Dr.Ehab Fathy Gabr Aboueladab (PhD in Biochemistry), Associate Prof.Dr. of Biochemistry, Damietta University, Faculty of Specific Education Damietta, New Damietta City, P.O.Box.34517, Egypt, Tel :002-057-2224444(HOME), 002-057-2403085(WORK), 002-0100-7834123(MOBIEL or HANDY), Email:firstname.lastname@example.org, ChatYahoo:email@example.com, http://www.labhoo.com,
Friday, September 28, 2012
In carbohydrate metabolism we will study, the types of carbohydrate, how they are digested and absorbed, then we will the cycles of carbohydrate oxidation which generate the energy required for life…. these cycles are:
Glycolysis= Glucose oxidation (i.e. break down of glucose to get energy).
Gluconeogensis= formation of Glucose form non-carbohydrate sources.
1) In the mouth, salivary amylase, hydrolyze starch partially into a mixture of dextrins and maltose.
2) In the stomach, salivary amylase continues hydrolysis of starch only for few minutes then stop because the pH becomes acidic due to the presence of the HCl in stomach and this is unfavourable condition for the amylase to work.
3) In the intestine, pancreatic amylase, completes the digestion of starch into maltose with little isomaltose and maltotriose which are then hydrolyzed in the intestine into glucose. Fructose and Galactose.
- The polysaccharides and oligosaccharides are not absorbable so, they must be converted to Monosaccharides.
- Monosaccharides are principally absorbed from theduodenumthen pass into the blood through the hepatic portal vein to the liver where:
Part of these monosaccharides is stored asglycogenand part is oxidised through glycolysis to obtain energy
Part is oxidised through the pentose phosphate pathway to regenerate NADPH which, together with glucose itself, is used in synthesis of such molecules as amino acids, nucleotides, fats and cholesterol
Part is oxidised to produce energy (ATP) which is used in the anabolism processes.
Before we know the mechanism by which the glucose is absorbed into the cells we must know the composition of the cell membrane through which the glucose pass.
this part will help any any one who don’t study biology or cell before to be able to understand biochemistry
Composition of the cell membrane:
==> As from the previous picture, the cell membrane is composed of 2 layers each of them consists of a layer of phospholipids.
==> Each phospholipid molecule consists of apolar hydrophilic head and a non-polar hydrophobic tail.
==> How the bilayer membrane is formed:And as we know, the extracellular and intercellular fluid are polar, therefore the polar heads are arranged towards the polar fluids and the non-polar tails arranged toward themselves inward the membrane where there are a hydrophobic interaction between them and thus forming a phospholipids bilayer membrane
==> And across the membrane there are transport proteins and receptor proteins
==> Function of the cell membrane:-
Protection of cells.
On the other hand, it represents a barrier which prevents entry of some molecules into cells such as polar molecules but it uses other mechanism by which the molecules enter the cells such as Na-K pump through which ions are transported into the cell.
In case of glucose:
Any polar molecules can’t pass through the inner non-polar layer of cell membraneso, how glucose enter the cell through the cell membrane while it is polar?
===> The answer is that glucose has 2 mechanism to enter the cells:
1) The passive diffusion or transport:
===>DEF:it transport of biochemical and other atomic or molecular substance across the cell membrane into the cell from higher concentration region to a lower concentration region without any need for energy. (i.e. the substance enter the cell only if its concentration outside the cell is more than its concentration inside the cell.)
===>It depends on the concentration gradient.
===> It doesn’t need energy.
===> Depends on the permeability of the cell membrane.
Simple diffusion:in which the substance are transported from higher concentration to lower concentration region through the phospholipids bilayer without any need for energy and without using the transmembrane proteins (carriers/transporters/channels/pores).
Facilitated diffusionor passive-mediated-transport:in which the substance are transported from higher concentration to lower concentration region through the phospholipids bilayer using the transport proteins and without any need for energy.
- Fructose and pentose use this mechanism.
- Glucose is transported by this mechanism into brain, kidney and liver.
2) Active diffusion or transport
===> DEF:it means transport of a substance from region of lower concentration to a region of higher concentration (i.e. against the concentration gradient) utilizing energy from ATP molecules and carrier proteins.
===> Itdoesn’t depend on the concentration gradient.
