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Dr. Munaf S. Dauod

Carbohydrates (CHO) Definition: Aldehyde or Ketone derivatives of the higher polyhydric alcohols or compounds which yield these derivatives on hydrolysis.

Classification: (mono, di, oligo, poly) saccharide.

Monosaccharides: Can be classified as trioses, tetroses, pentoses, hexoses and heptoses depending upon the number of carbon atoms, and as aldoses or ketoses, depending upon whether they have an aldehyde or ketone group. Aldehyde (-CHO) Aldoses Ketone (-C=O) Ketoses

Polysaccharides (glycans):

Homopolysaccharides (homoglycans): e.g. starch, glycogen, inulin, cellulose, dextrins, dextrans.

Heteropolysaccharides (heteroglycans): e.g. mucopolysaccharides (MPS) or glycosaminoglycans.

Function of CHO: 1) Chief source of energy (immediate and stored energy). 2) Constituent of compound lipids and conjugated protein. 3) Structural element like cellulose. 4) Drugs like cardiac glycosides and antibodies. 5) Lactating mammary gland (Lactose in milk). 6) Synthesis of other substances like fatty acids, cholesterol, amino

acids…etc. by their degradation products. 7) Constituent of mucopolysaccharides.

1) Stereo-isomerism

Stereo-isomers: D-form, L-form 2) Optical isomers (optical activity)

Enantiomers: dextrorotatory (d or + sign) Levorotatory (l or – sign) Racemic (d l)

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3) Cyclic structures or open chain

4) Anomers and Anomeric carbon

OH on carbon number 1, if below the plane then its -form, if above

the plane then -form.

Mutarotation: the changes of the initial optical rotation that takes place

in an aldohexose e.g. glucose (-form) when put (dissolved) in water and

the solution is put in optical path (plane polarized light). Sugar -glucose

will change into -form and a mixture of both ( and ). Glucose contains 4 asymmetrical carbon atoms no.s 2,3,4,5 whereas no.s 1 and 6 are symmetrical C atoms, which means that glucose has 16 isomers (8 of D-form and 8 of L-form) and according to OH position will have 16 D & 16 L, a total of 32 isomers.

Reactions: 1- Reducing action

2- Phenyl hydrazine osazone 3- Ester formation with H2SO4, H3PO4, Glc1P, Glc6P, Frc6P, glucosamine,

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glucose Sulphate. 4- Glucoside formation

e.g. Glc + Glc maltose Glc + Frc sucrose Glc + Gal Lactose

Glucose is found in nature in cyclic form either Pyranose and called

glucopyranose or furanose and called glucofuranose. Epimers of Glucose are: Galactose - orientation of OH on C no. 4 Mannose - orientation of OH on C no. 2 Allose - orientation of OH on C no. 3

5- Oxidation to produce sugar acids: (a) Aldonic acid e.g.

(b) Saccharic acid e.g. Aldaric acid

(c) uronic acid e.g.

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6- Reduction of sugars to form sugar alcohols: e.g.

Gal. forms Dulcitol ; Man. forms mannitol.

Sugar Derivatives: 1) Deoxy sugars: the oxygen of OH group has been removed. e.g. 2-deoxyribose found in DNA. 2) Amino sugars (contain an NH2 group) e.g. Glycosylamine (OH is replaced by NH2 group) like ribosylamine involved in purine synthesis. e.g. Glycosylamine like the two naturally occuring derviatives of Glc and Gal the glucosamine & galactosamine (OH on C-2 is replaced by NH2), found in MPS. 3) Amino sugar acids e.g. Neuraminic acid: found in nature in acylated derivative known as sialic acid (N- acetyl neuraminic acid, NANA) found in MPS and gangliosides. 4) Glycosides: CHO residue (glycone) linked to non-CHO residue (aglycone) by an acetal linkage by carbon 1. aglycone may be methyl alcohol, glycerol, phenol, sterol ... etc. If the sugar molecule (glycone) was Glc, Gal, then it’s called glucoside, galactoside. e.g. cardiac glycosides are important for its action on heart and antibiotics like streptomycin. Disaccharides: (mono + monosaccharides)

1) Maltose: Glc + Glc

It is linked by 1 4 glycosidic linkage and is -form, found in starch

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on hydrolysis by amylase.

2) Lactose: Glc + Gal

It is linked by 1 4 glycosidic linkage and is in -form, found in milk.

3) Sucrose: Glc + Frc

It is linked by 1 2 glycosidic linkage and is -glucopyranosyl - fructosfuranoside, found in cane (table sugar) and beet.

Oligosaccharides: 3-6 monosaccharides residues

1) Carbohydrate units that are attached to integral membrane proteins. 2) Attached to side chain O2 atom of Ser or Thr (aminoacids) by O-

glycosidic linkage, or nitrogen atom of Asn by N-glycosidic linkage. 3) N-linked oligosaccharides contain a common pentasaccharide core

consisting of 3 mannose and 2 N-acetyl glycosamine residues. 4) Glycoproteins function in molecular targeting and cell-cell

recognition. 5) Found in proteins like antibodies and coagulation factors and peptide

hormones. Polysaccharides:

A- Homopolysaccharides:

1) Starch-Polymer of Glc found in many plants, composed of two units Amylose and amylopectin, hydrolyzed by Amylase to give soluble starch Amylodextrin Erythro-dextrin Achrodextrin Maltose, then by maltase to give 2 Glc

2) Glycogen (animal starch) – polymer of Glc found in animals (storage form of Glc), found in the liver and muscles. Its synthesis from Glc is called glycogenesis, and its breakdown back into Glc is called Glycogenolysis.

3) Inulin-polymer of fructose found in tubers and roots of certain plants,

used for the determination of GFR (Glomerular Filtration Rate) and body water volume.

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4) Cellulose- polymer of Glc and its disaccharide is cellobiose made of

two D-Glc linked by β-glucosidic linkage (so its β-form) between C1 and C4.

5) Dextrin, it’s formed of starch hydrolysis used with maltose in infant

feeding.

6) Heteropolysaccharides (MPS) 1- Hyaluronic acid

(N-acetyl glucosamine + glucuronic Acid) acts as a cementing substance and as lubricant and shock absorbent, so it’s found in synovial fluid and skin, in free form or combined with proteins (so called ground substance of connective and other tissues). Hyaluronidase enzyme acts on it, and reduces its viscosity.

2- Chondroitin (N-acetyl galactosamine + Glucuronic acid) Found in the cornea of the eye and in the cranial cartilage.

Both hyaluronic acid and chondroitin are sulphate free.

3- Keratan sulphate Found in costal cartilages and Cornea (N-acetyl glucosamine + galactose) No uronic acid present.

4- Chondroitin sulphate Major MPS in the ground substance of mammalian tissue and cartilage, occurs combined with proteins (N-acetylgalactosamine

(sulphated) + Glucuronic acid). Cartilage, bone, cornea, tendons.

Iduronic acid may replace in some types of the 4 chondroitin sulphates (Types A, B, C, D).

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5- Heparin (sulphated MPS)

Anticoagulant; produced by mast cells of liver. Found in lungs, thymus, spleen, wall of large arteries, skin, and blood. Glucosamine + Glucuronic acid (Glc UA)

Iduronic acid (IDUA)

Combine to proteins as in proteoglycans

Iduronic acid is an isomer of glucuronic acid (an epimer) it is formed in liver from D-glucose

6- Neutral MPS. e.g. Blood group substances (blood groups A,B,O) contain peptides or aminoacids as well as CHO. Four monosaccharides and derivatives are found in all types of blood group substances; Gal, L-Fucose, Galactosamine (acetylated) & acetylated glucosamine. Mucopolysaccharidosis: Group of related disorders due to inherited enzyme defect. Deposits of MPS in tissues and excess excretion in urine. Six types are known and Clinical signs are skeletal changes, mental retardation, corneal clouding…etc. Iduronic acid is an isomer of glucuronic acid (an epimer).It is formed in liver from D-glucose.

