Biochemistry Flashcards
What are myoglobin and hemoglobin?
*Mb is a monomeric protein
(153 AA)
*Hb is a heterotetramer (a2b2)
Describe the structures of Mb and Hb
*Eight a -helices in Mb
and Hb b subunit
*Seven a-helices in Hb
a subunit
* The subunits contain
heme group
What are the functions of surface amino acids in Mb molecules?
Surface amino acids
prevent association
of Mb molecules
What are the functions of surface amino acids in Hb molecules?
Surface amino acids of Hb
provide hydrogen bonds and
non polar interactions with
other subunits
Describe the function of Hb
-Hemoglobin transports O2
from lungs to tissues
* Hemoglobin transports
CO2 from tissues to lung
Describe the function of Mb
- Myoglobin is an O2
storage protein in muscles
What are the forms of Hb?
–Adult hemoglobin (Hb A):
98% a2 b2
2% a2 d2
–Fetal hemoglobin (Hb F):
a2 g2 -Has more affinity to O2
–Embryonic hemoglobin (Hb e):
a2e2
Converted to Hb F after 6
months of gestation
–A clinically important form is
Hb A1c
It forms 4-6.5% of Hb A1.
Formed by conjugation of glucose with the N-terminal of b chains
Describe Hb A1c
-It forms 4-6.5% of Hb A1
-Formed by conjugation of glucose with the N-terminal of b chains
-As RBCs live for ~ 120 days, Hb A1c reflects how blood sugar is
controlled over the past 4 months.
What is heme?
*Heme is a prosthetic
group present in a
number of oxygen
transporting proteins
What is a heme group?
*It is a complex organic ring structure called
protoporphyrin with an iron atom in its center
*The iron is in the ferrous (Fe++) oxidation
state so it can bind O2 reversibly
-The fifth and sixth coordination
positions are perpendicular to
the porphyrin
How is heme positioned?
*Heme is positioned in a deep hydrophobic pocket to protect it from being oxidized to ferric (Fe+++)
which can not bind O2
What types of bonds are present?
Heme is non-covalently bound to globin proteins through proximal His residue.
When O2 binds, the electronic properties of
heme iron change. This accounts for the change of color from dark purple of
the deoxy form to the bright red of the oxy form
Describe the oxygen binding curves
*Mb has hyperbolic O2 binding curve,
PO50 = 5 mmHg
*Mb Binds to O2 very tightly, and
releases it slowly
*Hb has sigmoid O2 binding curve
*PO50 = 26 mmHg
*Hb has high affinity for O2 at high pO2 (lungs)
*Hb has low affinity for O2 at low pO2 (tissues)
what is heme?
Heme is the colored prosthetic group of hemoglobin and
a number of proteins called hemoproteins.
give examples of important hemeproteins
- Hemoglobin: Transport of oxygen in blood.
- Myoglobin: Storage of oxygen in muscle
- Cytochrome C: Involvement in electron transport chain (respiratory chain)
- Cytochrome P450: metabolism of xenobiotics
- Catalase: Degradation of hydrogen peroxide
- Tryptophan Pyrrolase: Oxidation of tryptophan.
- Cytoplasmic guanyl cyclase enzyme: formation of cyclic
GMP
describe the structure of heme
- Heme is composed of iron in the ferrous state attached
to the center of porphyrin ring. - Porphyrins are cyclic compounds formed by the linkage
of four pyrrole rings through 4 -HC= methenyl bridges
describe the structure of heme
- Heme is composed of iron in the ferrous state attached
to the center of porphyrin ring. - Porphyrins are cyclic compounds formed by the linkage
of four pyrrole rings through 4 -HC= methenyl bridges
Porphyrins can form complexes with metal ions bound to
the 4 nitrogen atoms of the pyrrole rings.
Examples :
1-Iron porphyrins as heme of hemoglobin
2- Magnesium-containing porphyrins as chlorophyll.
where is heme synthesised?
Heme synthesis occurs in most tissues except mature RBCs (have no mitochondria).
The major sites are:
A.Erythrocytic: Erythrocyte producing cells of bone marrow, which are active in hemoglobin synthesis.
B.Non-Erythrocytic: especially Liver, which synthesizes a
number of heme proteins (particularly cytochrome P450).
where does heme biosynthesis occur within the cell?
Heme biosynthesis begins and ends in the mitochondria, but 3 intermediate reactions occur in the cytoplasm.
The reactions are irreversible.
what is the first step of heme biosynthesis?
1- Synthesis of Delta Aminolevulinic acid:
-It starts in mitochondria by condensation reaction
between succinyl-CoA and glycine to produce delta-aminolevulinic acid (ALA).
