A – Retinol

1.     Comes from animal sources as retinol.

-       Important:

o   Vision

o   Reproduction

o   Growth

o   Epithelial tissue

o   Immune functions

-       Retinol oxidizes to retinoic acid

-       Vision depends on retinal which is the aldehyde derivate of retinol.

2.     Beta-carotene (provitamin A) comes from plants and oxidizes and is cleaved by intestine enzyme but the conversion to retinal is inefficient.

Biochemistry of A

Retinol (alcohol)  oxidize to retinal, aldehyde (interconvertible reaction)  retinoic acid is oxidized in liver (not interconvertible).

Functions of retinal

-       Vision (monochromatic)

-       Retinal exist as 11 cis retinal bound to opsin (protein) visual pigment rhodopsin

-       Rod cell contains outer segment filled with membrane discs. Each disc contains millions of rhodopsin molecules

-       Illumination = rhodopsin associated 11-cis retinal isomerize into all trans retinal  change in shape  electric signal is generated  optical nerve  vision center in brain.

-       Rhodopsin regenerates in dark.

Functions of retinoic acid

-       Hormone derived from retinal.

-       Transported by specific carrier proteins in blood.

-       Enters epithelial cells due to solubility in fats and lipids  binds to retinoic acid receptors (RAR)  responsible for epithelium regeneration by activation of gene transcription.

-       RAR is part of the family of transcriptional regulators.

Ø  Treatment of acute promyelocytic leukaemia, acne and skin aging.

Dosage

RDA: 900 retinol activity equivalents = 0,9 mg for male and 700 RAE = 0,7 mg female.

Deficiency

-       Nuctalopia (night blindness)

-       Loss of visual cells if deficiency is chronic.

-       Xerophtalmia: dryness of cornea and conjunctiva by increased keratin synthesis can lead to blindness because of scar tissue formation.

Toxicity

-       Hypervitaminosis A

-       > 7,5 mg/day of retinol

-       Early signs: dry skin and itching (pruritic)

-       Chronic: enlarged liver and cirrhotic, increased cranial pressure which mimic tumour.

-       Pregnancy: teratogenesis (malformation of fetus)

Sources

-       Animal products: liver, kidney, cream, butter, and egg yolk are good sources of preformed vitamin A.

-       Plant products: yellow, orange, and dark-green vegetables and fruits are good sources of the carotenes (provitamin A).

D – calciol

-       Sterol

-       Not required in diet

-       Synthesised through exposure to sunlight.

-       Dietary vitamin D  hormone 1,25-dihydroxycholecalciferol (active form of D3)

o   D3 (cholecalciferol) from animals

o   D2 (ergocalciferol) from plants. Has additional double bond and methyl group

Synthesis of hormone 1,25-dihydroxycholecalciferol

-       From endogenous source: 7-dehydrocholesterol from the skin convert to D3 once UV light reaches skin.

1.     Absorption of dietary vitamin D in the presence of bile.

2.     Carrier-mediated Transportation into the liver

3.     Hydroxylation by 25-hydroxylase in liver cells

4.     Transportation of 25-hydroxycholecalciferol into kidney 

5.     Hydroxylation by 1-hydroxylase in the cells of kidney

6.     Transportation by blood

-       From exogenous source: D2

o   Limiting steps:

1.     Absorption of dietary vitamin D.

2.     Activity of renal enzyme 1-hydroxylase (dependent on parathyroid hormone)

Mechanism of 1,25-dihydroxycholecalciferol

-       Hydrophobic hormone

-       Calcitriol is bound to carrier proteins (vitamin D binding protein and albumin)

-       Once in cell  binds to Vitamin D receptor (VDR)  calcitriol and receptor complex acts as transcription factor for mostly regulation of Ca ion homeostasis.

-       Target mainly: osteoclastocytes and osteoblastocytes, cells of intestinal mucosa

-       Increase Ca ion concentration in blood because of activation of ca absorption in intestine.

Dosage

RDA: 15 micrograms/day (1-70 years), 20 micrograms/day (70+)

Supplements for babies, due to lack of vitamin D in milk.