Sialic Acid: Neuraminic acid, it is an amino sugar acid and structurally an aldol

condensation product of pyruvic acid and D-Mannosamine.

Neuraminic acid is unstable and found in nature in the form of acetylated derivatives known as sialic acid (N-acetyl Neuraminic acid – “NANA”)

Neuraminic acid sialic acids occur in a number of muccopoly-saccharides (MPS) and in glycolipids like Gangliosides.

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CO2+H2O

Mannosamine + pyruvic acid Condensation neuraminic acid

acetylation N-acetylated neuraminic acid (NANA) or sialic acid.

Sialic acid is found in mucin and blood groups substances.

For its synthesis, Mannose sugar and pyruvic acid (end product of glycolysis) are required.

Sialic acid level in the blood is considered to be a tumor marker. Its level is increased in cases of tissue destruction due to certain diseases. Its level is decreased after surgical removal of the tumor to its normal levels. If carcinoma is reactivated (back again), its level is again increased so patients should test blood sialic acid every 3-4 months.

Biological Energy

It’s the energy of anabolism and catabolism that results from chemical changes occurring in the organism. Anabolic reactions: energy is needed to synthesize a compound from its simplest component. Such energy requiring reactions are known as endergonic reactions. Catabolic reactions: energy is released from breaking down the compound or substance, such type of energy releasing reactions are known as exergonic (energy liberating) reactions, this released energy can be used to do work. Source of energy: Ultimate source for all living matters is the sunlight that converts CO2 and H2O into CHO (starch). Starch converts it into glucose (in the body) which give energy (E) on oxidation. This energy can be stored in many compounds, mainly in the nucleotide (ATP)

Plants Photosynthesis

Glucose Starch

Sunlight

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Starch Body

Glucose Oxidation (O2)

Energy (E) ADP + Pi + E ATP

The process of coupling ADP, Pi and E is known as oxidative phosphorylation. Kinetic energy is also stored in other nucleotide compounds: e.g. GTP: is used to supply energy in Protein synthesis. CTP: is used to supply energy in Lipid synthesis. UTP: is used to supply energy in polysaccharide synthesis.

The above compounds contain two energy-rich or high energy phosphate bonds. Base Ribose P P P Base may be Adenine, Guanine, Cytosine or Uracil. Other nucleotides that store energy in its phosphate bond are ADP, GDP… etc. These contain only one high energy phosphate bond and therefore of less energy. Other compounds that store energy as phosphate bond are: Creatine phosphate (in muscle), Glyceraldehyde-3-phosphate and phosphoenol pyruvate (Formed in glycolysis). Acetyl CoA (active acetate) This compound formed by β-oxidation of fatty acids and glycolysis is important in the citric acid (Kreb’s) cycle. The energy is in its thioesters bond. O CH3C SCoA

ATP acts as universal currency for energy in cells

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Carbohydrate Metabolism

1. Carried out in every cell in the body 2. In the cytoplasm (cytosol) •Glycolysis •Glycogenesis •Glycogenolysis

and others. 3. In the Mitochondria (membranes and matrix) • citric acid cycle •

Electron Transport chain.

Others are:

Hexose monophosphate shunt (HMP shunt).

Glucosamine pathway.

Uronic acid pathway. Note: Gluconeogenesis is both cytosolic and mitochondrial. After digestion and absorption, glucose in the blood circulation enters the

cell and become phosphorylated (i.e. activation with phosphate given by ATP).

This phosphorylation occurs on cell membranes by the action of two enzymes: 1- Specific enzyme glucokinase (GK) in the liver. 2- Non-specific enzyme hexokinase in the liver and other extrahepatic

tissue.

Glucose-6-phosphate is formed as a product. Glc-6-P is an important compound at the junction of several metabolic pathways. The reaction is irreversible. (See the figure)

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Figure

of Glc phosphorylation

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Differences between HK and GK

HK GK

1- Nonspecific; can phosphorylate any of the hexoses

2- Found in all tissues

3- Found in fetus and adults liver

4- Allosteric inhibition by Glc-6-P

5- Low Km (0.1 mM) high affinity for glucose

6- Not very much influenced by diabetic state or fasting

7- Not induced by Glc feeding

8- Insulin doesn’t have effect on it.

9- Its Main function is to make Glc available to tissues for oxidation at lower Glc level

1- Specific for Glc only

2- Found in liver only (hepatic enzyme).

3- Found in adult liver only. 4- Not inhibited by Glc-6-P 5- High Km (10 mM)

Low affinity for Glucose 6- Depressed in fasting and in

diabetes. GK is deficient in patients of DM changes according to nutritional status.

7- Induced by Glc feeding after fasting.

8- Insulin stimulates it inducible enzyme

9- To clear Glc from blood after meals and at blood levels > 100 mg/dl.

Both enzymes require Mg+2 or Mn+2 as cofactors by forming complexes with ATP. The reaction of phosphorylation is irreversible because the energy in Glc and ATP is higher than the energy in Glc-6-P, so there Is loss of energy and reaction goes forward only. Glc-6-P cannot provide the reaction with energy that is required for its reversion and the production of ATP+Glc.

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1-Glycolysis: Glycolytic pathway or Embden Meyerhof pathway.

Cytosolic used by all tissues for breakdown of glucose to give energy (in the form of ATP) and intermediates for other metabolic pathways.

Aerobic (in Presence of oxygen) Glc Pyruvate. Anaerobic (in absence of oxygen) Glc Lactate.

Glc (6-C) is lysed by a series of 9 reactions into 2 molecules of pyruvate (3-C) or lactate (3-C).

Stages of Glycolysis: Stage 1: Energy requiring stage

Note: Two ATP are used for one mole of glucose. Stage 2: Energy producing stage ○1 ○2

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○1- Inhibited by iodoacetate ○2- Inhibited by arsenate ○3- Inhibited by fluoride

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Regulatory Enzymes HK = Hexokinase PFK = Phosphofructokinase PK = Pyruvate Kinase

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The phosphorylation that occurs at the expense of inorganic phosphate (Pi) of a compound is known as substrate-level phosphorylation. e.g. Glycrald. 3-P 1,3 bis PG 1,3 bis PG 3 PG PEP Pyruvate Bis is of two parts; Bi =ثنائي, while s = “separated” (i.e. on different locations) Glycerald. 3-P converts into 2,3 bis PG or 2,3 BPG or 1,3 DPG and is present in most cells at low concentrations, but in the RBCs (erythrocytes) it is at high concentration (4 mM) which is equal to hemoglobin. It acts as a regulator of oxygen transport, stabilizing the deoxygenated form of hemoglobin.

Glycrald. 3-P 1,3 bis PG 2,3 bis PG 3PG

Mutase Phosphatase

Glycolysis and RBC Metabolism: 1- Mature RBCs or Erythrocytes contain NO mitochondria, so they are

totally dependent on glycolysis for ATP production. 2- ATP is required for the ATPase-ion transport system which is

necessary to maintain the proper biconcave shape of the erythrocyte membrane.