-It is catalyzed by ALA synthase in the presence of
Pyridoxal phosphate. (coenzyme of Vit B6)
It is the rate-controlling enzyme of Heme synthesis.
what is ALA synthase?
It is the rate-controlling enzyme of Heme synthesis.
describe the second step of heme biosynthesis
-ALA leaves the mitochondria to cytoplasm where two molecules of ALA are condensed by the enzyme
ALA dehydratase
(zinc-containing enzyme)
to form porphobilinogen (PBG) and remove two molecules of water.
-This enzyme is sensitive to inhibition by heavy metal ions as lead that replace zinc.
list all the steps of heme biosynthesis
1- Synthesis of Delta Aminolevulinic acid
2-Formation of Porphobilinogen PBG in cytoplasm
3-Condensation of 4 PBG to form the first Porphyrinogens
4-modification of the side chains
5- oxidation of the rings
6-Insertion of iron by enzyme Ferrochelatase in mitochondria
what is ALAS?
the rate limiting enzyme
- There are 2 ALA synthase isomers, each produced
by different genes and controlled by different
mechanisms. - ALAS1 is found in all tissues, whereas ALAS2 is
erythroid-specific. - Loss of function mutation in ALAS2 results in
sideroblastic anemia and iron overload.
Describe the effect of heme on ALAS
Repression (at gene level)
❑ When porphyrin production exceeds the availability of the proteins that require it, heme accumulates.
❑The excess heme is converted to hemin (hematin) by oxidation of Fe2+ to Fe3+
❑Hemin inhibits synthesis of the enzyme ALAS1 by
repressing transcription of its gene, decreasing synthesis and stability of mRNA of the enzyme and decreasing its import into mitochondria (in bone marrow ALAS2 is controlled by the availability of intracellular iron)
Describe the effect of drugs on ALAS activity
The activity of ALASI in liver (not in Bone marrow) can be increased by certain drugs such as barbiturates and steroids or other compounds such as insecticides and carcinogens.
1) These drugs are metabolized in liver by
cytochrome P450, increasing consumption of heme containing proteins.
2) So, heme concentration decreases with removal of its inhibitory effect on ALA synthase gene
Describe the effect of hypoxia on heme synthesis
In the erythropoietic tissues
Heme synthesis is affected by Hypoxia that increases ALA synthase activity by increasing Erythropoietin hormone
Describe the effect of lead on ALA
Lead has an inhibitory effect on ALA dehydratase and Ferrochelatase.
The body has enough iron available but cannot incorporate it into hemoglobin. Lead poisoning causes Sideroblastic anemia.
What are sideroblast?
Sideroblasts are atypical
nucleated erythrocytes with
granules of iron accumulated in perinuclear mitochondria
What is Porphyria “The Vampire Disease”?
These are rare group of genetic disorders of heme synthesis resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors that have unfairly branded many sufferers with the term “vampire”.
These poor souls are
-extremely sensitive to sunlight that can easily result in burns and abrasions, and so they prefer darkness.
-They suffer from acute attacks of abdominal pains and vomiting.
-Their urine may have a purplish-red color leading some to wrongly believe that it results from drinking blood.
- They may have increased hair growth, and with repeated damage, their skin tightens and shrinks.
-When this occurs around the mouth, the canine teeth appear to be more prominent, and suggestive of fangs.
what are globins?
*The globins are a family of
globular proteins, which are
thought to share a common
ancestor.
how did globins first originate?
*Globins originated first as one gene in one chromosome.
* As with many genes, different types of mutations occurred so that the globin gene underwent some molecular events to produce the current diversity of the
polypeptide chains present.
what is the first step of globin evolution?
Duplication
First, a duplication process occurred to this gene on the same chromosome to produce two identical copies of the gene.
- Mutations occurred to both genes so that some dis-similarity between them occurred, however because they still have more than 95% similarity in their nucleotide sequence, they are still expressing proteins that have almost the same amino acid sequence and molecular structure (alpha helices folded the same way).
A molecular event called “transposition” occurred so that these two copies of the
gene became on two different chromosomes (chromosome 11 and
chromosome 16)
Describe the alpha gene family
- The α-gene cluster on chromosome 16 contains two genes for the α-globin chains.
- It also contains the ζ gene that is expressed
early in development as an α-globin-like component of embryonic hemoglobin
Describe the beta gene family?
- A single gene for the β-globin chain is located on chromosome 11.
- There are an additional four β-globin-like
genes:
➢the ε gene (which, like the ζ gene, is expressed early in embryonic development),
➢two γ genes (Gγ and Aγ that are expressed in HbF),
➢δ gene that codes for the globin chain found in the minor adult hemoglobin
HbA2.
what are hemoglobinopathies?