===> It needs energy.
===> This mechanism applied by the cell to accumulate high concentration of molecules that the cell needs such as ions, glucose and amino acids.
===>Doesn’t depend on the permeability of the cell membrane because this mechanism transports the substance through the transport proteins and carrier proteins.
===> It has 2 types:
Primary active transport: in which the cell uses chemical energy such as ATP.
Secondary active transport: in which the uses electrochemical gradient such as sodium and potassium dependent ATP bump
===> It is the mechanism by which glucose is transported from intestinal tract
Glucose transportation into the cell by insulin action:
===> When the blood glucose level is high, the nervous system sends signals to the beta-cells of the pancreas to secrete insulin.
===> Then insulin binds to its receptors on the outside surface of the cell membrane causing conformational changes to the receptors (where the receptors are soft protein) leading to conformational changes to the cell membrane and opening of protein gates which called glucose transporter (GLUT) leading to entrance of glucose inside the cell.
===> Opening of these gates lead to activation or deactivation of some enzymes that responsible to glucose oxidation.
===> Insulin binding to the receptor is a reversible process because it will leave the receptor after delivering the message.
===> This process is regulated by the central nervous system.
===> Then after the glucose level returns normal; the insulin leave the receptor and no glucose will enter the cell
===> But in some cases the B-cells is highly activated secreting more insulin which make the glucose to enter the cell and its level is lowered causing hypoglycaemia
===>The receptors of the insulin are specific for insulin and are distributed in all tissue with different concentration where the receptor concentration increases in the tissue:
Which utilize glucose as the main source of energy such as brain and cells of the nervous system, red blood cells, muscles,…..etc
At which the blood supply is low such as the peripheral tissues.
What is the difference between the receptors and enzymes?
Just delivering messages sent by the nervous system into the cell (just letter box)
Doesn’t catalyse any reaction
Catalyse reactions and doesn’t deliver any messages.
Why glucose is stored in the liver and muscles as glycogen and not as it is (i.e. glucose)?Because glycogen is solid polymer that is compacted and occupy a small size inside the cell which prevents cell membrane rupture by the pressure on the cell membrane.While if the glucose is stored as glucose, it will take large size which then cause pressure on the cell membrane and then rupture of the cell membrane.
What is the factor that determines the amount of glucose that enters the cell?The amount of glucose after meal where the glucose must be maintained at the normal level and the excess must enter the cells
Is the metabolism of fats can be used in formation of glycogen?The answer is yes because the result of oxidation of fat is formation of glycerol which can enter the gluconeogensis process in which glucose is formed.So, if glucose can be formed from fats thus glycogen can be form fats
===> Glucose is aldose sugar and has 2 isomers: (D-glucose and L-Glucose)
It has 2 chiral carbon atoms (i.e. the carbon atom that carry 4 different groups); the carbons number 2 and 3The (L)-glucose is the isomer that is utilized by the cells. WHY?Because the amino acid molecules which forms the enzyme present in the L- form which make the enzymes in the L- form, therefore the L-glucose is the more suitable substrate which is the more matching with the binding groups in the active site than the D-glucoseProteins molecules also have L- and D- forms because all has a chiral carbon except glycine which doesn’t carry 4 different groups.────────────────────────────────
How the scientists classified the isomers into L- and D- forms?They use the glyceraldehyde as a standard as follow:- Each isomer has a chiral carbon (i.e. the carbons atom which carries 4 different groups)- The isomer that has L- and D- forms are called enantiomers────────────────────────────────
Differentiate between the following terms:
1. Enantiomers:Are 2 stereoisomers that are mirror images of each other that are “non-superposable” (not identical), much as one’s left and right hands are “the same” but opposite
2. Epimers:Epimers are diastereomers that differ in configuration of only one carbon atom and they are non-superposable, and non-mirror images of one anotherThe glucose molecules are non-mirror image to each other but aren’t identical because they differ in one carbon atom.
3. Anomers:In carbohydrate chemistry, an anomer is a special type of epimer because it is a stereoisomer of a cyclic saccharide that differs only in its configuration at the hemiacetal or hemiketal carbon, also called the anomeric carbonThe cyclic structure of glucose has 2 anomers because the hydroxyl groups orientation differes on the hemiketal carbon atom.
