2.Iron Heme Hb

7/31/2019 2.Iron Heme Hb

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2.nd semester

2.nd lecture Biochemistr of IronMetabolism

2012/02/14

Dr Rka Tth Rvszn

Biochemistry and Molecular Biology Department

Lectures for 2nd year Physiotherapyst


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COMPULSORY READING:

Lecture presentations with short explanations are available on the web page ofthe de artment: htt ://bmbi.med.unideb.hu .

Username: student , password: student2011.Downloads/

Educational materials in English/Physiotherapists/

Biochemistry

FURTHER READINGS:

Biochemistry and Molecular Biology Syllabus III. (ed .by Prof Lszl Fss) chapter 5.1. th

. -

1075.p)

Harvey, Ferrier: Biochemistry 6th ed. (Lippincott, 2011) chapter 21. Haem metabolism

Supplementary

Most important obligatory

2


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CONTENTS

I. IRON METABOLISM

1. Introduction

.

3. Transport of iron and Storage of iron

4. Regulation of iron metabolism: hepcidin

II. HEME METABOLISM

1. Biosynthesis of heme, Porphyrias

2. Degradation of heme, Jaundice

III. HEMOGLOBIN, MYOGLOBIN

1. Structure of hemoglobin

2. Polymorphism of globins

. , , , ,

4. Abnormal hemoglobins: Sicle cell anemia, MetHb, HbA1c

3


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IRON METABOLISM


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Iron is an essential metal for humans, involved in

metabolism and transport of oxygene

but free iron is dagerous both iron deficiency anaemia (affects over 30% of the

wor s popu a on an emoc roma os s ron over oa

are dangerous

of iron absorption from the diet

5


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Iron is involved in the metabolism and

ron con a n ng pro e ns:

Hem containing proteins --myoglobin

Electrons transporters- cytochromes in the electron transport chain

2

NADPH oxydaseTryptophan pyrrolase

a a ases egra e 2 2NO synthetase

FeS cluster proteins (electron transport,succinate DH, aconitase) Iron in the catalytic centre various oxidoreductases (RR,

Homogentisate oxidase, Lys, Pro, Phe, Tyr hydroxilase)

, ,

lactoferrin) 6


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free iron generates reactive oxygen species (H2O22OH

-)

forms complexes with anions, which are precipitated

body

7


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Human iron metabolism

Iron metabolism is the set of chemical reactions maintaining human homeostasis of iron. Iron

is an absolute requirement for most forms of life, including humans and most bacterialspecies. Because plants and animals all use iron, iron can be found in a wide variety of food

sources (meat, liver, dried leguminoses, dried fruits, fortified flour, cereals).

,

acceptor. However, iron can also be potentially toxic. Its ability to donate and accept

electrons means that if iron is free within the cell, it can catalyze the conversion of hydrogen

peroxide into free radicals (Fenton reaction). Free radicals can cause damage to a wide

variety of cellular structures, and ultimately kill the cell. In addition, free iron causes

distorsion in the structure of macromolecules and forms complexes with anions, which are

precipitated within the cells. To prevent that kind of damage, all life forms that use iron, bind

. ,

ability to do harm.Iron containing proteins

Most well-nourished people in industrialized countries have 3-4 grams of iron in their bodies.

Of this, about 2.5 g is contained in the hemoglobin needed to carry oxygen through the

blood. Another 400 mg is devoted to cellular proteins that use iron for important cellular

processes like storing oxygen in the muscle (myoglobin), performing energy-producing redox

,

enzymes having Fe in the catalytic centre). 3-4 mg circulates through the plasma, bound to

transferrin. Some iron in the body is stored. Physiologically, most stored iron is bound by

ferritin molecules. The largest amount of ferritin-bound iron is found in cells of the liver

8

hepatocytes, the bone marrow and the spleen. The liver's stores of ferritin are the

primary physiologic source of reserve iron in the body.