Energy or ATP production:

1- Anaerobic Glycolysis 2 moles ATP used Glc Glc 6P Frc 6P Frc 1,6 bis P 2ATP 2 moles ATP produced ( 1,3 bis PG 3 PG) 2 moles ATP produced (PEP Pyruvate) 4ATP net ATP produced is 4-2= 2ATP

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Produced by substrate-level phosphorylation

2- Aerobic Glycolysis 2 moles ATP used Glc Glc 6P Frc 6P Frc 1,6 bis P 2ATP 2 moles ATP produced ( 1,3 bis PG 3 PG) 2 moles ATP produced (PEP Pyruvate) 4ATP net ATP produced is 4-2= 2ATP Produced by substrate-level phosphorylation Plus 2 NADH that give 4 moles ATP through the Glycerol 3-Phosphate shuttle

(NADH Glycerol 3-P FADH2 4 ATP) ETC

Net ATP produced is 2+4 = 6ATP

Glycerol 3-Phosphate shuttle

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Malate Shuttle: NADH produced in glycolysis is extra-mitochondrial (cytosol), whereas ETC is mitochondrial. NADH is impermeable into mitochondrial membrane it transfers reducing equivalents via substrate pairs linked by suitable dehydrogenases by Shuttle Systems. These dehydrogenases should be present on both sides of the membrane

1- Glycerol phosphate shuttle 2 ATP are yielded of 1 NADH FADH2

2- Malate shuttle 3 ATP are yielded of 1 mole NADH (cytosolOxidized

1 mole NADH (Mitochondria).

Diseases associated with impaired glycolysis: 1- Pyruvate kinase deficiency

A genetic defect that leads to low ATP production, decreased RBCs stability and swelling and lysis that results in Hemolytic anemia

2- Hexokinase deficiency A genetic defect in RBCs that lead to low 1,3 bis PG and 2,3 bis PG. 2,3 bis PG binds hemoglobin (Hb) and lowers its affinity to oxygen and normally allows Hb to release O2 in tissue capillaries. But in these patients Hb has high O2 affinity, leading to Hemolytic Anemia.

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Fate of pyruvate or pyruvic acid, the end product of Aerobic glycolysis:-

1- Conversion to lactate (lactic acid) by Lactate dehydrogenase (LDH) and NADH in anaerobic glycolysis.

2- Conversion to alanine (Ala)

This occurs in reversible transamination reactions of amino acids metabolism

3- Conversion to acetyl CoA

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Pyruvate DeHydrogenase Complex (PDC or PDH):- 1- Provide a link between Glycolysis and citric acid cycle. 2- Oxidizes pyruvate to give CO2 and Acetyl CoA. 3- Acetyl CoA is the substrate for citric acid cycle. 4- The reaction is irreversible. 5- Located within the matrix of mitochondria. 6- PDC is of 4 distinct enzymatic activities and require 5 coenzymes (TPP,

CoA, NAD, FAD, and lipoic acid). 7- PDC is inhibited by Dietary Deficiency of thiamine (vitamin B1) and

arsenite or mercuric ions.

Regulation of Glycolsis:

1) Allosteric activation or inhibition of GK, HK, PFK & Pk. e.g. phosphorylation and dephosphorylation. This is short term influence (minutes-hours).

2) Hormonal influence on the amount of enzyme synthesized. This is long term influence by increasing 10-20 fold the enzyme activity and takes hours-days.

3) Well-fed state (after a meal of CHO) or high insulin High enzyme activity.

4) Starvation or diabetes low enzyme activity. 5) PFK-activated by cAMP, AMP, Frc 6P, Pi and frc 2,6 bisP (in liver).

-inhibited by Citrate & ATP. 6) PK-activated by Frc 1,6 bisP -Inhibited by ATP, glucagon, epinephrine. The enzyme Pyruvate dehydrogenase complex (PDC) -Activated by CoA, NAD+, Insulin, ADP and Pyruvate. -Stimulated in the well-fed state. And it’s inhibited in starvation and DM.

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Abbreviations of Biochemical compounds and Enzymes and their full names:- ATP = Adenosine Triphosphate ADP =Adenosine Diphosphate Glc = Glucose G6P= Glucose 6 Phosphate CTP = Cytidine Triphosphate GTP = Guanosine Triphosphate UTP = Uridine Triphosphate PEP = Phosphoenolpyruvate Frc 6P = Fructose 6 Phosphate Frc 1,6 bis P = Frc 1,6 Bis Phosphate ATPase = Adenosine Triphosphatase Gly3P = Glycerol 3 phosphate NAD+, FAD, NADH & FADH2 – coenzymes GK = Glucokinase HK= Hexokinase ETC = Electron Transport Chain Glycerald. 3P = Glyceraldehyde 3 Phosphate DHAP = Dihydroxy Acetone Phosphate 1,3 Bis PG = 1,3 Bis PhosphoGlycerate 3PG = 3 Phosphoglycerate 2PG = 2 Phosphoglycerate 2,3 bis PG = 2,3 bis phosphoglycerate LDH = Lactate Dehydrogenase PFK = PhosphoFrcutokinase ALT = alanine amino transferase

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Citric Acid Cycle (CAC) *Kreb’s cycle or Tricarboxylic acid cycle (TCA)+

Series of enzymatically catalyzed reactions that form a common pathway for the final oxidation of all metabolic feuls (carbohydrate, lipids and proteins giving Glc, free fatty acids, ketone bodies and amino acids which are catabolized to Acetyl CoA, the substrate of the CAC).

These reactions occur within the mitochondrial matrix.

Amphibolic pathway (i.e. involved in both anabolic and catabolic processes (reactions)).

Provides much of the energy for respiration. The electrons generated by this cycle in the form of reducing equivalents NADH and FADH2 are

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transferred to the electron transport chain (ETC) and produce ATP by oxidative phosphorylation.

Net reaction of CAC is : Acetyl CoA + 3NAD+ + FAD + GDP +Pi + 2H2O 2CO2 + 3NADH + FADH2 + GTP + 2H+ +CoA

Reactions:-

Inhibited by: Fluoroacetate

arsenite malonate

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Energy or ATP production:

3NADH Oxidative phosphorylation

3×3 ATP = 9

1FADH2 Oxidative phosphorylation

1×2 ATP = 2 GDP+Pi GTP ATP = 1

Substrate level phosphorylation

1 mole Acetyl CoA 12 ATP

1 mole Glc 2 mole pyruvate 2 mole Acetyl CoA 2 mole Acetyl CoA 24 ATP

Regulation of CAC: The 3 important regulatory enzymes are:

1- Citrate synthase: Inhibited by ATP, NADH, succinyl CoA and Acetyl CoA derivatives.

2- Isocitrate dehydrogenase: inhibited by NADH and ATP, Activated by ADP.

3- α-Ketoglutarate dehydrogenase: inhibited by ATP, GTP, NADH and Succinyl CoA.

Another regulator is the availability of ADP (more ADP more ATP).

Abbreviations: NADH = reduced nicotinamide adenine dinucleotide NAD+ = oxidized nicotinamide adenine dinucleotide FAD = flavin adenine dinucleotide (oxidized) FADH2 = flavin adenine dinucleotide (reduced) OAA = oxaloacetate CoA = coenzyme A

Mitochondrial Electron Transport Chain (ETC) and Oxidative phosphorylation: ETC or respiratory chain is the final common pathway in aerobic cells by which electrons derived from various substrates are transferred to oxygen. It is a series of highly organized oxidation-reduction enzymes and reaction

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are represented as Reduced A + Oxidized B Oxidized A + Reduced B The enzyme use NAD+ or FAD as electron acceptor cofactors (coenzymes). Oxidative phosphorylation is the main source of energy in aerobic cells, it is the process whereby the free energy that is released when electrons are transferred along the ETC, is coupled to the formation of ATP from ADP + Pi -In intact mitochondria, electron transport and phosphorylation of ADP are tightly coupled reactions. -In damaged mitochondria, these reactions may occur unaccompanied and free energy is released as heat (i.e. No ATP production).