In the first lecture, we study carbohydrate digestion and know that the end of digestion is production of Glucose, Fructose and Galactose
In presence of O2, oxidation of glucose is complete in mitochondria where pyruvate enters the kerb’s cycle and the electron transport chain to complete the oxidation of glucose resulting in a high amount of energy.
In absence of O2, pyruvate is converted into lactate in the cytoplasm giving a small amount of energy but it is important to some tissue.
Occurrence of glycolysis is of physiological importance in:
Tissues with no mitochondria such as RBCs, cornea and lens.
Tissues with few mitochondria: Testis, leucocytes, medulla of the kidney, retina, skin and gastrointestinal tract
Tissues undergo frequent oxygen lack: skeletal muscles especially during exercise.
Where they depend only on the glycolysis not on kerbs cycle and the electron transport chain
===> A phosphate group is transferred from ATP molecule to the carbon number 6 in the glucose molecule forming glucose-6-phosphate, thus this step is energy consuming.
===> Glucose-6-phosphate is an intermediate forming an important branch point in the metabolism.
===> This step is fast irreversible step
2. Enzymes stimulating this step:
===> The enzymes used are either glucokinase or hexokinase enzymes which are responsible for entry of glucose into the cell and phosphorylation of glucose which lead to glucose trapping inside the cell, therefore this step is irreversible because if it is reversible the glucose-6-ph will return to glucose and could exit from the cell again.
===>They are activated after a carbohydrate rich meal for 2 hours to lower the glucose blood level.
NOTE:Kinase enzyme always add phosphate group on the substrate
What is the difference between Hexokinase and glucokinase? (Click here to open)
1. Event: (formation of fructose-6-phosphate from glucose-6-phosphate)
Isomerization of glucose-6-phosphate to fructose 6-phosphate, I.e. a conversion of an aldose into a ketose byphosphoglucose isomerase.
It doesn’t need energy because it occurs spontaneosly.
2. Enzymes stimulating this step: phosphoglucose isomerase.
NOTE:Isomerase enzyme always catalyzes the structural rearrangements.
a. Event: (Phosphorylation of Fructose-6-phosphate to fructose-1,6-bisphosphate)
- Phosphorylation of Fructose-6-phosphate by ATP to fructose-1,6-bisphosphate (F-1 ,6-BP)
- This step is irreversible.
b. Enzymes stimulating this step:by phosphofructokinase (PFK)
a. Event: (cleave of 6-carbon sugar into two 3-carbon fragments)
- Splitting of fructose-1,6-bisphosphate into tow 3-carbon fragments:
Glyceraldehyde 3-phosphate (GAP)
Dihydroxyacetone phosphate (DHAP)
- Reversible under intracellular conditions
b. Enzymes stimulating this step:
- Aldolase. (This enzyme derives its name from the nature of the reverse reaction, an aldol condensation).
a. Event: (isomerisation of (GAP) to (DHAP))
- Isomerisation of Glyceraldehyde 3-phosphate (GAP) to Dihydroxyacetone phosphate (DHAP)
- It is fast reversible step.
b. Enzymes stimulating this step: triose phosphate isomerase (TPI or TIM).
c. Importance of this step:
To get full energy from glucose molecules in other words to get energy from the 6 carbons. EXPLAIN?
- The cell can’t oxidize DHAP to get energy from it , thus it will be lost as a waste product.
- Thus, the cell will obtain energy from GAP only (i.e. from 3 carbon of the glucose), not from the 6-carbons.
- But, the GAP will continue the glycolysis and give energy, while DAHP is not
- Thus, in order to get energy from the 6-carbons of glucose, the DHAP must be converted to GAP
After this step we get 2 molecules of Glyceraldehyde 3-phosphate (GAP):
Tow moleculse of Glyceraldehyde 3-phosphate (GAP) is converted into two molecules of pyruvate producing 4 molecules of ATP.
a. Event: (Oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate)
Conversion of glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate (1,3-BPG), a reaction catalyzed byglyceraldehyde-3-phosphate dehydrogenase.
b. Enzymes stimulating this step: glyceraldehyde 3-phosphate dehydrogenase.
Note that, the phosphate used in this step is inorganic phosphate.