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Iron distribution in the human bod

g

hemoglobin 2.5 68

myoglobin 0.15 4transferrin 0.003 0.1

ferritin, tissue 1.0 27

ferritin, serum 0.0001 0.004

enzymes 0.02 0.6

Iron requirement (if the absorption efficiency is ~10%):,

Iron sources: meat, liver, leguminoses, fruits

9


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Overview of iron metabolism

dietary iron gut absorption plasma transferrin transport

receptors on iron-requiring cells

absorption

internalization, acidification

intracellularsynthesis of iron proteinsutilization

mobile iron poolemog o n, myog o n,

cytochromes, etc.)

ferritin

s orage(mainly in liver)

hemosiderinNo physiologic excretion mechanism!But iron is highly recycled!10


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11


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How does the body get its iron?

Most of the iron in the bod is hoarded and rec cled b the

reticuloendothelial system (macrophages) which breaks down aged

red blood cells. However, people lose a small but steady amount bysweating and by shedding cells of the skin and the mucosal lining of

the gastrointestinal tract. The total amount of loss for healthy people

day for men, and 1.52 mg a day for women with regular menstrual

eriods. Peo le in develo in countries with astrointestinal

parasitic infections often lose more. This steady loss means that

people must continue to absorb iron. They do so via a tightlyregulated process that under normal circumstances protects against

iron overload.

12


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Absorbing iron from the diet

Like most mineral nutrients, iron from digested food or supplements is

almost entirely absorbed in the duodenum by enterocytes of theduodenal lining. These cells have special molecules that allow them to

move ron n o e o y. oo ng o oo ac a es e rea own o

ligands attached to iron. To be absorbed, dietary iron must be in its2+ .

of vitamin C. In addition, a ferric reductase enzyme on the

enterocytes' brush border, Dcytb, reduces Fe3+ to Fe2+. A protein

called divalent metal transporter 1 (DMT1), which transports all kindsof divalent metals into the body, then transports the iron across the

en erocy e s ce mem rane an n o e ce .

These intestinal lining cells can then either store the iron as ferritin (in

sloughed off into feces) or the iron can move it into the body, using a

transporter protein called ferroportin. Ferroportin transports Fe2+,

13but tranferrin carries Fe

3+

, so iron has to be oxidized by hephaestin onthe capillary surface of enterocytes for further transport.


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Absorption of iron

Andrews (2005) N. Engl. J. Med., 353, 2508-2509.


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Absorption of iron

Stomach Small intestine

+

low pH,

ferroxidasesCerulo-

plasmin

Fe3+

vitamin Cand/or Steap homolog

ferrireductases?

HCP1

(heme carrierprotein 1) 15


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16


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Structure of the transferrin (Tf)

Tf is an 80 kDa serum glycoprotein synthesized mainly by the liver. Tf is bilobal in

Structure of iron binding site of transferrin

.

high affinity.

Iron is coordinated by Tyr, Asp and His residues. The binding of iron also needs an anion

which is usually carbonate (CO 2-). The charge on the anion is balanced by arginine side

chain. The iron-binding capacity of transferrin is strongly pH-dependent: high-affinity

binding occurs at pH 7.4 (Ka ~ 1023 M1), but no binding occurs below pH 4.5. In a healthyindividuals only ~30% of Tf binding sites are saturated.


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Structure of transferrin receptor 1 (TfR1)

TfR1 is a homodimeric glycoprotein

that consists of two 90 kDa subunits

671 aa

n e y su e on s.

The Tf-TfR1 complex occurs

.

The TfR1-Tf interaction is reversible

S-S

Transmembrane

28 aa

iron content of transferrin.

61 aa

TfR2 Role: sensing iron stores. It isconstantly expressed on some iron sensing

cells, such as hepatocytes and enterocytes

18(no IRE in its mRNA).


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How do cells get their own iron?

Most of the iron in the bod is located on hemo lobin molecules of red blood cells.