ETC and oxidative phosphorylation take place in the inner mitochondrial membrane (coupling membrane). This membrane has a highly selective permeability for specific substances ATP and other nucleotides, pyruvate, succinate, α-ketoglutarate…etc. The sources of electrons are NADH & FADH2

ETC is organized into 4 complexes:

1- Complex I : The point of entry into ETC for electrons (e-) from

NADH. The enzyme is NADH-dehydrogenase and electrons move to FMN to give FMNH2 which give electrons to coenzyme Q or Q. (ubiquinone) to be QH2. Rotenone (insecticide) is an example of inhibitors of this complex.

2- Complex II : The point of entry to ETC from succinate into FAD to

give FADH2. The enzyme is succinate dehydrogenase. FADH2 give (e-) to Q to become QH2. Carboxine is an example of an inhibitor.

3- Complex III : The quinone (Q) reduced form. I.e. QH2 gives (e-) to

cytochrome b (its iron ion ferric convert to ferrous Fe+3 Fe+2). Then (e-) moves into cytochrome c (again its iron ion (Fe+3 Fe+2).

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The enzyme is cytochrome reductase, Antimycin is an inhibitor.

4- Complex IV : The (e-) move into cytochrome aa3 (its copper ion Cu+2

cupric is reduced into Cu+ cuprous and this (cytochrome aa3) reduces ½ molecular oxygen (½ O2) into H2O (water) as a final product of ETC. The enzyme is cytochrome oxidase and is inhibited by carbon monoxide (CO) which competes with oxygen; others are H2S, Azide and cyanide.

Case - cyanide (CN) poisoning:

CN binds heme of cytochrome oxidase inhibiting the enzyme and blocking respiration. The almond odor is noted on the patient’s breath. It is fatal if not treated as early as possible by giving nitrites or sodium bicarbonate or thiosulfate or ventilation on 100% oxygen.

The Chemiosmotic or Mitchell’s theory: It’s the most widely accepted theory of oxidative phosphorylation. The free energy generated by the transport of (e-) by (ETC) is used to produce ATP. The electron carriers act as pumps of H+ (Hydrogen ions) across the membrane (proton pump) moving from matrix towards the inter-membrane space. H+ passes through pores or channels in the membrane back towards the matrix by the enzyme ATP synthase. This enzyme is responsible for the synthesis of ATP from ADP + Pi (This is Complex V). 2ADP + 2Pi + 2H+ 2ATP + 2H2O The enzyme complex ATP synthetase is also called ATPase, because the isolated enzyme also catalyzes the hydrolysis of ATP to ADP & Pi. Oligomycin is an example of inhibitors of this enzyme.

Control of Oxidative Phosphorylation: 1- Availability of ADP Or both 2- Availability of substrates 3- Availability of oxygen 4- The capacity of electron chain itself.

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Uncouplers of oxidative phosphorylation are compounds that cause normal ETC but no production of ATP. e.g.: 2,4 dinitrophenol (2,4 DNP) and dicumarol.

Energy production from 1 mole Glc oxidized aerobically: Glycolysis 1 Glc 2 Pyruvate (2+4) = 6 ATP

2 Pyruvate 2 Acetyl CoA (2×3) = 6 ATP

CAC 2 Acetyl CoA 4CO2 + H2O = 24 ATP Total = 36 ATP Therefore for every mole of Glc oxidized completely through glycolysis, citric acid cycle and electron transport chain… 36 ATP are produced aerobically. The energy is released for each electron pair that passes through the chain. This energy is coupled to the formation of ATP at three (3) sites. These coupling sites are at: Complex I to give 1 mole ATP. Complex III to give 1 mole ATP. And complex IV to give 1 mole ATP. NADH give 3 ATP And FADH2 give 2 ATP

Inhibitors: Complex I – Retenone Complex I – Carboxine Complex III – Antimycin Complex IV – CO, H2S, Azide and cyanide

Uncouplers: 2,4 DNP and dicumarol

The sources of electrons are NADH and FADH2

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Substrates that feed NADH into the electron transport chain give 3 ATP Succinate which enters through FADH2 into ubiguinone (Q) miss the first phosphorylation site of complex I give 2 ATP

Complex V It’s a phosphorylation site with pores, proteins enter the channels in the bilayer membrane into it (subunit F1).

Protein subunit Fo + F1 make the complex V (Adenosine Triphosphate Synthase or synthetase) (ATPase).

Glycogen Metabolism:

1- Glycogen is a storage form of Glc. It provides Glc. Once needed by the body, so it acts as a source of it.

2- Glycogen structure is highly branched very large polymer of Glc linked by α-1,4 glycosidic linkage and branches by α-1,6 glycosidic bonds at about every 10 residues. It is found in the Cytosol as granules.

3- Major sites of storage are muscle and liver. (Concentration is higher in the liver than in the muscle, but

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the amount is larger in the muscle). Liver – releases Glc. From glycogen into blood Muscle- No release of Glc from Glycogen but uses glycogen for its own energy needs.

4- Duration of liver glycogen storage is 12 hrs.(i.e. enough for about 12 hours) then gluconeogenesis starts.

Glycogenesis (Glycogen synthesis)

1- Synthesis of uridine diphosphate glucose (UDP-Glc), the precursor of glycogen.

2- Synthesis of glycogen molecule: a- Formation of the amylose chains.

The synthesis of new glycogen requires the presence of existing glycogen chains and Glc residue from UDP-Glc. The Glc residues are added (successively) to the C-4 terminus of an existing glycogen chain in α-1,4 glycosidic linkages. This reaction is the rate-limiting step in Glycogen synthesis.

b- Formation of branched chains and further growth. 1- Segments of amylose chain are transferred onto the C-6 OH

group of neighboring chains forming α-1,6 linkages. this is done by the branching enzyme glucosyl – 4:6 transferase.

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2- Seven (7) – residue segments of amylase terminal chains are

transferred to a C-6 OH group of a glucosyl residue that is 4 residues away from an existing branch. A terminal branch must be at least eleven residues in length before a segment is transferred from it.

3- Genetic defect in the branching enzyme leads to Type IV glycogen storage disease (Anderson disease). Infants born with this disease suffer cirrhosis and failure of the liver and hepatosplenomegaly. Most affected infants die by 2 years of age.

Glycogenolysis (glycogen degradation or use)

1- Phosphorlytic cleavage of the terminal α-1,4 glycosidic bond.

This cleavage reaction, which is the rate-limiting step in glycogenlosysis, gives Glc-1-P and a glycogen chain that is smaller by one glucose unit. Glycogen (n residue) + Pi Glycogen (n-1 residue) +Glc 1P Glycogen phosphorylase

Glycogen phosphorylase – dimeric enzyme and needs pyridoxal phosphate as a coenzyme.

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Genetic defect of Glycogen phosphorylase: a- Type V glycogen storage disease ( McArdle disease) in muscles.

Patients suffer from skeletal muscle cramps and show low blood lactate level during exercise.

b- Type VI glycogen storage disease (Her’s disease) in liver. Patients suffer hepatomegaly, hypoglycemia acidosis and growth retardation.