The oxidation of the aldehyde to an acid is coupled to the reduction of NAD+ to NADH/H+
Importance of 1,3-bisphosphoglycerate:
- During the glycolysis inside the RBCs, part of the 1,3-bisphosphoglycerate is converted into 2,3-bisphosphoglycerate by the enzyme bisphosphoglycerate mutase
- 2,3-BPG decreases the affinity of Haemoglobin for Oxygen (i.e. decreases the attachment of O2 to haemoglobin) thus the oxygen can leave hemoglobin easily and this is good in case of oxygen shortage because cells will be able to get their needs of O2 easily even if the oxygen supply is low.
Clinical and physiological aspects of 2,3-BPG
smokers and people who live in high altitude, where the Hb increase in each RBC and the number of RBCs increase and increase the amount of 2,3-bisphosphoglycerate leading to decrease the affinity of Hb for O2 causing dissociation of O2 form Hb easily into the blood capillary and thus the cells takes its need easily.
Fetus gets oxygen form the mother thus fetal Hb has a high affinity for O2 because it has a plenty of O2 from his mother, thus competing with the Hb of the mother because the oxygen leave the Mother Hb because of it low affinity and attach to the Fetus Hb which has a high affinity for the O2.
The sotred blood for blood transfusion has diminished levels of 2,3-BPG, thus before transfusion of inosine or the glycolytic substrate dihydroxyphosphate to regenerate 2,3-BPG before blood transfusion because in absence of 2,3-BPG suffocation اختناق occurs.
a. Event: (Conversion of 1,3-bisphosphoglycerate to 3phosphoglycerate)
- Conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate
- This step gproduce one ATP molecule.
b. Enzymes stimulating this step: phosphoglycerate kinase.
Formation of ATP from transferring a Phosphate group to ADP from organic substrate is calledSubstrate level phosphorylation.
Formation of ATP from by transferring a Phosphate group to ADP from NAD or FAD in the electron transport chain is calledOxidative phosphorylation.
a. Event: (Conversion of 3-phosphoglycerate to 2-phosphoglycerate)
Shifting the phosphate group from the carbon number 3 to the carbon number 2
a. Enzymes stimulating this step: phosphoglycerate mutase.
The importance of this step is to make the compound suitable for the active site of the enolase enzyme.
NOTE:Mutase shifting of a functional group from one position to another within the same molecule
a. Event: (2-Phosphoglycerate is dehydrated to form phosphoenolpyruvate)
The dehydration of the alcohol produces a double bond between carbons 2 and 3 and creates a high-energy enol phosphate linkage.
b. Enzymes stimulating this step: enolase.
a. Event: (Conversion of phosphoenol pyruvate to pyruvate)
Conversion of phosphoenol pyruvate to pyruvate producing one ATP molecule.
It is irreversible step.
b. Enzymes Stimulating this step:Pyruvate Kinase (transfer Phosphate group from phosphoenolo-pyruvate to ADP molecule to produce one ATP Molecule)
فالخطوة دى برضو اتكون عندنا kinase يعنى هينقل مجموعة فوسفات وبالفعل هو نقل مجموعة فوسفات من الphosphoenolo-pyruvate الى جزئ ADP عشان يدينا جزء واحد من الATP.
We know that we get from STAGE (I) 2 molecules of glyceraldehyde-3-phosphate and we know that each molecules when continue glycolysis in the STAGE(II) give 2 ATP molecule, thus the 2 molecule of glyceraldehyde-3-phosphate give 4 ATP molecules.
Also we know that the steps number 1 and 3 consume 2 molecule of ATP
Thus, the net product of ATP during glycolysis is 2 ATP
4 ATP form Stage (II) – 2 ATP from Stage (I) = 2 ATP
Insulin: Stimulates synthesis of all key enzymes of glycolysis. It is secreted after meal (in response to high blood glucose level).
Glucagon: Inhibits the activity of all key enzymes of glycolysis. It is secreted in response to low blood glucose level.