A group of genetic disorders caused by production of a structurally abnormal hemoglobin molecule/ synthesis of insufficient quantities of normal hemoglobin; or rarely both
What mutation causes sickle cell anemia?
Sickle cell anemia is an Autosomal Recessive disease
in which a mutation occurs in b- chain:
E6 (polar negatively charged Glutamic Acid)
is changed to
V6 (hydrophobic Valine)
what kind of disease is sickle cell anemia?
Autosomal Recessive
what type of individuals are affected by sickle cell anemia?
–Being Autosomal Recessive disease, Symptoms occurs mainly in homozygous individuals.
–Heterozygous individuals (trait) present symptoms only in Hypoxic conditions.
how does sickle cell anemia occur?
-Valine, as a hydrophobic amino acid, binds to a
hydrophobic pocket in deoxy-Hb
-This type of hemoglobin is called Hemoglobin S
-HbS Polymerizes to form long filaments when oxygen tension is reduced
-This causes sickling of cells
- Such sickled cells frequently block the flow of blood in the narrow capillaries.
This leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually death (infarction) of cells in the vicinity of the blockage.
why does sickle cell anemia offer an advantage against malaria?
The Fragile sickle cells
cannot support the
parasite
What causes HbM?
substitution of proximal
histidine by tyrosine causing
oxidation of Fe++ to Fe+++ forming methemoglobin
What causes HbC?
substitution of two
glutamate residues in the b chain by lysine causing Mild hemolytic anemia.
What is Hb SC?
hemoglobin SC disease
(Hb S + Hb C)
What is Thalassemia?
decreased rate of
synthesis of one or more globin chains
a-Thalassemia: decreased rate of synthesis of a-globin chains
b-Thalassemia: decreased rate of synthesis of b-globin chains. g or d chains will increase.
how many copies of the a-globin gene are present in the genome?
4 copies (two on each
chromosome 16)
what happens when one/multiple a-globin genes are defective?
–If one of the four genes is defective, the individual is termed a silent carrier of α -
thalassemia, because no physical manifestations of the disease occur.
–If two α -globin genes are defective, the individual is designated as having
α-thalassemia trait.
–If three α -globin genes
are defective, the individual has Hb H (β4 ) disease—a mildly to moderately severe hemolytic anemia.
–If all four α -globin genes
are defective, Hb Bart (γ4)
disease with hydrops
fetalis and fetal death
occurs, because α -globin chains are required for the
synthesis of Hb F.
what is B-thalassemias?
Decreased rate of synthesis of B-globin chains. The y or g chains will increase.
Increase in HbA2 and HbF occurs.
There are only two copies of the B-globin gene in each cell.
-Individuals with one defective B-globin gene - B thalassemia minor
-Individuals with both genes defective - B thalassemia major (Cooley anemia)
what happens to infants born with B-thalassemia major?
They become severely anemic, usually during the first or second year of life due to ineffective erythropoiesis. Skeletal changes as a result of extramedullary hematopoiesis are also seen. These patients require regular blood transfusion.
What is iron and how is it distributed in the body?
- Iron is one of the Micro (trace) elements.
- The total body iron range between 3-5 g and is distributed as:
1- Hemoglobin iron: 75% of the total body iron.
2-Fixed tissue iron: e.g. Myoglobin, cytochrome C (in respiratory chain), catalase and peroxidase enzyme.
3-Labile tissue iron: (changeable) stored
iron in the form of ferritin and small amount of hemosiderin.
4- Plasma iron: transport form of iron (transferrin)
what is the daily iron requirement?
*The average daily iron requirement is 15-20 mg/day.
*Only 3-6% of iron intake is absorbed into the blood, which is equal to iron lost by the sloughing of cells (0.6 mg/day).
* Absorption is increased by increasing demands.
How is iron homeostasis maintained?
- To maintain iron homeostasis, the amount of iron absorbed should be equal to iron excreted.
- Iron homeostasis depends on control of iron
absorption from the intestine. Iron losses are
generally unregulated.
What are the two types of iron and how are they sourced?
Heme iron:
Easily absorbed and used by the body. Found in meat, fish and poultry
Non-heme iron: Poorly absorbed and used. Found in vegetables, fruit and grains
Describe Iron Absorption?
- Heme is absorbed by intestinal cells in the duodenum by a heme carrier protein 1 (HCP1).
- In enterocytes, Fe+2 is released from heme by a heme oxygenase enzyme.
- Non heme Iron is present in the diet in the Fe3+ state.