2- Removal of branch chains: This is catalysed by the “debranching enzyme system”, it has 2 enzymatic activities: a- 1,4 1,4 glucantransferase (glucosyl transferase)

In this step, 3 Glc-residues from a branch are transferred onto a chain terminus, leaving a single residue on C-6.

b- α-1,6 glucosidase (amylo-6-glucosidase). In this step, a single residue on C-6 is removed to give a free glucose molecule. In lysosomes, another enzyme, α-1,4 glucosidase is involved in debranching.

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Disorders due to genetic defect in debranching enzymes: a- Type II glycogen storage disease (Pompe’s disease):

Defect in α-1,4 glucosidase of lysosomes Glycogen accumulates General nervous system problems, enlarged heart and later failure of heart and lung.

b- Type III glycogen storage disease (Cori-Forbes disease):

Defect in debranching enzyme system, also heart and lung problems stunted growth (growth stop), enlarged liver, hypoglycemia and acidosis.

Glycogen storage disease: caused by genetic deficiencies of certain enzymes of glycogen metabolism accumulation of glycogen and/or inability to use that glycogen as a fuel source.

Recently more than 10 glycogen storage diseases have been found out, most common ones are Type I, Type III, Type IV of which Type I incidence is about 1/20,000 person. Regulation of glycogen metabolism:

A- Hormonal In liver Glucagon (stimulates glycogenolysis, reduces glycogenesis) Insulin (stimulates glycogenesis) In muscle Epinephrin (promotes glycogenolysis, inhibits glycogenesis) Insulin (stimulates glycogenesis, reduces glycogenolysis) Glucagon/insulin ratio is important in regulating Gly metabolism Insulin is an anabolic hormone

B- Level of cAMP (a.k.a.1 covalent modification, i.e. phosphorylation) Adequate level Glycogenolysis is increased High Low level Glycogenesis is increased

1 a.k.a = also known as

34

Effect of epinephrine and/or glucagon: epinephrin Adenylate cyclase (Ia) adenylate cyclase (a) Ia = Inactive a = active ATP cAMP Phospho diesterase Protein kinase (Ia) protein kinase (a) After the activation of protein kinase, there are 2 pathways:

1- Protein kinase (a) Glycogen synthase I Glycogen synthase D (Active) (Inactive) Dephosphorylated phosphorylated

Inhi bits UDP-Glc Glycogen

2- Protein kinase (a) Phosphorylase kinase phosphorylase kinase (inactive) (active) Ca+2

Phosphorylase b phosphorylase a (inactive) (active) This reaction can be reversed stim ulates By phosphatase enzyme Glycogen Glc-6-P

Glycogenesis Stops

Glycogenolysis Starts

35

This regulation is called Cascade regulation of Glycogen synthase activity and Glycogen phosphorylase activity (Glycogenesis and Glycogenolysis).

Cascade = Type of an amplification system, where one molecule or few of circulating hormones like Epinephrin or glucagon can activate another molecule(s) to produce a larger number of cAMP which cause activation of protein kinase and so on…

36

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37

Alternative pathways of CHO metabolism

Pentose Phosphate Pathway (PPP): (Hexose monophosphate shunt; HMS

or HMP) Function: provides a source of NADPH for reductive biosynthesis and ribose

5-phosphate for nucleic acid synthesis, and provides a route for the use of pentose and their conversion to fructose 6-phosphate (Frc 6-P) and Glyceraldehyde-3-phosphate (Glycer. 3-P).

Location: a) In tissues, the pathway is most active in the liver, mammary

glands, adipose tissue and adrenal cortex. b) Within the cell, the enzymes of this pathway are located in the

cytoplasm. Reactions: 1)

RBCs need NADPH production to keep glutathione in the reduced state glutathione (tripeptide: Gly-Cys-Glu) (Glycine-Cysteine-

Glutamate) Reduced Glutathione is needed to maintain the integrity of the RBCs (Erythrocytes) membrane. Glc 6PD or G6PD deficiency RBCs hemolysis and hemolytic anemia. (Due to genetic deficiency) In people with low G6PD, certain drugs, like Aspirin, and the antimalarial primaquine that act as oxidizing substances of reduced glutathione ( to

form

oxidized glutathione), can cause hemolytic Anemia

.

Favabeans can cause Favism, hemolytic effects of ingesting this type of

38

beans in some people with G6PD deficiency in the Mediterranean region.

2)

Hemolytic anemia may lead to a type of Jaundice known as Hemolytic Jaundice.

3) 4)

5)

*the enzyme is transketolase, coenzyme is Thiamine Pyrophosphate (TPP)

Chronic thiamine deficiency defective Transketolase and leads to Wernicke-Korsakoff syndrome. Symptoms are:

Weakness or paralysis

Impaired mental function 6)

7)

39

Summary of reactions: 3Glc-6-P + 6NADP+ 2Frc-6-P + 3CO2 + 6NADPH + 6H+

Regulation of the pathway: 1) NADP+ concentration is the major factor in regulating Glc-6-P

reactions (G6PD) reactions. 2) NADPH is a competitive inhibitor. 3) Key enzyme is G6PD. 4) G6PD is activated in starvation and DM and inhibited by carbohydrate

feeding. induced by insulin. 5) Activated in the presence of TTP.

Notes: 1) The first three reactions are Oxidative + irreversible 2) The rest are non-oxidative + reversible. 3) Glyceraldehyde-3-P and Frc-6-P are intermediates of Glycolysis. 4) The pathway needs one ATP and provides no ATP unlike glycolysis and CAC. 5) The pathway provides Ribose. 6) It’s a route for conversion of hexose into pentose and pentose interconversion 7) Provides NADPH, which is used for synthesis of FAs, Steroids, glutathione…etc.

40

41

Uronic acid pathway (Glucuronic acid cycle) 1- The Glc-6-P is converted into Glc-1-P that takes high energy

UTP and becomes UDP-Glc by the enzyme UDP-Glc dehydrogenase (NAD+ is required).

2- UDP gluclucuronic acid is produced and conjugated with Glucosamine and Galactosamine to form mucopolysaccharides. Utilized in detoxification by conjugation with Benzene (phenol), Bilirubin and other steroids.

3- UDP-glucuronic acid loses UMP and becomes D-glucuronic acid 1-P less, then loses Pi (phosphate) to become D-glucuronic acid, which may also come from diet (like meat) and this convert to L-glucuronic acid by reductase.

4- L-glucuronic acid is either converted into: a- L-gulonolactone which is converted into 2 keto L-

gulonolactone that results in ascorbic acid (vit. C) this step is absent in human, primates (Monkeys) and guinea pigs, because of enzyme absence, So no vit. C is formed. But in other animals the enzyme’s present and thus vit. C can be sunthesized.

b- L-Xylulose is formed by decarboxylation and then reduced by NADPH to L-xylitol (alcohol) which loses H+ to NAD to become NADH and D-Xylulose results by a reductase enzyme, so if this enzyme is deficient then L-Xylulose accumulates in the blood, causing “Essential Pentoseuria”.

D-Xylulose converts to Xylulose 5-P by kinase and by HMP shunt Ribose-5-P is produced then Frc-6-P and Glyceraldehyde-3-P are formed and so pyruvic acid that’s oxidized to CO2, H2O and energy is produced.

42

43

METABOLISM OF FRUCTOSE : Fructose is considered as an inverting sugar, because fructose is strongly levorotatory and changes (inverts) the weaker dextrorotatory action of Sucrose. SOURCES:

1- Hydrolysis of Sucrose (by Sucrase). 2- Fruits & Honey.

MAJOR PATHWAY:

1- fructose + ATP Fructokinase

Frc-1-P + ADP

Fructokinase : (liver, kidney, intestines) is specific for fructose, not affected by feeding-fasting cycles nor by insulin levels, which may explain why fructose is cleared from the blood of diabetic patients at a normal rate.