Deficiency:

-       Rickets: incomplete mineralization of bones

-       Ostemalacia: demineralization of bones

Sources: Fish and plant oils, fatty fish, liver, eggs and non-processed milk

Toxicity

-       100,000 IU = 2,5 mg  loss of appetite, nausea, thirst, weakness, hypercalcemia (since absorption of Ca ions increase and can deposit in soft tissue)

-       Only seen with supplements because excess of Vitamin D in skin converts to inactive form.

E – tocopherols

-       8 naturally occurring compounds.

o   4 tocopherols, differ in number of methyl groups.

o   4 tocotrienols

-       Tocopherols have isoprene chain attached.

-       E acts as antioxidant in lipids  binds to free radicals, stop formation of peroxides.

Deficiency

-       Oxidative damage to cell membrane due to peroxidation of phospholipids.

-       Rear with deficiency

Source

-       Seed oil, liver, egg

Dosage

RDA: 15 mg/day

Antioxidant mechanism

Vitamin K

-       Exist in several forms:

o   Plants: phylloquinone (vitamin K1)

o   Intestinal bacteria: a family of menaquinones (vitamin K2)

-       Essential cofactor (for gamma glutamyl carboxylase) for carboxylation of glutamic acid residues in proteins from blood coagulation and bone formation.

Biochemical function

-       Coagulation factors (proteins) are synthesized and secreted from liver as inactive proteins.

-       Activated by post transitional modification (glutamyl carboxylation)

-       Gamma-carboxyglutamyl act as Ca ion binding centers.

-       Calcium attachment at Gamma-carboxyglutamyl residue favors binding of coagulation factors to membranes of damage endothelium of BV and platelets.

-       Binding is essential for activating coagulation factors.

Dosage

-       90 – 120 micrograms/day

Source

-       spinach, broccoli, cabbage, beans, asparagus

Deficiency

-       Antibiotics can decrease amount of bacteria producing Vitamin K

-       Bleeding is main symptom.

-       Anticoagulant warfarin prevents normal regeneration of vitamin K. It also perturbs blood coagulation although amounts of vitamin K are not decreased.

Origin of fatty acids

-       Fats: Neutral lipids, molecules are esters of glycerol and long chain carboxy acids.

-       Fatty acids: long chain carboxylic acids released from fats after their hydrolysis.

Sources:

-       Dietary fats: products from digestion are delivered with blood lipoproteins (chylomicrons) and hydrolyse in the blood for donation of fatty acids to tissue.

-       Adipose tissue: Store of endogenous fats. After enzymatic breakdown (endogenous lipolysis), fatty acids are delivered to the blood.

o   Bile salts emulsify fats which gives intestinal lipases the opportunity to degrade TAG  fatty acids are absorbed and converted into TAG  Transported by chylomicron through lymph system  lipases release free fatty acids and glycerol into myocyte or adipose.

Free fatty acids in tissue?

Ø  Breakdown (depends on vascularity, mitochondria and tissue morphology)

Ø  TAG (triglyceride) accumulation in adipocytes. (they cannot breakdown fatty acids).

Ø  Incomplete breakdown of fatty acids in liver  ketone bodies are formed.

Breakdown

1.     Beta-oxidation,

2.     acetyl-CoA decomposition in Krebs cycle,

3.     Oxidation of NADH and FADH2 by ETC.

Ø  Some cells can oxidize glucose and Fatty acids simultaneously.

Ø  FA oxidation is less common.

Prior to beta-oxidation fatty acid activation is required.

-       Free fatty acids are inactive.

-       Anabolism and catabolism begin with activation by conjugation with CoA.

o   Carried out by acetyl-CoA synthetase on the outer membrane of mitochondria.

o   Acyl-S-CoA is activated fatty acid. And 2 ATP is used in hydrolysis.

First event of decomposition

Ø  Acyl-unit transfer into mitochondria:

o   done by carnitine shuttle, comprised by isoenzymes of CPT I and II and mobile carrier carnitine.

o   When it enters the mitochondria  beta-oxidation.