Cooking of food facilitates breakdown of ligands attached to iron, helping to release it in the gut. - It is reduced to Fe2+ by ferrireductase enzyme
- The reduction of ferric iron to ferrous iron is facilitated by HCl in the stomach and vitamin C (ascorbic acid)
- Iron is transported into the enterocyte with a proton via
Divalent metal transporter (DMT-1).
What are the fates of Absorbed Fe2+?
*Absorbed Fe2+ from heme or non heme sources may have the following fates:
I. Fe2+ iron is oxidized again inside intestinal cells to Fe3+ ions by ferroxidase. Then the ferric ions combine with apoferritin (mucosal cell protein) to form ferritin for temporary storage.
II. Fe2+ is transported out of the enterocyte by the basolateral membrane protein (ferroportin), oxidized by Cu-containing
membrane protein (hephaestin), and taken up by the plasma transport protein transferrin (2 Fe3+ / transferrin molecule).
Ferroportin protein (the only known exporter of iron from cells to blood) is regulated by the hepatic peptide hepcidin that induces internalization and lysosomal degradation of ferroportin.
What is Hepcidin?
*It is a hormone released from the liver when iron
levels in the body become too high.
*It inhibits further iron passage from the small intestine epithelial cell into the blood by attaching and
inhibiting the ferroportin transporter
*A deficiency of hepcidin causes tissue iron overload.
*Hepcidin has a central role in iron hemostasis (its
transcription is suppressed when iron is deficient).
What are the excretory characteristics of Iron?
- 1-The body has no mechanism of excretion of
iron - 2- Only minimal amounts of iron are lost by:
*Bleeding
*Shedding of cells of intestine
*Shedding of cells of skin
So Total body Iron is regulated at the level of absorption by enterocytes of the duodenum
what is mucosal block?
The intestinal content of ferritin is limited so once saturated with iron, the absorption of the iron is inhibited.
what foods decrease iron absorption?
phytic acid, oxalates, and phosphates in diet decrease iron absorption.
calcium in dairy foods
What factors increase iron absorption?
- Requirements of body: An increase rate of erythropoiesis (e.g. after hemorrhage)
- Vitamin C helps reduction of ferric to ferrous and
forming soluble complex. - Gastric HCl helps absorption, so absorption decreases in achlorhydria and partial gastrectomy.
why should tea and milk be avoided during meals?
Tea, milk and other dairy products reduce iron absorption, and that is why you should avoid drinking milk and tea with meals.
leave at least an hour before and after a meal containing iron.
What is Transferrin?
In the plasma, Fe2+ is changed to Fe3+and bound to apotransferrin forming the iron transport protein,
transferrin (Tf).
Tf is a glycoprotein, synthesized in the liver.
What is the function of transferrin?
Tf transports iron (2 mol of Fe3+ per mol of Tf) in the
circulation to sites where iron is required.
Tf binds to receptors in cell membrane of erythroblasts,
ingested by endocytosis, and iron is delivered to the
mitochondria, where heme is synthesized.
What is total iron binding capacity (TIBC)?
- Represents the capacity of transferrin to bind iron.
- It measures the free transferrin that is ready to carry Iron.
- Normally, the protein is only one third (35%) saturated with iron.
- Low TIBC levels usually indicate high levels of iron in the blood.
- High TIBC levels typically indicate low levels of iron in the blood.
Describe cellular uptake of iron
There are receptors (Transferrin receptors) on the
surfaces of many cells for transferrin (bone marrow
cells, placenta). Transferrin binds to these receptors and is internalized by receptor-mediated endocytosis.
The acid pH inside the lysosome causes the iron to
dissociate from the protein (Apotransferrin), that is not degraded within the lysosome. Instead, it reenters the plasma to pick up more iron to cells in need.
The translation of the mRNA of transferrin receptors and ferritin is regulated by iron regulatory proteins and iron responsive elements that depend on iron level in the cell.
How is iron stored?
1- Ferritin: is the predominant storage form of iron in cells. It is present in most cells especially in intestinal mucosa, liver, spleen and bone marrow.
- Apoferritin (the protein part of ferritin) can take 4000-4500 ferric ions / molecule to form ferritin.
- Normally, there is a little ferritin in human plasma. However, in patients with excess iron (e.g. hemochromatosis), the amount of ferritin in plasma is markedly elevated.
- In iron deficiency anemia it is markedly decreased.
- Ferritin is the primary intracellular iron-storage globular protein. It keeps iron in a soluble and non-toxic form.
2- Hemosiderin:
When iron is in excess, the storage capacity of the apoferritin is exceeded. This leads to iron deposition adjacent to ferritin spheres in the form of brown aggregated deposits, called hemosiderin.
How is iron excreted?