Deficiency leads to Essential fructosuria.

2- Frc1-P Aldolase B

DHAP + Glyceraldehyde

Aldolase B is the cleavage enzyme, it’s found in and predominant in the liver, while aldolase A is found in all tissues. Aldolase B deficiency Hereditary fructose intolerance. Increased Frc-1-P (pathological changes and high osmotic pressure)

Water retention

Jaundice, vomiting, hyperbilirubinemia and liver enlargement

This case occurs in infants after ablactation, because the food given contains sucrose and this provides Fructose, the same symptoms are noticed and this condition is called “Sequestering of phosphate”.

Sequestering of phosphate: phosphate is attached covalently to an inorganic molecule and is therefore no longer available to participate in

44

α-Lactalbumin (protein B) + β-D- Galtransferase (protein A)

other essential metabolic reactions.

Pi ATP from ADP + Pi (especially in Liver which metabolites most dietary Fructose)

3- Interconversion of DHAP and Glyceraldehyde

DHAP may be converted to Glycer. 3-P by an isomerase enzyme (See Glycolytic pathway).

Glyceraldehyde may be phosphorylated to glycer. 3-P by ATP and a kinase enzyme glycolysis & gluconeogenesis.

or reduced to Glycerol gluconeogenesis or oxidized to glycerate serine biosynthesis. METABOLSIM OF LACTOSE : A disaccharide of Glc and Galactose (Gal). Source: milk biosynthesis in human ‘’the mammary gland’’.

1- UDP-Glc epimerase UDP-Gal

UDP-Gal + D-Glucose Synthetase

Lactose + UDP

45

UDP-Gal Glycolipids, Glycoproteins & Glycosaminoglycans.

Prolactin: a hormone that increases the rate of synthesis of α-lactalbumin Lactose synthesis.

Protein B of the synthetase enzyme is abundantly found in milk.

2- Lactase (β-galactosidase) hydrolyzes lactose to glucose and galactose and is found in the small intestine.

Its deficiency is called “alactasia”, results in Lactose intolerance which is either genetic or acquired disorder, Its symptoms are diarrhea and flatus.

In infants, milk which is their primary food is not tolerable and lactose –

free formula is used instead (Soya milk). In adults, the condition is less serious and is treated by avoiding milk

products.

46

Metabolism of Galactose:

1- Gal + ATP galactokinase

Gal-1-P + ADP

Galactokinase Deficiency: it leads to Galactosemia and Galactosuria. In the lens of the eye, Gal is reduced to the sugar alcohol dulcitol (galacitol) by aldose reductase and causes an osmotic effect that lead to the development of Cataracts.

2- Gal-1-P Glc-1-P

Gal-1-P uridyl transferase deficiency Classical Galactosemia. Symptoms: Cataracts, mental retardation and liver cirrhosis.

3- UDP-Gal is converted to UDP-Glc by an Epimerase enzyme 4-epimerase deficiency Galactosemia

Cases:

Galactosemia: a male infant exhibits difficulty to feed, diarrhea, vomiting, and failure to thrive (grow). At 5 days of age, exhibits mild jaundice.

Glycosuria: (Reducing surgar in urine) but not glucose, i.e. Galactosuria.

Galactose-free diet: no milk is given, and for older people not even milk products.

Glactose is important in Cerebrocytes, Brain & Cartilage.

In case of milk deficiency in food, Gal can be synthesized in body by Glc-6-P Glc-1-P UDP-Glc UDP Gal.

Gal in Blood diffusion of Gal in Eye lens H2 reductase

Galacitol. Galacitol is impermeable and accumulates in lens osmotic pressure and H2O retention Myopia; swelling of the eye and damage of lens tissue causing cataracts.

47

Gal-transferase (protein A) + Lactalbumin (protein B)

protein A

Gal is necessary for milk lactose production in mammary gland. The process is done by the action of Galactosyl transferase enzyme which needs α-lactoalbumin as a cofactor, this cofactor is synthesized in mammary gland in the last 3 months of pregnancy (i.e. 7th, 8th and 9th) when the progesterone level is decreased.

UDP-Gal + Glc Lactose (normal in lactating mammary gland) UDP-Gal + N-acetyl Glc N N-acetyl lactosamine (N-acetylglucosamine)

Question:- Can lactose be synthesized directly from Glc? Answer:- Yes, Here’s how: Glc 6-P UDP-Glc, by glycogenesis can be converted to UDP-Gal by isomerase enzyme, then UDP-Gal Forms lactose. Lactose synthase or synthetase (UDP-Gal: Glc galactosyl transferase) is composed of two proteins: A and B. Hormonal control of lactose synthesis:

Prior to and during pregnancy, the mammary gland synthesizes N-acetyl lactosamine.

o During pregnancy, the steroid hormone, Progesterone inhibits the synthesis of protein B.

After birth, progesterone levels drop significantly, stimulating the synthesis of the peptide hormone prolactin which in turn stimulates α-lactalbumin (protein B) synthesis. The resulting regulatory protein B forms a complex with the enzyme, protein A, changing the specificity of that transferase so that lactose, instead of N-acety lactosamine, is produced.

Ingestion of large quantities of fructose has profound metabolic consequences.

48

Fructose undergoes more rapid glycolysis in the liver than does Glc, because it bypasses the regulatory step catalyzed by phosphofructokinase, this allows fructose to flood the pathways in the liver, leading to enhanced fatty acid synthesis, increased esterification of fatty acids and increased VLDL secretion, which may raise serum triacylglyceroles and ultimately raise LDL-cholestrol concentrations. In extrahepatic tissues, HK catalyzes the phosphorylation of most hexose sugars, including fructose, but Glc inhibits the phosphorylation of fructose, since it’s a better substrate for HK. Nevertheless, some Frc. Can be metabolized in the adipose tissue and muscle. Fructose is found in seminal plasma. The presence of Sorbitol dehydrogenase in the liver, including the fetal liver, is responsible for the conversion of sorbitol into fructose. This pathway is also responsible for the occurrence of fructose in seminal fluid

Diets high in sucrose or in high-fructose Syrups (HFS) used in manufactured foods and beverages lead to large amounts of fructose (and glucose) entering the hepatic portal vein.

49

Conversion of Glc to Frc. By way of Sorbitol (polyol pathway)

Large amounts of Glc enter these cells during times of hyperglycemia (uncontrolled D.M.) Elevated Glc and adequate supply for NADPH Sorbitol, which can’t pass (trapped). When Sorbitol dehydrogenase is low or absent, e.g: Kidney, lens, retina, nerve cells: Sorbitol accumulates Strong osmotic effects cell swelling (due to H2O retention).

50

Some pathologic alterations associated with diabetes can be attributed to this phenomenon.

Metabolism of Fructose

: deficiency: 1) Deficiency of Fructokinase: leads to Essential

Fructosuria. 2) Deficiency of Aldolase B: leads to Hereditary Fructose Intolerance.

: An enzyme called Sorbitol dehydrogenase

51

: note: DHAP & Glycerald. 3P maybe degraded by glycolysis or may

be substrates for aldolase and hence glyconeogensis, which is the fate of much of the fructose metabolized in the liver.

: HK = Hexokinase

: Aldolase A: found in all tissues.

Aldolase B: Predominant in the liver.

: an enzyme called phosphotriose Isomerase.

Gluconeogenesis: Synthesis of a new glucose or CHO from non-carbohydrate precursors like lactate, pyruvate, glycerol, some amino acids called glucogenic AAs & odd no. F.A. Location: liver, kidney (main site) and less in the intestine. Function: 1- During starvation or periods of limited CHO intake, when the level of liver glycogen is low. Glycogen supplies are enough for 10-24 hours, Gluconeogenesis is important in keeping the blood sugar (glucose) concentration.