Beta-Oxidation

-       Under each cycle, covalent bond is broken after the second or beta carbon atom in acyl unit.

-       4 enzymes specific to the number of C atoms in the unit.

-       In a single cycle, acetyl-unit is released as acetyl-CoA.

-       Number of acetyl-CoA molecules released in all cycles of B-oxidation is twice less than the number of C atoms in fatty acid subjected to breakdown.

-       Number of cycles of B-oxidations required to decompose fatty acid, is equal (n-1), where n is the number of acetyl-CoA molecules.

Stages:

1.     Oxidation of 2^nd and 3^rd C atom

2.     Adding of water molecule to double bond

3.     Oxidation of 3^rd C atom

4.     Breakdown of bond between C 2 and 3.

5.     Continuous until acyl unit is decomposed into acetyl-CoA.

Energy yield in FA oxidation depends on number of C atoms in fatty acid molecule and saturation level of fatty acid.

Oxidation of unsaturated fatty acids.

1.     Beta-oxidation occurs in a regular way until double bond is approached.

2.     Isomerization into trans-configuration from cis.

3.     One round of beta-oxidation is done by one reaction shorter  FADH2 is not produced. Loss of 2 ATP.

Oxidation of odd chain containing FA

-       Milk & ruminant.

-       In beta oxidation, these fatty acids are decomposed in a regular way until 3C atoms molecule of propionyl-CoA is formed.

-        3 sequential reactions occur:

o   First needs biotin.

o   Third needs B12

-       Oxidation of propionyl-CoA results in small energy output.

Ketone body synthesis

Ø  FA oxidation produce big amounts of acetyl-CoA which has to be broken down in mitochondria.

Ø  Liver: activity of Krebs cycle can be diminished because of oxaloacetate withdrawal for gluconeogenesis.

Ø  Excess of Acetyl-CoA can convert to ketone bodies.

b.     In blood of healthy individuals, concentration of ketone bodies is up to 2 mg/100 ml of plasma (the mean value is about 1 mg/100 ml or less than 0.6 mmol/L).

c.     Acetoacetate + 3-hydroxybutyrate are the principal acid ketone bodies in a healthy individual.

d.     Acetone is a spontaneous product of acetoacetate decarboxylation occurring under high concentrations of acetoacetate (in starvation and DM). This is not an acid.

Synthesis in liver mitochondria

Ketone bodies are formed when both acetoacetate and D-B-hydroxybutyrate are high!

Consumption of ketone bodies

-       70-98% consumed by extrahepatic tissue.

-       2-30% excreted with urine.

-       Brain + heart is principal consumers.

-       High amounts are produced under starvation.

-       Ketonuria: ketones in urine

-       Ketonemia: High ketone bodies concentration

Consumption in mitochondria

Ketonemia

Ø  0.6 - 1.5 mmol/L  recheck blood glucose and ketones in 2-4h

Ø  1.5 – 3.0 mmol/L  risk of ketoacidosis

Ø  > 3.0 mmol/L  Emergency treatment

Synthesis of fatty acids and their derivates

Non-essential fatty acids

-       All saturated fatty acids with even number of C-atoms in the chain, can be synthesized in humans.

-       These fatty acids are not obligatory in human diet.

-       Some unsaturated fatty acids come from saturated when enzymes make doble bonds after carbon atoms number 9, 6, 5, and 4 in molecules of fatty acids.

Essential fatty acids

-       Unsaturated fatty acids containing double bonds further than carbon atom number 10.

-       They must be included in human diet. There are two essential fatty acids linoleic (omega-6) and α-linolenic acids (omega-3).

Characteristics of FA synthesis

-       Occurs:

o   Under insufficient dietary intake of lipids

o   Excess of glucose in blood

-       Occurs in smooth ER of hepatocytes by multienzyme complex (FA synthase or palmitic acid synthase).

-       Fatty acid chains are constructed by adding two carbon atoms which come from acetyl-CoA. Glucose-derived acetyl-CoA is a preferable substrate for this synthesis.