- The high binding capacity of iron to macromolecules,
leads to absence of free iron salts. - Normally the body guards its own iron content and
there is no physiological excretory mechanism. - So adult male loses about 1-2 mg / day which is
replaced by absorption. - Loss of iron occurs only through the normal
shedding of tissues: - Epidermis
- Gastrointestinal mucosal cells in stools
- Hair
- Menstrual blood in females
What is iron overload? (symptoms/ treatment)
- Can occur due to accidental ingestion of iron tablets:
Acute Fe poisoning
- It is one of the most common causes of poisoning deaths in children under the age of 6 years.
- The lethal dose in children is 200–300 mg/kg body weight and approximately 100 g in adults.
- High doses of Fe supplements cause gastrointestinal symptoms especially constipation although nausea, vomiting, and diarrhea may occur.
- Iron overload is treated by an iron chelator.
- Overload can also occur due to genetic defects:
Hereditary hemochromatosis
* Caused by mutations to the high iron gene (HFE).
* Excess iron is deposited in liver, pancreas, heart and skin; damaging their cells and inducing hyperpigmentation and hyperglycemia (Bronze diabetes).
* Serum iron and transferrin-saturation are elevated.
* Treatment is by phlebotomy or use of Fe chelators.
What are vitamins?
Vitamins are organic compounds occurring in small quantities in different natural foods.
Vitamins show the following general characters:
They are essential factors that must be supplied in the diet.
They are not synthesized by humans.
They are needed in small quantities (trace amounts) for normal growth, differentiation and maintenance of normal cellular function.
Deficiency of vitamins result in various metabolic diseases.
They are required for the proper utilization ‘metabolism’ of carbohydrates, lipids and proteins as they can serve as coenzymes for many metabolic enzymes.
They cannot be used as fuel (not oxidized to produce energy) and they do not enter in the structure of tissues
What are the causes of vitamin deficiency?
Inadequate dietary intake.
1) Malabsorption: e.g. biliary obstruction, pancreatitis.
2)Dysfunction or absence of proteins required for
absorption, transport, activation or utilization
causes deficiency (e.g., activation of folic acid and
vitamin D3, absorption of B12).
3) Increased excretion as in kidney diseases.
4) Treatment with some drugs; destruction of intestinal microorganisms by antibiotic therapy can produce symptoms of vit K deficiency.
Other drugs can form complexes with specific vitamins and affect their absorption or excretion (e.g.
the Anti tuberculous drug isoniazid can affect vitamin B6).
Describe fat soluble vitamins (solubility, absorption, carrier proteins, storage, excretion, deficiency, treatment)
Major vitamins: A, D, E and K
Solubility in fat: Soluble
Water solubility: Not soluble
Absorption: Requires the presence of lipid and bile salts.
Carrier proteins: Required
Storage: Stored in liver
Excretion in urine: Not excreted
Deficiency: Manifested only when stores are depleted
Treatment of deficiency: Single large doses may prevent deficiency.
Describe water-soluble vitamins
Major vitamins: B complex group and C
Solubility in fat: Not Soluble
Water solubility: soluble
Absorption: Absorption is simple
Carrier proteins: Not Required
Storage: No appreciable storage; excreted
Excretion in urine: Excreted
Deficiency: Manifests rapidly as there is no storage
Treatment of deficiency: Regular dietary supply
is required
Wha are the water soluble vitamins
–Vitamin C and Ascorbic Acid
–B family:
Energy Related:
Thiamin (B1)
Biotin
Riboflavin (B2)
Niacin (B3)
Pantothenic acid
Hematopoietic:
Folic Acid
Vitamin B12
Others:
Pyridoxine (B6)
Describe the structure of Vitamin B12
Vitamin B12 has a complex tetrapyrrole ring structure (corrin ring) to which a cobalt ion is added to its center.
Cobalt is essential for activity, it is the cause of the red color.
What groups can be attached to cobalt (vitamin b12)?
–Methyl group (methylcobolamin),
–Hydroxyl group (hydroxycobolamin),
–CN (cyanocobolamin),
–Adenosyl group (adenosylcobolamin)
What are the sources of VB12
Vitamin B12 is exclusively gained from animal
sources.
It is absent from plant sources.
In animals it is conserved in the liver as
adenosylcobolamin, methylcobolamin or
hydroxycobolamin.
-Liver and yeast are good sources
what glycoprotein receptor is required for intestinal absorption of vitamin B12?
intrinsic factor
what happens after vitamin B12 absorption?
After absorption it is transported bound to a
plasma protein called transcobolamin.
Describe Vitamin B12 storage
It is the only water soluble vitamin that is stored in the liver. Up to 6 years supply of B12 can be stored in the liver.
what is Deoxyadenosylcobolamin?