2- During long exercise, lactate is used as precursor (when O2 in the muscles Lactic acid is formed transported by RBCs to liver Gluconeogenesis).

3- Odd number fatty acid (odd no. F.A.) e.g. propionic acid (3-carbons) is

formed by oxidation of longer F.A. (9c) and by oxidation 2-C are removed (9C 7C 5C 3C) propionic acid converts into propionyl CoA.

52

LDH

Glycolysis

LDH

Gluconeogenesis

NAD+ NADH+

Substrates for Gluconeogensis: 1- Lactate or lactic acid, increased during extended exercise and

transported to the liver in the Cori cycle or lactic acid cycle and converted to pyruvate. Muscle Blood Liver

Pyruvate Lactate Lactate Lactate Pyruvate Glc into blood.

Lactate Pyruvate

2- Alanine (Ala) Muscle Blood Liver

Glc

Pyruvate Ala Ala Ala

Pyruvate

Transamination reaction Glc

3- Glycerol: formed in adipose tissue by lipolysis of Triacylglyceride (TAG) and released into the blood and then to the liver to be phosphorylated to Glycerol 3-P then to DHAP and Glyceraldehyde 3-P.

4- AminoAcids (Glucogenic AAs) Several AAs (in addition to Alanine) may be converted to pyruvate or CAC intermediates like α-ketoglutarate (α-KG), succinyl CoA, fumarate which are metabolized to oxaloacetate (OAA). OAA is an intermediate in gluconeogenesis.

Glucogenic pathway: The pathway of gluconeogenesis is the reversal of glycolytic pathway. But irreversible step in glycolysis must be passed and these are:

53

Malate Dehydrogenase

GDP+CO2

1- The phosphorylation of Glc by glucokinase or hexokinase. 2- The conversion of (Frc 6-P) to (Frc 1,6 BP) by phosphofructokinase

(PFK). 3- The conversion of PEP to pyruvate by pyruvate kinase.

Reactions of Gluconeogenesis:

1- Pyruvate carboxylation (occurs in matrix) Pyruvate

2- OAA Malate

This reaction occurs in both matrix and cytosol. OAA is reformed in cystol and can be converted to phosphoenol pyruvate(PEP).

3- OAA PEP

4- PEP Frc 1,6 BP This conversion occurs by sequential reactions catalyzed by 6 enzymes (simply the reverse of glycolytic ones).

5- Frc 1,6 BP Frc 6-P Frc 1,6 BPase is the major regulatory enzyme in gluconeogenesis (an allosteric enzyme, activated by citrate and inhibited by AMP and Frc 2,6 BP).

Pyruvate carboxylase (Biotin, mg+2)

CO2, ATP ADP

OAA

NADH NAD+

GTP

PEP Carboxykinase

Fructose 1,6 bis phosphatase

(Frc 1,6 BPase)

54

6- Frc 6-P Glc 6-P

7- Glc 6-P Glc + Pi

Glc 6-Pase is found in liver, kidney, small intestine but not in muscles, So muscles do no contribute Glucose into the blood.

Energy Consumption: The conversion of 2 moles of pyruvate to 1 mole Glc uses 4 moles ATP and 2 moles GTP = 6 moles ATP (GTP like ATP has high-energy phosphate bonds) Regulation of Gluconeogenesis:

1- Stimulated by glucagon, glucocorticoids, Epinephrine and cAMP. 2- Stimulated by substrates like Glucogenic AAs. 3- Stimulated in starvation since Acetyl CoA acts as allosteric activator of

pyruvate carboxylase. 4- Inhibited by Insulin and after CHO feeding.

Clinical disorder of Gluconeogensis: Example: multiple carboxylase deficiency, a genetic defect in a synthetase enzyme, responsible for attaching Biotin to pyruvate carboxylase and Biotinase responsible for removing Biotin from enzyme protein. How can AAs convert into Glc in gluconeogenesis? AAs can undergo deamination to give one of the component of CAC or any other compound that undergo certain reactions to give Glc. How??

1) Ala ,Cys, Gly, Ser, Thr – give pyruvic acid by deamination. 2) Asp, Asn – give OAA (oxaloacetic acid). 3) Glu – give α-ketoglutaric acid (α-KG) a compound of CAC cycle to

give malate that leave mitochondria into cystol and ends in Glc as seen in the figure. Other AAs are Arg, Gln, His, Pro.

4) Val, Met, Thr, Ile – give succinyl CoA. 5) Phe and Tyr – give fumarate.

Glc 6-Pase

Glucose 6 phosphatase

isomerase

55

Carboxylation

Questions:- How can Asp form Glc? How can Glu form Glc? How can Tyr form Glc? How can Met form Glc? Note: you can make more questions in such way. Question: How can even number F.A. give Glc? How can odd number F.A. give Glc? Answer: Even number F.A. oxidize by B-oxidation process to give acetyl CoA (i.e. 2 carbons). Acetyl CoA acts as an allosteric activator of Pyruvate carboxylase. But for odd number F.A. like propionic acid or longer one (9-C), Glc is produced as follows: Odd no. FA (9-C) 7-C 5-C 3-C (propionic acid) Propionic acid + CoA Propionyl CoA D-methyl malonyl CoA L-Methyl malonyl CoA Succinyl CoA Succinate Malate Glc.

Vit. B12 Isomerase

Oxidation

56

57

*Key enzymes -Carboxylase -Carboxykinase -Frc 1,6 bis Pase -Glc 6 Pase Regulation: Stimulation by: Glyucagon, Epinephrine Glucocorticoids cAMP and substrates like Glucogenic AAs Starvation Inhibited by: Insulin + CHO intake

Ethanol may replace CHO (carbohydrates) as an energy source when it’s ingested in large amounts.

1) Oxidation to acetate in the liver Ethanol is oxidized in the liver by Cytosolic Alcohol dehydrogenase to acetaldehyde. CH3CH2OH + NAD+ CH3CHO + NADH + H+

2) Acetaldehyde is further oxidized to acetate by a mitochondrial Aldehyde dehydrogenase. CH3CHO + NAD+ + H2O CH3COO- + NADH

3) Much of the acetate produced from ethanol leaves the liver and is converted to Acetyl CoA and then CO2 by other tissues.

4) Acetyl CoA may also be formed in the liver and used as a precursor

58

for lipid biosynthesis.

5) Ethanol may also be oxidized by a microsomal Cytochrome P450 oxidase, which is induced by ethanol. In the body ethanol is oxidized to Acetaldehyde then acetic acid which is converted to active acetic acid (or acetate), i.e. Acetyl CoA, which enters the CAC and gets converted into ATP, CO2 and H2O.

That 1 gm alcohol gives 9 calories energy. How does Disulfiram (a drug used in treatment of chronic alcoholism) work? This drug inhibits the action of Aldehyde dehydrogenase by competing with NAD+ to the binding site of enzyme and so increases the level of acetaldehyde in blood causing symptoms of vomiting, thirst, sweat and headache.

A person may ingest methyl alcohol (methanol) by mistake, so what happens? Methanol is oxidized by liver alcohol dehydrogenase to give formaldehyde and this is oxidized to formic acid (formate): CH3OH HCHO HCOOH -Formaldehyde causes retinal damage and blindness. -Formic acid causes acidosis (coma and death). Methanol toxicity is treated by giving ethanol as an antidote. It competes with methanol at the dehydrogenase enzyme causing a delay of methanol metabolism and its excretion in urine is increased.