When red blood cells reach a certain age, they are degraded and engulfed by

specialized scavenging macrophages. These cells internalize the iron-containing

hemo lobin, de rade it, trans ort iron via ferro ortin molecules into the blood,

which is then transported by the transferrin molecules to the cells expressing

transferrin receptors. Most of the iron used for blood cell production comes from

this cycle of hemoglobin recycling.

All cells use some iron, and must get it from the circulating blood. Since iron is

tightly bound to transferrin, cells throughout the body have receptors for

transferrin-iron complexes on their surfaces and takes them up by receptor

mediated endocytosis. Once inside, the cell transfers the iron to ferritin, the

internal iron storage molecule, and recycles of complex of apotransferrin-TrfR

back to cell surface where release apotransferrin to blood.

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Receptor mediated endocytosis

- -

binding of loaded transferrinapotransferrin release

to its receptor

cell membrane

clathrin-coated pits

internalization into

coated vesicles

2-15 minutes

recycling of complex

of apotransferrin-TfR1

endosomal pH drop:

iron release

intracellular iron pool 20


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21


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Structure of ferritin

24-mer of light chain 24-mer of heavy chain(L-chains catalyse the formation of iron core (H-chains have ferroxidase activity)

err n s a wa er-so u e mo ecu e cons s ng o su un s a orms

a hollow sphere that houses up to 4,500 atoms of iron. Each subunit is

one of two isoforms, the heavy (21 kDa) and light (19 kDa) subunits.

err n a es up an re eases ron rom s nner core roug

hydrophilic channels found in the apoferritin shell. The core containscrystal-like Fe(III)-hydroxide-phosphate.

22


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The ratio of heavy-to-light subunits of ferritin

pIMw (kDa)

4.6

HeLa

H24L0 550

Heart

Kidney

Liver

5.7

H0L24 460

ironiron

Nat. Apoover oaover oa

23


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Hemosiderin

Hemosiderin is a water-insoluble iron-protein aggregates present in

lysosomes and is a by-product of ferritin degradation through incomplete

. ,

marrow. Iron stored in hemosiderin is more inaccessible and less

effective in producing free radicals than iron stored in ferritin. 24


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metabolism


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Iron is such an essential element of human life, in fact, that humans have nophysiologic regulatory mechanism for excreting iron. Human bodies tightly

re ulate iron absor tion and rec clin and revent iron overload solel b

regulating iron absorption.

Those who cannot regulate absorption well enough get disorders of iron overload

haemochromatosis . In these diseases, the toxicit of iron startsoverwhelming the body's ability to bind and store it. Haemochromatosis, is a

hereditary disease characterized by excessive absorption of dietary iron resulting

in a pathological increase in total body iron stores. Excess iron accumulates in

tissues and organs disrupting their normal function. The most susceptible organs

include the liver, adrenal glands, the heart and the pancreas; patients can present

with cirrhosis, adrenal insufficiency, heart failure or diabetes mellitus. Iron

overload may be also the consequence of repeated blood transfusions, or

diseases that affect the gastrointestinal tract such as Crohns or celiac disease.

Since so much iron is required for hemoglobin, iron deficiency anemia is the firstan pr mary c n ca man es a on o ron e c ency. xygen ranspor s so

important to human life that severe anemia harms or kills people by depriving their

organs of enough oxygen. Iron-deficient people will suffer or die from organ

26electron transport.


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Main lo ic of human iron

metabolism regulation

1. humans have no physiologic regulatory mechanism

for excreting iron, but we continually loose iron,

or bleeding (enteral infections)

2. human bodies tightly regulate iron absorption andrecycling

3. human bodies prevent iron overload solely by

.