Deoxyadenosylcobolamin is the coenzyme for
conversion of methylmalonyl~CoA to
succinyl~CoA which is a member of the
citric acid cycle.
what is Methylcobolamin?
Methylcobolamin is the coenzyme in the combined
conversion of (1) homocysteine to methionine,
(2) methyltetrahydrofolate to tetrahydrofolate
what are the conditions associated with Vitamin B12 deficiency?
–Malabsorption (lack of the intrinsic factor) due to
atrophy of gastric mucosa, achlorhydria, or
after total gastrectomy.
–Debilitated patients.
–Vegetarian people. If plants are contaminated
with microorganisms, supply of B12 will be
sufficient.
what are the manifestations of vitamin B12 deficiency?
It includes hematological manifestation in the
form of Pernicious anemia or Megaloblastic anemia
& neurological manifestations in the form of
itching sensation, memory loss, and may be pains.
why do pernicious and megaloblastic anemia occur in vitamin B12 deficiency?
This occurs due to Impaired DNA synthesis and prevention of cell division
with the accumulation of immature erythrocytes in the circulation.
why do neurological manifestations occur with vitamin B12 deficiency?
*Neurological disorders are due to progressive
demylination of nervous tissue.
*This may be secondary to deficiency of
methionine leading to defective methylation.
*Folate will be trapped as methyltetrahydrofolate
(folate trap) will lead to impaired purine and
pyrimidine synthesis.
how is vitamin b12 deficiency treated?
High levels of supplemental folate can overcome the megaloblastic anemia but not
the neurological disorders.
-Hence caution must be utilized in using folate to treat megaloblastic anemia.
Describe the structure of folic acid
Folic acid or folate consists of the base pteridine attached to one molecule of each p-aminobenzoic acid (PABA), and glutamic acid
can folic acid be synthesised?
Animals are not capable of
synthesizing PABA or
attaching glutamic to
pteridine.
It must be supplied in diet
–Liver, yeast and green leafy
vegetables are the major
sources.
Describe the metabolic role of folic acid
The biologically active form is the tetrahydrofolate.
It is the carrier of activated one carbon units that are essential for the synthesis of choline, serine, glycine, methionine and purines.
Describe the effect of drugs on folic acid
Sulfa drugs inhibit synthesis of folic acid in bacteria (antibiotics).
Trimethoprim, and methotrexate are inhibitors of folate reductase (anticancer chemotherapeutic agents).
what are the daily requirements of folic acid?
–400 µg/day to be increased to 800 µg/day during pregnancy and lactation.
Recommended to be supplemented to ladies before pregnancy.
how does folic acid deficiency occur?
It may occur due to increased demand, poor absorption, vitamin B12 deficiency, interference with metabolism of folic acid.
Deficiency leads to Megaloblastic anemia.
what is glycolysis?
Glycolysis is the sequence of
reactions that convert glucose into pyruvate (or lactate) with the concomitant production of small amount of ATP
where does glycolysis occur?
Glycolysis occurs in the cytosol
-Glycolysis occurs in all tissues
does glycolysis occur in the presence of oxygen?
- Glycolysis can proceed either in presence and in absence of O2
Describe the number of platelets in the body
Number: 150,000- 400,000/mm³.
Increase in number: Thrombocytosis
Decrease in number: Thrombocytopenia
what is the lifespan of thrombocytes (platelets)?
10 days. Senescent platelets are phagocytosed by macrophages primarily in the spleen.
what is the origin of platelets?
The platelets are cell fragments derived from the megakaryocytes in the bone marrow
Describe the morphology of platelets (LM)
Size: 2 - 4 µm in diameter.
Shape: oval disk shaped (cell fragments).
Nucleus: They lack nuclei.
Describe the morphology of platelets (EM)
The cell membrane coated by glycocalyx (glycoproteins and glycolipids) to be involved in platelets adhesion.
Describe the cytoplasm of platelets
Cytoplasm: divided into 2 regions:
Granulomere: central granular region
Hyalomere: Peripheral clear region
Describe the hyalomere
: It is the peripheral part. It shows:
a) Marginal bundle consisting of:
10 -15 microtubules arranged parallel to each other forming a ring within the hyalomere. They assist in maintaining the discoid form of the platelets.
Actin and myosin microfilaments helping contraction of the
platelets during retraction of blood clot.
b) Two tubular systems:
Open canalicular system: which is invagination from the cell membrane, facilitating platelets’ uptake of factors from plasma.
Also, this system facilitates rapid degranulation upon activation and Ca release.
Dense tubular system: which may be remnants of endoplasmic reticulum of megakaryocytes (stores Ca ions)
What are the granules in the granulomere?