Chronic Ethanol Ingestion: can cause fatty liver.

Fatty liver occurs in conditions in which there’s an imbalance between hepatic triacylglycerol synthesis and secretion of VLDL. Other conditions are diseases such hepatitis, and uncontrolled diabetes

59

mellitus. Is ethanol synthesized in the human body? A: No, but in microorganisms it’s formed from the conversion of Pyruvate by two reactions.

Example of a microorganism that synthesizes ethanol is Yeast. Alcohol (Ethanol) induces hypoglycemia. Due to inhibition of gluconeogenesis.

Biochemical lesion of CHO metabolism in RBC

RBC are synthesized in bone marrow and have a range of life span of 120 days. They function in carrying O2 to tissue and transport CO2 to lungs. A mature RBC has:

1- Glycolytic pathway to provide energy and 2,3 Diphosphoglyceric acid (2,3 DPG) which play a role in the delivery of O2 to tissues.

2- HMP shunt to yield NADPH which help in maintaining the SH group of a) Glutathione b) sulfhydryl-containing proteins in reduced state. Such proteins function as enzymes, glyceraldehyde 3P dehydrogenase in glycolysis and as part of the membrane system in the RBC.

The normal production and release of RBC is under control of glycoprotein hormone Erythropoietin (EPO). The mechanism of action is through stimulation of mRNA and protein synthesis system, Erythropoietin is produced in the plasma by the action of the Erythropoietic factor which comes from the liver and the kidneys.

60

Drugs can cause a decrease in the number of circulating RBC either through (a) Impaired production as in Bone Marrow Aplasia due to treatment with chloramphenicol, or due to (b) destruction of Red cells (hemolysis) caused by a variety of chemical compounds. This occurs in persons with deficiency of one or more of the enzymes associated with CHO metabolism as glucose-6-Phosphate dehydrogenase (G6PD). There are several factors that may contribute to hemolysis of RBC:-

1) Damage to the cell membrane due to oxidation of SH group in the membrane.

2) Inactivation of Gly3P dehydrogenase due to oxidation of SH group in this enzyme.

3) Degradation of Hb and shifting of equilibrium between HbO2* and Met.Hb** due to the lack of the enzyme Glutathione peroxidase which acts on H2O2 to convert it into H2O and oxidized glutathione.

4) Biochemical lesion in synthesis of G.SH

5) Glutathione reductase:

a) Flavoprotein deficiency as in B2 deficiency leads to RBC hemolysis. b) Other deficiency in the production of NADPH

These factors are all interrelated, e.g:

1) Lack of Glyc 3PDH would cause interruption of glycolysis. 2) A decrease of NADH (Lack of NADH) may result in an increase in Met-

Hb due to decrease in the reaction:

61

*HbO2 : Oxyhemoglobin or oxygenated Hb **Met-Hb : hemoglobin in which iron is in the ferric form Fe+3 or oxidized form. It can’t carry oxygen, it’s brown and is normally oresent in very low plasma concentrations. Drugs such as Sulphonamides may increase Met-Hb, the Symptoms of Methemoglobinemia are due to hypoxia which causes Cynosis & increased respiratory rate

3) Also a decrease in production of 2,3 DPG which lowers the affinity of Hb to O2 and releases it. This compound is formed from 1,3 DPG by Gly 3P DH. Glc 6P DH deficiency is an inherited (as a sex-linked factor) inborn error of metabolism. Glc 6P DH deficiency- Some drugs given to some people may cause hemolytic anemia like: a) Antimalarial drug-Primaquine. b) Sulfonamide & sulfones. c) Analgesics-acetanilide. d) Antibacterial-nitro furagon. e) Ingestion of vicia fava bean, nephthaline, phenyl hydrazine.

G6PD converts G6P to 6-phosphogluconic acid and by NADP+ as cofactor which becomes NADPH. NADPH is used to oxidize GSSG by donating hydrogen so reduced glutathione is formed, i.e. GSH. GSH gives its hydrogen to H2O2 so H2O is formed and if no enzyme is present then H2O2 will accumulate inside the cell causing oxidation of the cell membrane.

62

Glutathione maintains the integrity of the SH group in enzyme, Hb, protein and cell membrane. Notes Erythropoietin: (EPO) a glycoprotein hormone, controls erythropoiesis (RBCs production) also known as hematopoeitin or hemopoietin, used as a drug, i.e. the exogenous EPO (rHu EPO) therapy, mainly produced in kidney and less in liver. Then acts as RBCs precursor in Bone Marrow. Stimulus: lowered O2 supply to tissues (Hypoxia) and Anemia. rHu EPO: recombinant human EPO used in treatment of patients with Renal Failure on hemodialysis.

Metabolism of Aminosugars

Amino sugars: (glucosamine, mannosamine, galactosamine, and their derivatives). Glucosamine 6-Phosphate is the precursor of all hexosamine residues in glycosaminoglycans.

63

A summary of interrelationships in metabolism of aminosugars

Amidination

Activation

Conjugation with Glucuronic acid NANA = N-acetylneuraminic acid Glc NAc = Acetyl glucosamine Glc N = glucosamine Man NAc = Acetyl mannosamine Gal NAc = Acetyl galactosamine UTP = Uridine triphosphate

64

UDP = Uridine Diphosphate Gln = Amino Acid Glutamine Glu = Amino Acid Glutamate Glycosaminoglycans or mucopolysaccharides are important in the formation of Heparin, Hyaluronic acid, chondroitin sulfate…etc. Aminosugars like Glucosamine, Galactosamine…etc. and their derivatives are constituents of these mucopoly-saccharides. For example: Glucosamine and Glucuronic acid are constituents of chondroitin sulfate which is found in the cartilage. The cartilage is very important for growth and mineralization to form the bones. So how are glucosamine and its derivatives formed? These compounds come from Glc 6P which comes from:

a) Glucose b) Amino acid metabolism c) Glycogen

Glc 6P is aminated by glutamine (Gln) by transaminase to form Glucosamine 6P (Glc N6P) which is converted by mutase to Glc N1P that reacts with UTP forming UDP-GlcN that may conjugate with glucuronic acid to form Heparin (anti-coagulant). Glc N6P may come from diet. Acetylation of Glc N6P by Acetyl CoA by transacetylase gives N-Acetylglucosamine 6P (N-Glc NAc 6P) that converts into N-Acetylglucosamine 1P (N-Glc NAc 1P). N-Glc NAc 1P Is activated with UTP forming UDP-N-acetyl glucosamine that is conjugated with glucuronic acid to form hyaluronic acid needed in Synovial fluid, eye, placenta…etc. If chondroitin sulfate is needed, the enzyme epimerase converts Glc to Gal and so UDP-Gal NAc is formed and conjugate with glucuronic acid to produce chondroitin sulfate. If Sialic acid (N-acetyl neuraminic acid=NANA) is needed, the enzyme epimerase converts Glc to Mannose so Glc NAc 6P converts to Man NAc 6P and this reacts with pyruvic acid to form Sialic acid which enters in the formation of Rh factor and Gangliosides.

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Metabolism of Mannose: The body utilize glucose as a major source of energy. Other monosaccharides, Fructose, Galactose and Mannose are not used directly but have to be converted to glucose (Glc) or other intermediates that are part of the Glycolytic pathway. Mannose + ATP Mannose 6P Man 6P Glc 6P The second reaction is reversible so that if Mannose is needed then the reaction goes to the left and Mannose is obtained and used for sialic acid synthesis with pyruvic acid coming from Glycolysis.

Isomerase

Hexokinase

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