In the lecture no. 2 we saw that the end of the glycolysis is pyruvate and also NADH which is obtained in step 6, but we don’t know the fate of pyruvate and NADH; will they be waste products or will they consumed in any way? ………. this what we see in the lecture no. 3
also, we will study the importance of glycolysis and the diseases resulted from the deficiency of the 3 enzymes that catalyze the 3 irreversible steps
We know before that in the step 5, Dihydroxyacetonephosphate (DHAP) and glyceraldehyde-3-Phosphate (G-3-P) are produced and also we know that the cell doesn’t consume DHAP, so, DHAP is converted into G-3-P……….thus,the stage 2 of glycolysis starts with 2 G-3-P and thus we get 2 NADH and 2Pyruvate from the stage 2 (the end of glycolysis) >>>>>> and we know that the conversion of DHAP to G-3-P occurs to get the energy from the 6 carbons of glucose
But also pyruvate has 3 carbon and the cell doesn’t use, …….. so, what if the fate of pyruvate? …..Will the cell get rid of it as waste product and lose the energy of 3 carbons? ….. this what we will see in this section.
in case of NADH, What is its fate? ….. it should be converted to NAD+ which is needed in the step 6 and if this conversion doesn’t occur, glycolysis can’t be repeated because there will not be NAD+……. so NADH should be oxidized through Krebs cycle and Electron transport chain or by fermentation in case of oxygen shortage (we will study this in the next lectures).
1) Fate of Pyruvate:
Pyruvate will be oxidized to get the energy of the rest 3 carbons …. in 2 ways:
Oxidation in presence of Oxygen through krebs cycle (Tricarboxylic acid cycle [TCA]) and Electron Transport chain (ETC) in Mitochondria.
Oxidation in absence of Oxygen to be converted into lactate
a) Under aerobic conditions (in presence of oxygen):
In aerobic conditions, pyruvate proceeds through the tricarboxylic acid (TCA) cycle (=Kerbs cycle) to complete glucose oxidation by its conversion to acetyl CoA and oxaloacetate which are the principle intermediates for of kreb’s cycle according to the following:
i. Oxidation to acetyl-CoA:
With presence of oxygen, pyruvate is oxidized to acetyl-CoA (acetyl coenzyme A) producing much more energy by Pyruvatedehydrogenase via oxidative decarboxylation reaction as follow:
Acetyl-CoA is a major fuel for krebs cycle as well as a building block for fatty acid synthesis in mitochondria.
ii. Carboxylation to oxaloacetate:
Conversion of pyruvate to oxaloacetate (OAA) by pyruvate carboxylase is an important, because it provide the citric acid cycle with OAA intermediate and provides substrate for gluconeogenesis and krebs cycle.
b) Under anaerobic conditions:
If no oxygen is available or in case of short supply of oxygen, cells can obtain energy through the process of anaerobic respiration such as fermentation producing lesser energy than aerobic respiration in the form of fewer ATP molecules and it is not efficient process.
In an aerobic condition, glycolysis is repeated when the NAD+ is regenerated in the kerbs cycle and electron transport chain by oxidising NADH resulting in high energy, but in case of anaerobicrespiration glycolysis is self-functioning through regeneration of NAD+ by the conversion of Pyruvate to lactate by lactate dehydrogenase through lactic acid fermentation process without producing any ATP molecules.
This conversion of Pyruvate to lactate in anaerobic conditions is promoted by the accumulation of NADH and the depletion of NAD+ which is needed as an electron acceptor so that glycolysis can continue
In microorganisms, NAD+ is regenerated through conversion of Pyruvate to ethanol by ethanoldehydrogenase through the alcohol fermentation.
Tissues that use lactic fermentation:
1) RBCs: because it doesn’t contain mitochondria because aerobic oxidation of pyruvate occurs through Kerbs cycle and electron transport chain which occurs in mitochondria
2) Muscles: during heavy exercise the cells deplete oxygen and thus there is a shortage of oxygen supply, thus the muscle get energy through anaerobic glycolysis and regenerate NADH through the lactic acid fermentation (anaerobic respiration).
2) Fate of NADH:
a) In aerobic conditions:
NADH is re-oxidized in the electron transport pathway and kerbs cycle, making 6ATP molecules in oxidative phosphorylation, thus in the aerobic respiration, the cell get 8ATP;
8ATP = 6 ATP form the electron transport chain + 2 ATP from glycolysis .
we will study kerbs cycle and electron transport chain in the next lectures.
b) In anaerobic conditions:
NADH is re-oxidized by lactate dehydrogenase (LDH) or alcohol dehydrogense either of which provide NAD+ for more glycolysis.