(genetically or coupled to other diseases such as

develops. 27


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Summary of human iron metabolism

dietary iron gut absorption plasma transferrin transport

receptors on iron-requiring cellsExport of iron via

ferroportin

internalization, acidificationEngulfment of dead

intracellularsynthesis of iron proteinsutilization

by macrophages

mobile iron poolemog o n, myog o n,

cytochromes, etc.)

ferritin

s orage(mainly in liver)

Loss of iron

hemosiderinNo physiologic excretion mechanism!But iron is highly recycled!28


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The body regulates iron levels by

regulating absorption of iron inenteroc tes

Factors affecting iron absorption

.

2. the extent to which the bone marrow is producing

3. the concentration of hemoglobin in the blood

4. the oxygen content of the blood


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Re ulator roteins of iron

metabolism

-HFE

-Hepcidin

30

The liver is the central regulator of iron homeostasis Research over the last decade


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The liver is the central regulator of iron homeostasis. Research over the last decade

has confirmed that the liver is the primary site of expression of many of the molecules

res onsible for the re ulation of iron homeostasis. The hereditar hemochromatosis

(abnormal accumulation of iron) associated molecules HFE (hefaestin), hepcidinexpressed at high levels in the liver. Mouse models of HH, where the genes have been

disrupted or mutated all result in hepatic iron overload. Constitutive over-expression of

hepcidin (negative regulator) in the liver results in iron deficiency anemia. Liver-specific

deletion of HFE in mice recapitulates the phenotype of HH.These studies all suggest a

major role for the liver in iron metabolism.Our bodies' rates of iron absorption appear to respond to a variety of interdependent

factors, including total iron stores, the extent to which the bone marrow is producing new

red blood cells, the concentration of hemoglobin in the blood, and the oxygen content of

the blood. We also absorb less iron during times of inflammation. Recent discoveries

demonstrate that hepcidin regulation of ferroportin is responsible for the syndrome ofanemia of chronic disease.

e o y regu a es ron eve s y regu a ng eac s eps o a sorp on o ron n

enterocytes. This is achieved within the crypt cells, which sense the availability of

iron by taking up iron via both transferrin receptor 1 and 2.The affinity of transferrin-

- , .

with TfR1 in such a way that binding of HFE to the TfR enhances its affinity for iron-

transferrin, resulting in an increase of cellular iron uptake. Depending on the amount of

, , ,

uptake of the iron, will express the appropriate amount of the Dcytb, DMT1 and

ferroportin .31

I i fl i t t t i d t i d


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Iron influx into enterocyte is determined

-Stomach

Fe2+

Small intestine

villus

cell

(enterocyte) Fe3+

low pH,

vitamin Cand/or Steap homolog

ferrireductases?

ferroxidasesCerulo-

plasmin

,

responsible for the uptake

of the iron from gut, will

express the appropriate

(heme carrier

protein 1)

Stomach Small intestine

,

ferroportin proteins.

crypt

cell

Fe2+

low pH,vitamin C

ferroxidasesCerulo-

plasmin

cell)Fe3+

an or eap omo og

ferrireductases?

HCP1(heme carrier

protein 1)

32

cryp ce s, sense e ava a y o ron

by taking up iron via transferrin receptor 1,

2 helped by the HFE protein.


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Iron influx into enteroc te is determined

by the set-point of precursor cells

In response to iron deficiency anaemia:

villus cells produce more Dcytb, DMT1 and ferroportin.

Villus cell produce less Dcytb, DMT1 and ferroportin.

33


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Hepcidin, a circulating peptide hormone, is the master regulator

of s stemic iron homeostasis, coordinatin the use and stora e

of iron with iron acquisition. This hormone is primarily produced

by hepatocytes in response to iron overload or inflammation. Its a negat ve regu ator o ron entry nto p asma. epc n

functions to reduce serum iron levels by reducing intestinal iron

types and achieves this by binding to the iron exporterferroportin on the surface of cells and inducing its

internalisation and degradation. Ferroportin is distributed

throughout the body on all cells which store iron. Thus,

regu a on o erropor n s e o y s ma n way o regu a ng e

amount of iron in circulation.