Alpha granules (majority):
- Fibrinogen
- PDGF
- coagulation factors.
- Platelet factor 4
Delta granules:
-ADP
- ATP
- Ca
- Serotonin
Lambda granules:
Lysosomes
What are the 2 phases of glycolysis?
First phase; Energy–investment phase: 2 ATP molecules consumed.
-The first 5 reactions of glycolysis
Energy–generation phase
(pay off):
10 ATP molecules produced
What are the reactions of glycolysis?
1) Formation of glucose 6 phosphate by hexokinase or glucokinase
*Irreversible reaction
2) Formation of fructose 6 phosphate
catalyzed by phosphoglucoisomerase
*Reversible reaction
3) Formation of fructose 1,6 biphosphate
catalyzed by phosphofructokinase-1 (PFK-1)
*Irreversible reaction
main regulatory enzyme of glycolysis
4) Formation of dihydroxyacetone phosphate (DAHP) and glyceraldehyde 3-phosphate (GAP)
by adolase
*Reversible reaction
5) Interconversion of DHAP to GAP by triose phosphate isomerase
6) Oxidation of GAP to 1,3 - biphosphosphoglycerate (1,3- BPG) by GAP dehydrogenase
7) Synthesis of 3- phosphoglycerate by phosphoglycerate kinase
*Reversible reaction
– substrate level phosphorylation - 2 ATP per glucose molecule
8) Formation of 2- phosphoglycerate by phosphoglycerate mutase
9) Formation of phosphoenolpyruvate (PEP) by enolase
10) Formation of pyruvate by pyruvate kinase
*Irreversible, regulatory reaction
what is the fate of pyruvate under aerobic conditions?
Oxidative decarboxylation of pyruvate into acetyl CoA by pyruvate dehydrogenase
- Important reaction in tissues with high oxidative capacity (cardiac muscle and heart)
Pyruvate dehydrogenase irreversibly converts pyruvate into acetyl coA, a fuel for the TCA cycle
What is the fate of pyruvate under anaerobic conditions?
Pyruvate is reduced to lactate by lactate dehydrogenase in order to recycle NADH back to NAD+
Which cells are lactate forming cells?
1) Cells of lens and cornea of the eye, kidney medulla, testes, leukocytes and RBCs because those tissues are all poorly vascularized or lack mitochondria
2) In exercising skeletal muscle:
-NADH production exceeds the oxidative capacity of the respiratory chain.
-This results in an elevated NAD/NAD+ ratio, favoring reduction of pyruvate to lactate.
-Lactate accumulates in the muscle, causing a drop in the intracellular pH, causing cramps.
- Much of this lactate diffuses into the bloodstream and can be used by the liver to make glucose.
what are constitutive enzymes?
Enzymes that are always produced in constant amounts without regard to the physiological demand or the concentration of the substrate
What determines the direction of the lactate dehydrogenase reaction?
Depends on the 1) relative intracellular concentrations of pyruvate and lactate and the 2) ratio of NADH/NAD+ in the cell
What is the energy yield of anaerobic glycolysis?
A net of two ATP molecules per glucose molecule. No net production or consumption of NADH
What is the energy yield of aerobic glycolysis?
Direct net gain of 2 ATP molecules and 2 NADH molecules per glucose molecule.
- Each NADH molecule is oxidized into NAD+ by the ETC in the mitochondria, producing 3 ATP for each NADH molecule.
-Net of 8 ATP molecules for each glucose molecule
what are the 3 regulatory enzymes of glycolysis?
1) Gluco or hexokinase
2) Phosphofructokinase (PFK-1)
3) Pyruvate kinase
They are irreversible enzymes and their activities increase in low cell energy levels (Decreased ATP and increased AMP)
Their activities decrease in high energy levels (increased ATP and decreased AMP)
what are the characteristic features of glycolysis in RBCs?
Synthesis of 2,3-biphosphoglycerate in RBCs
1) Some of the 1,3-BPG is converted into2,3-biphosphoglycerate by the action of biphosphoglycerate mutase.
-The 2,3-BPG is present at high concentrations in RBCs and serves to increase O2 delivery from oxyhemoglobin
2) The 2,3 BPG is hydrolyzed by a phosphate to 3-phosphoglycerate that can be further metabolized by glycolsis
3) Using the 2,3 BPG shunt, the phosphoglycerate kinase reaction is bypassed with subsequent loss of 2 ATP from oxidation of each glucose molecule
what is Pyruvate Kinase deficiency?
Mature RBCs lack mitochondria and are completely dependent on glycolysis for ATP
How does PKD cause hemolytic anemia?
The hemolytic anemia observed in PKD is a consequence of the reduced ATP production by glycolysis.