During vigorous exercise, the cell needs high energy leading to high rate of glycolysis which then consumes all oxygen in the muscle resulting in shortage of oxygen and thus it will regenerate NAD+ through the lactic acid fermentation.
But accumulation of lactate (lactic acid) in muscle increase acidity and decrease the pH causing muscle cramp and muscle fatigue.
So, accumulation of lactic acid is harmful for the cells, thus the cells must remove the excess lactic acid
The muscle remove the excess lactic acid when the O2 is restored by the high rapid respiration during resting stage and this occur through the lactic acid cycle (also called Cori cycle) in which the lactic acid enter the blood circulation and move to the liver in which lactate is converted to Pyruvate by the lacticdehydrogenase which then is Converted to glucose through gluconeogensis and then glucose enter the circulation to move to the muscle to give it the required energy and the cycle will repeated again in case of shortage of O2
And in the resting stage, without continuing exercise, the Pyruvate may enter the kerbs cycle and the electron transport chain in mitochondria and produce high energy
2) Wight loss of patients suffer from cancer:
The cancerous cell needs high amount of energy because it reproduce rapidly and thus glycolysis is ten times faster in solid tumors than in the non-cancerous tissue and also the solid tumors lack the blood connection and thus lack oxygen supply, thus it use the lactic acid fermentation and the Cori cycle in which the lactate formed in the tumor cell goes to the liver through the blood to converted to Pyruvate by the lactate dehydrogenase and then Pyruvate is converted to glucose through gluconeogensis to produce glucose which then pass to the blood then to the cancer cell
But here there is an energy dissipation because high energy used in the form of 6-phosphate groups to form glucose in liver to get low energy in the form of 2 phosphate groups through glucose oxidation in the cancer cells and this energy dissipation cause weight loss.
This leads to decreasing production of the ATP which is require for the Na/K pump leading to shutting down these pumps causing malformation of the RBCs and then destruction of the RBCs and thus leading to haemolysis causing haemolytic anaemia
Excessive RBC destruction in spleen and haemolysis lead to jaundice (elevated bilirubin) and increased reticulocyte count
2) Hexokinase deficiency:
Leads to haemolytic anaemia due to decrease ATP production. The mechanism is similar to that of PK deficiency.
3) Lactic acidosis:
Lactic acidosis occurs whenever production of lactate exceeds its utilization.
Lactic acidosis is characterized by a pH < 7.36 and lactate level > 5mmol/L
Lactate is produced in all tissues. Skeletal muscle, brain, red blood cells, and renal medulla are responsible for the majority of the production.
Causes of lactic acidosis:
- It results from increased formation or decreased utilization of lactate.
- Increased formation of lactate as in:
Severe muscular exercises.
Decreased utilization of lactate in tissues: it occurs in cases of anoxia or lack of oxygen. This is because oxygen is essential for conversion of lactate into pyruvate, which proceeds into acetyl CoA, and Krebs’cycle. Tissue anoxia may occur in conditions that impair blood flow e.g. myocardial infarction, angina pectoris, respiratory disorders,’ and anaemia.
4) Oxygen Deprivation in Heart Attack and Stroke:
- Myocardial infarction: interruption of the blood (O2) supply to a portion of the heart leading to blood clotting within the heart.
- Stroke: interruption of the blood (O2) supply to a portion of the brain.
The main problem lack of (O2), is to block of aerobic metabolism (ETS) and induction of anaerobic metabolism (Glycolysis) Leading to lactic acid acidosis.
5) Consequences of O2 Limitation:
Disruption of osmotic balance (ion pumps) leading to entry of water to the cells causing Swelling of cells and organelles and increased permeability.
Acidification (anaerobic lactic acid production), high level of activity for leaked lysosomal enzymes.
How to diagnose Myocardial Infarction in lab:
- The lactate dehydrogenase had 5 forms, LDH 1,2,3,4, and 5
- The LDH1 is the present in the heart and it presence is indication to lactic acid formation and so, indication of shortage of (O2) supply.
LDH5 is induction to hepatitis.
Dr.Ehab Aboueladab, Tel:01007834123 Email:firstname.lastname@example.org,email@example.com