34

Regulatory pathways of hepcidin


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Regulatory pathways of hepcidin

Chua et al. (2007) Crit. Rev. Clin. Lab. Sci., 44, 413-459.

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Hepcidin has antimicrobial properties

high iron levelin patients having hemochromatosismakes them more susceptible for microbial infection

inflammation

n ec onMacrophage Hepatocyte Hepcidin

-

Iron release from enterocytes

and macro ha eslow iron level

36


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Iron and bacterial protection

A proper iron metabolism protects against bacterial infection. If bacteria are to

survive, then they must get iron from the environment. Disease-causing bacteriado this in many ways, including releasing iron-binding molecules called

siderophores and then reabsorbing them to recover iron, or scavenging iron from

hemoglobin and transferrin. The harder they have to work to get iron, the greater

a metabolic price they must pay. That means that iron-deprived bacteria

reproduce more slowly. So our control of iron levels appears to be an importante ense aga ns ac er a n ec on. eop e w ncrease amoun s o ron, e

people with hemochromatosis are more susceptible to bacterial infection.

To obtain a more perfect protection during bacterial infection, cytokines (such as

- re ease rom e n amma on s es, w n uce e re ease o epc n.

(Hepcidin alone is antifungal, and was discovered in urine during a screen forantimicrobial peptides.) Hepcidin functions to reduce serum iron levels, thus

.

this mechanism is an elegant response to short-term bacterial infection, it can

cause problems when inflammation goes on for longer. Since the liver produces

,the result of non-bacterial sources of inflammation, like viral infection, cancer,

auto-immune diseases or other chronic diseases. When this occurs, the

37

chronic disease, in which not enough iron is available to produce enough

hemoglobin-containing red blood cells.

Haemochromatosis: disorders of iron overload


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Haemochromatosis: disorders of iron overload

Hemosiderin is deposited allover the body. Haemochromatosis a hereditary

disease characterized by excessive absorption of dietary iron resulting in a

atholo ical increase in total bod iron stores. See models . Excess ironaccumulates in tissues and organs disrupting their normal function. The most

susceptible organs include the liver, adrenal glands, the heart and the

pancreas; patients can present with cirrhosis, adrenal insufficiency, heart failure or

diabetes mellitus. (Iron overload may be also the consequence of repeated blood

transfusions, or diseases that affect the gastrointestinal tract such as Crohns orceliac disease.) 38

Crypt-programming model of hemochromatosis


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Crypt-programming model of hemochromatosis

39

Li h idi d l f h h t i


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Liver hepcidin model of hemochromatosis

40


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Regulation of ironme a o sm a e eve o

What is regulated?


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What is regulated?

Ferritin concentration Number of tansferrin rece tor

depends on iron amount inside of the cell

.

not necessary to uptake more iron, so less transferrin

receptor is required to expressed on the cell surface,

but more ferritine is required to store excess iron

2, When iron level is low inside the cell:

No need to express storage protein (ferritine), but more

42

paralely an a recyprocal way by the same regulatoryprotein!

In human cells the best characterized iron sensing mechanism is the result of translational


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In human cells, the best characterized iron-sensing mechanism is the result of translational

regulation of mRNA of proteins involved in iron metabolism: transferrin receptors, and for

ferritin.

When iron level is low inside the cell an iron sensing protein (IRE-BP, a FeS cluster

protein) binds to special mRNA sequences of ferritin and transferrin receptor mRNA and

receptor synthesis (by stabilising its mRNA).

When iron level is high iron binds to the iron sensing protein (IRE-BP) the protein changes

shape with the result that the it can no longer bind the ferritin and transferrin receptormRNA, as a consequence the result is just the oposite seen above, so transferrin is readily

translated , but no transferrin made. (Interestingly, in iron-bound state the IRE-BP functions

as a cytosolic aconitase.)