-       As a substrate, acetyl-CoA has 2 functions:

o   1 molecule of acetyl-CoA (2C) initiates syntheses if fatty acid.

o   Acetyl-CoA is converted to three C-atoms containing malonyl-CoA, which donates 2C-atom increase the length of C-atom chain in synthesized palmitic acid molecule.

-       Under fatty acid synthesis, reduction of C-atoms occurs in expense of NADP. NADPH comes from the pentose phosphate pathway.

Stages:

Ø  Priming stage: synthesis of malonyl CoA from acetyl-CoA

o   Occurs in cytosol of liver cells.

o   Acetyl-CoA is transported through citrate shuttle from the mitochondria.

o   Acetyl-CoA carboxylase is a complex enzyme containing biotin as a coenzyme.

1.     Regulation: Citrate (+), Palmitic acid (-)

2.     Reversible phosphorylation: insulin trigger dephosphorylation and activates it, glucagon does opposite.

3.     Amount of acetyl-CoA is regulated at level of gene transcription.

Ø  Initiation stage: Binding acetyl- and malonyl-group to acyl-carrier protein of FA synthase

Ø  Condensation stage: Formation of 4 C-atoms containg Butiryl-unit in active site of FA synthase

Ø  Elongation stage: Increase in the length of fatty acid occurs in expense of malonyl-unit which donates two C-atoms. Elongation stage occurs until sixteen C-atom chain is produced in the active site of fatty acid synthase.

Ø  Reduction stage: After the addition of each two-carbon atoms unit, the growing chain undergoes two reduction reactions that require NADPH.

Ø  Release of palmitic acid from FA synthase

Characteristics of fatty acid synthase

-       Fatty acid synthase is a large enzyme composed of two identical subunits (i.e. highly folded polypeptide chains).

-       Each subunit has seven catalytic activities and an acyl carrier protein (ACP) segment in a continuous polypeptide chain.

-       The ACP segment contains a phosphopantetheine residue that is derived from the cleavage of coenzyme A.

-       The key feature of the ACP is that it contains a free sulfhydryl group (from the phosphopantetheine residue).

-       The two dimers associate in a head-to-tail arrangement, so that the phosphopantetheinyl sulfhydryl group on one subunit and a cysteinyl sulfhydryl group on another subunit are closely aligned.

Palmitic acid synthesis in FA synthase complex.

1.     Acetyl-CoA donates acetyl-unit to ACP (phosphopantethein component - P) in the active site.

2.     Transfer of acetyl-unit to SH-group of cysteine residue in the active site.

3.     1^st: Binding of malonyl-unit from malonyl-CoA to vacant ACP in the active site.

4.     2^nd: Condensation acetyl and malonyl units with acetoacetyl-unit formation at ACP and CO2 liberation.

5.     3^rd: Reduction of acetoacetyl-unit at ACP using NADPH.

6.     4^th: Dehydration with formation of unsaturated acyl-unit.

7.     5^th: Reduction of unsaturated acyl-unit into butyryl unit attached at ACP.

8.     Then, all processes are repeated, 1st reaction is transfer of butyryl-unit onto cysteine residues and binding of new malonyl-unit onto ACP,2nd reaction is condensation of acetyl-unit with malonyl-unit followed by CO2 liberation, then reduction, dehydration and second reduction occur to produce 6 C atoms containing fatty acid unit attached to ACP.

Termination of fatty acid synthesis

-       End product of FA synthase is balmitoyl-unit which stays attached to ACP in the active site.

-       Palmitoyl-thioesterase domain the active site breaks down the bond between SH group of phosphopatethein and palmitoyl unit releasing palmitic acid (palmitate) into cytosol.

The overall reaction equation of palmitic acid synthesis from acetyl CoA:

8 Acetyl-CoA (2C) + 14 NADPH + 13H+ + 7ATP → Palmitate (16C) + 8 CoA-SH + 6 H2O + 14 NADP+ + 7 ADP + 7 Pi

Regulation

Central regulatory point: acetyl-CoA carboxylase.