-ATP is required to fuel the ion pumps necessary for the maintenance of the flexible, biconcave shape of the RBCs that allows them to squeeze through the narrow capillaries.
-The resulting alterations in the RBC membrane lead ultimately to hemolytic anemia.
what are the effects of PK deficiency?
The effects are restricted to RBCs and include mild to severe Non spherocytic hemolytic anemia, with the severe form requiring transfusions.
- Almost all individuals with PKD have a mutant enzyme that shows abnormal properties such as altered kinetics
-Individuals heterozygous for PK deficiency have resistance to the most severe forms of malaria
what determines the severity of PKD?
-Severity depends on the 1) degree of enzyme deficiency and on the 2) extent to which RBCs compensate by synthesizing increased levels of 2,3-BPG, which increase O2 delivery from oxyhemoglobin to the tissues improving anemia.
Define HMP pathway
It is a direct oxidative pathway where glucose is oxidized directly at
C1 & C3 without previous cleavage (into two halves) as in glycolysis.
No ATP consumed or produced
site of HMP pathway in the cell
cytosol
Importance of HMP pathway
1) The HMP provides a major portion of the body’s NADPH, which functions as a biochemical reductant.
2) It also produces ribose 5-phosphate, required for the biosynthesis of nucleotides.
3) It provides a mechanism for the metabolic use of five carbon sugars obtained from the diet or the degradation of structural carbohydrates.
what are the 2 phases of the HMP pathway
Reactions are divided into 2 phases:
Irreversible Oxidative phase
Reversible Non-Oxidative phase
what is the end result of the oxidative phase of the HMP pathway?
It consists of 3 reactions that lead to the formation of:
-Ribulose-5-phosphate
-CO2
-Two molecules of NADPH
for each molecule of glucose-6-phosphate oxidized
what is the first step of the irreversible oxidative reactions of HMP Pathway?
Glucose 6 phosphate is oxidized to 6-phosphogluconolactone by Glucose 6 phosphate dehydrogenase (G^PD).
-This leads to removal of hydrogen and reduction of NADP+ to NADPH.
–This is the rate limiting step of the pathway
what is the first irreversible oxidative reaction of the HMP pathway?
Glucose 6 phosphate is oxidized by G6PD to 6-phosphogluconolactone.
-This leads to the removal of hydrogen and the reduction of NADP+ to NADPH
-This is the rate limiting step
what is the second irreversible oxidative reaction of the HMP pathway?
6-phosphogluconolactone is hydrolyzed by hydrolase to 6-phosphogluconate
what is the third irreversible oxidative reaction of the HMP pathway?
6-phosphogluconate undergoes another step of oxidation and decarboxylation by 6-phosphogluconate dehydrogenase to form ribulose 5-phosphate and CO2. This is accompanied by the production of a second NADPH.
Describe regulation of ppp at the level of the oxidative phase
Key enzyme is G6PD:
-Induced by insulin In well fed
-Allosterically inhibited by
Excess NADPH
Describe regulation of ppp at the level of the non-oxidative phase
Need for NADPH —> oxidative phase is encouraged
- shift towards glycolytic pathway
Need for pentoses—> starting material obtained from glycolytic pathway
- non-oxidative phase is reversed
NADP in Reductive Biosynthesis
• NADPH can be used in reactions requiring an electron donor, such as fatty acid and steroid syntheses.
NADPH in reduction of Hydrogen peroxide
The cell has several protective mechanisms that minimize the toxic potential of these compounds including enzymes and chemicals.
In the red cells, glutathione (GSH) is used to reduce H202 to form water, catalyzed by the enzyme glutathione peroxidase. This reaction needs selenium as a metal cofactor. The oxidized glutathione (GS-SG) produced, is reduced again catalyzed by glutathione reductase enzyme which needs NADPH as a hydrogen donor. The reaction also needs riboflavin as a coenzyme. In the absence of NADPH, H202 will oxidize the lipid moiety of the red cell membrane, leading to lyses of the red cells and hemolytic anemia.
NADPH is also important for keeping the iron of hemoglobin in the ferrous state and preventing methemoglobin (MetHb) formation.
What are ROS
• Reactive oxygen species (ROS) such as hydrogen peroxide are formed from the partial reduction of molecular oxygen.
• ROS are formed continuously as byproducts of aerobic metabolism, through reactions with drugs and environmental toxins, or when the level of antioxidants is diminished, all creating the condition of oxidative stress.
• The highly reactive oxygen intermediates can cause serious chemical damage to DNA, proteins, and unsaturated lipids and can lead to cell death.
-ROS have been implicated in a number of pathologic processes, including reperfusion injury, cancer, inflammatory disease, and aging.