- -, .

more transferrin receptors make it easier for the cell to bring in more iron from transferrin-

iron complexes circulating outside the cell. But as iron binds to more and more IRE-BPs,

they change shape and unbind the transferrin receptor mRNA. The transferrin receptor

mRN is rapidly degraded without the IRE-BP attached to it. The cell stops producing

transferrin receptors. When the cell has obtained more iron than it can bind up with ferritin or

heme molecules, more and more iron will bind to the IRE-BPs. This will initiate ferritin

.

(Detailed mechanism: the special mRNA sequences (called iron response elements=IRE)

located at different ends of the two mRNAs. If it is located at the 5 end, binding of IRE-BP

43

n ts trans at on o t e m . t s ocate at t e en , t protects m rom

degradation leading to more protein synthesis. )


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-

IRE

44

SUMMARY


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SUMMARY

+ e

-Fe

45

Utilization of iron


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Utilization of iron

-

hemoglobin, myoglobin, cytochromes, oxidases, peroxidases

- ron-su ur c us er pro e ns:

ferredoxin, succinate dehydrogenase, aconitase, etc.

- Other iron containing proteins:

amino acid hydroxylases (Phe, Tyr, Pro, Lys), acid phosphatase,

homo entisinate diox enase, ribonucleotide reductase

46


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HEME / HAEM METABOLISM

Structure of heme


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Structure of heme

COOHCOOH

NN

Fe

Fe-protoporphyrin IX

Porphyrins are cyclic compounds that readily bind metal ionsusually Fe2+or Fe3+,

and formed by the linkage offour pyrrole rings through methenyl bridges.

The most prevalent metalloporphyrin in humans is heme, which consists of one

48

errous e ron on coor na e n e cen er o e e rapyrro e r ng o pro o

porphyrin IX.

Heme is the prosthetic group for hemoglobin, myoglobin, the cytochromes, catalase,

nitric oxide s nthase and eroxidase.


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49

Tetrapyrrole biosynthetic pathways


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py y p y

(5-aminolevulinate)(in most bacteria and plants)

(in most eukaryotes)

corin ringporphyrin ring

50

Porphyrins are cyclic compounds that readily bind metal ionsusually Fe2+or Fe3+.

The most prevalent metalloporphyrin in humans is heme which consists of one ferrous (Fe2+) iron


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The most prevalent metalloporphyrin in humans is heme, which consists of one ferrous (Fe2+) iron

.

Heme is the prosthetic group for hemoglobin, myoglobin, the cytochromes, catalase, nitric oxidesynthase, and peroxidase. These heme proteins are rapidly synthesized and degraded. (For

example, 67 g of hemoglobin are synthesized each day to replace heme lost through the normal

urnover o ery rocy es. oor na e w e urnover o eme-pro e ns s e s mu aneous

synthesis and degradation of the associated porphyrins, and recycling of the bound iron ions.

Structure of porphyrins

Porphyrins are cyclic molecules formed by the linkage of four pyrrolerings through methenyl bridges(F

Slide : ). Three structural features of these molecules are relevant to understanding their medical

significance.

1. Side chains: Different or h rins var in the nature of the side chains that are attached to each

of the four pyrrole rings:

Uroporphyrin contains acetate (CH2COO) and propionate(CH2CH2COO) side chains,Coproporphyrin contains methyl(CH3) and propionate groups,

= , , .

The methyl and vinyl groups are produced by decarboxylation of acetate and propionate side

chains, respectively.

2. Distribution of side chains: The side chains of porphyrins can be ordered around the

e rapyrro e nuc eus n our eren ways, es gna e y oman numera s o . n y ypeporphyrins, which contain an asymmetric substitution on ring D (see Figure21.2), are

physiologically important in humans. (Protoporphyrin IX is a member of the Type III series.)