-       Its product malonyl-CoA inhibits CPTI  the FA oxidation does not occur when FA synthesis continues.

-       Hormones which act through cAMP, promote phosphorylation, therefore activity of acetyl-CoA decarboxylase decrease.

Elongation of C-atom chain in palmitic acid. Synthesis of long chain fatty acids

-       Although palmitate, a 16-carbon, fully saturated LCFA (16:0), is the primary end product, it can be further elongated by the addition of two-carbon units to the carboxylate end primarily in the smooth endoplasmic reticulum (SER).

o   This is process of elongation.

o   Elongation is catalyzed by microsomal fatty acid elongation system (elongase), which comprises several enzymes.

-       Before elongation, palmitic acid is subjected to activation by conjugation with CoA by a help of palmitoyl-CoA synthetase:

-       Malonyl-CoA serves as the donor of the two-carbon units, and NADPH acts as a reductant in the same way as if FA synthase.

-       The major elongation reaction that occurs in the body involves the conversion of palmityl- CoA (C16) to stearyl-CoA (C18). Very-long-chain fatty acids (C22 to C24) are also produced, particularly in the brain.

o   Requires a system of separate enzymes rather than a multifunctional enzyme.

-       Any type of fatty acid (saturated and unsaturated) can be subjected to elongation.

-       In brain cells, elongation generates very long chain fatty acids needed for myelination of nerve fibers.

Synthesis of non-essential unsaturated fatty acids

-       In smooth endoplasmic reticulum of cells, unsaturated fatty acids are produced outside FA synthase.

o   The enzyme is desaturase.

-       Synthesis of unsaturated fatty acids uses NADH as a source of electrons, which reach fatty acid via iron containing cytochrome b5 by a help of an enzyme cytochrome b5 reductase. When fatty acid accepts a pair of electrons, double bond is formed.

-       Oxygen is consumed.

-       Desaturases are specific regarding C atom in account to carboxyl-group (5- , 6- , and ‚9- desaturases).

-       There is no desaturase which can act on C atom more distant than C10.

-       The most common non-essential unsaturated fatty acids include palmitoleic and oleic acids.

Metabolism of essential fatty acids

Acts as:

-       Substrates for synthesis of complex lipids (e.g. phospholipds) needed to form and reconstruct of cellular membranes (plasma membrane and endo-membranes in mitochondria, endoplasmic reticulum, Golgi complex).

-       Substrates for synthesis of eicosanoids (molecules essential for specific cell functions).

-       Role of essential fatty acids in synthesis of eicosanoids.

-       Primarily, linolic and alpha linoleic fatty acids act as substrates for further elongation and desaturation to produce polyunsaturated fatty acid with 20 C-atoms in the chain - arachidonic acid.

Synthesis of eicosanoids

-       Eicosanoids: group of 20 C atoms containing compounds synthesized from arachidonic and other polyunsaturated fatty acids.

-       Synthesis occurs in many tissue cells.

-       Act to regulate tissue functions:

o   inflammation, pain, smooth muscle contraction, regulation of urine formation in the kidney, blood clotting, bronchoconstriction and relaxation, and etc.

Synthesis of prostaglandins, thromboxanes and leucotrients from arachidonic acid

Principles of arachidonic acid action in eicosanoid synthesis

Inactivation of eicosanoids

-       Prostaglandins and thromboxanes are rapidly inactivated.

o   Their half-lives (t1/2) range from seconds to minutes.

-       Inactivated by oxidation of the 15-hydroxy group, critical for their activity, to a ketone. The double-bond at carbon 13 is reduced. Subsequently, both B- and Omega-oxidation of the nonring portions occur  producing dicarboxylic acids that are excreted in the urine.

-       Active TXA2 (tromboxane 2) is rapidly metabolized to TXB2 by cleavage of the oxygen bridge between carbons 9 and 11 to form two hydroxyl groups. TXB2 has no biologic activity.

Summary: oxidation of eicosanoids is a principal way of their inactivation.