and memory

• The fatty acid composition of phospholipids can deteriorate with aging and disease.

Regulation of synthesis• The fatty acid distribution at the C-1 and C-2 positions of

glycerol within phospholipids is continually in flux, owing to phospholipid degradation and the continuous phospholipid remodeling that occurs while these molecules are in membranes.

• Phospholipid degradation results from the action of phospholipases. There are various phospholipases that exhibit substrate specificities for different positions in phospholipids. In many cases the acyl group which was initially transferred to glycerol, by the action of the acyl transferases, is not the same acyl group present in the phospholipid when it resides within a membrane. The remodeling of acyl groups in phospholipids is the result of the action of phospholipase A1 and phospholipase A2

LIPID PROFILE

• Is a presentation of concentration of different lipid components in blood.

• Normally it involves determination of capillary blood cholesterol and triglyceride of fasting and non-fasting subject.

• Concentration of the lipid component is determined using a specific test strips and GCT meter

• Low and high readings are indicative to some form of health state. ** refer manual for details

Lipid Metabolic Disorder

• Abnormalities in the enzymes in lipid metabolism result in 2 types of disorder.

1. Lipidosis : case when there is accumulation of specific fatty substances due to abnormalities in the enzymes that are involved in assimilation of the specific fatty substances eg. Gaucher's Disease, Tay-Sachs Disease, Niemann-Pick Disease Fabry’s Disease; rare case: Wolman's disease, sitosterolemia, Refsum's disease

2. Fatty acid oxidation disorder : When body is unable to properly convert fats into energy due to abnormalities of enzymes in the fatty acid oxidation pathway. Eg medium chain acyl-CoA dehydrogenase (MCAD) deficiency

• Gaucher's Disease, most common. - accumulation of glucocerebrosides in liver and

spleen, most common in Ashkenazi (Eastern European) Jews leads to enlargment of the organs and brownish pigmentation of skin.

- Accumulations of glucocerebrosides in the eyes cause yellow spots called pingueculae to appear in the eye

- Accumulations in the bone marrow can cause pain and destroy bone.

- 3 types :

i) Type 1, the chronic form, with symptom of enlarged liver and spleen and bone abnormalities. More common among adults

ii) Type 2, develops in infancy; infants with the disease have enlarged spleen and severe nervous system abnormalities and usually die within a year.

iii) Type 3, the juvenile form, can begin at any time during childhood. Children with the disease have an enlarged liver and spleen, bone abnormalities, and slowly develop progressive nervous system abnormalities. Children who survive to adolescence may live for many years.

Gaucher's disease can be treated with enzyme replacement therapy

• Tay-Sachs disease : accumulate gangliosides in tissues, most common in families of Eastern European Jewish origin.

At early age, children with this disease become progressively retarded and appear to have floppy muscle tone. Spasticity develops and is followed by paralysis, dementia, and blindness.

Patient usually die by age 3 or 4. Tay-Sachs disease can be identified in the fetus by chorionic villus sampling or amniocentesis. The disease cannot be treated or cured.

• Niemann-Pick disease: accumulation of sphingomyelin or cholesterol; has several forms, depending on the severity of the enzyme deficiency and thus accumulation of sphingomyelin or cholesterol. The most severe forms tend to occur in Jewish people. The milder forms occur in all ethnic groups.

The most severe form (type A), children fail to grow properly and have multiple neurologic problems. These children usually die by age 3

Type B, disease develops fatty growths in the skin, areas of dark pigmentation, and an enlarged liver, spleen, and lymph nodes; may be mentally retarded.

Type C, disease develops symptoms in childhood, with seizures and neurologic deterioration.

Some forms of the disease can be diagnosed in the fetus by chorionic villus sampling or amniocentesis. After birth, the diagnosis can be made by a liver biopsy None of the types can be cured.