3. Porphyrinogens: These porphyrin precursors (for example, uro-porphyrinogen) exist in a

51

chemically reduced, colorless form, and serve as intermediates between porphobilinogen and

the oxidized, colored protoporphyrins in heme biosynthesis

Overview of heme synthesis


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y

Gly + Suc-CoA

HEME

8

mitochondrionmitochondrion

Fe2+

pyridoxal

phosphate

protoporphyrin IX1

7

-aminolevulinic acid (ALA) protoporphyrinogen IX

6

Porphobilinogen (PBG)

uroporphyrinogen IIIcoproporphyrinogen III3 4

5

-

cytoplasmcytoplasm

The organs mainly involved in heme synthesis are the liver and the bone marrow.52

Biosynthesis of heme (1)


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The major sites of heme biosynthesis are the liver, which synthesizes a number of heme

proteins (particularly cytochrome P450 proteins), and the erythrocyte-producing cells of the bone

marrow, which are active in hemoglobin synthesis. (Over 85% of all heme synthesis occurs in

erythroid tissue.) In the liver, the rate of heme synthesis is highly variable, responding to

.

contrast, heme synthesis in erythroid cells is relatively constant, and is matched to the rate of

globin synthesis.

The initial reaction and the last three steps in the formation of porphyrins occur in mitochondria,whereas the intermediate steps of the biosynthetic pathway occur in the cytosol. (Slide. ).

(Mature red blood cells lack mitochondria and are unable to synthesize heme.)

.

porphyrin molecule are provided by glycine (a nonessential amino acid) and succinyl

coenzyme A (an inter-mediate in the citric acid cycle) that condense to form ALA in a

reaction catalyzed by ALA synthase (ALAS) .This reaction requires pyridoxal phosphate

(PLP) as a coenzyme, and is the committed and rate-limiting step in porphyrin biosynthesis.

(There are two isoforms of ALAS, 1 and 2, each controlled by different mechanisms.

Erythroid tissue produces only ALAS2,the gene for which is located on the X-chromosome.

- .2. Formation of porphobilinogen: The condensation of two molecules of ALA to form

porphobilinogen by Zn-containing ALA dehydratase (porphobilinogen synthase) This enzyme

is extremely sensitive to inhibition by heavy metal ions, for example, lead that replace the

53

zinc. This inhibition is, in part, responsible for the elevation in ALA and the anemia seen in

lead poisoning.


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Reaction catalyzed by ALA synthase is the rate-limiting reaction

, .

Aminomethyl -bilane

e y ra ase

55Side chains: A: acetyl; P: prppionyl; M. methyl; V: vinyl.

Conversion of Uroporphyrins to Coproporphyrins


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-

| CH3

2

CH2 |

Acetyl- Methyl-

(A)4x

56

Conversion of Coproporphyrins to Protoporphyrins


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COO-

CH2

2

CH2

| |

| |

Propionyl-

(P)Vinyl-

(V)

2x

57

Steps of Heme Synthesis (7)


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ro oporp yr nogen ox ase conver s e me y ene r ges e ween

the pyrrole rings to methenyl bridges.58


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Names of Porphyrins


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, . ., .

substituents found on the ring, and the number denotes how they are arranged.

WORDS: uroporphyrin, coproporphyrin, protoporphyrin

AP MP MPV

, , ,

In series I the substituents repeat in a regular manner: AP AP AP AP.

Series II does not occur in natural systems.

In series III the order of substituents in ring IV is reversed: AP AP AP PA.

Series IV does not occur in natural systems.

Porphyrin vs Porphyrinogen

.

The porphyrins contain a system of conjugated double bonds all around the tetrapyrrole ring,

which makes the porphyrins more stable than the corresponding porphyrinogens. 60

Regulation of Heme Synthesis


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synthesis of new enzyme cytoplasmcytoplasm

-ALA synthase

Gly + Suc-CoA

HEME

8

m oc on r onm oc on r on

Fe2+

-

pyridoxal

phosphateALA

synthase

1

7

-am no evu n c ac

2

6

porphobilinogen

uroporphyrinogen IIIcoproporphyrinogen III

aminomethyl-bilane

3 4

5

61

Regulation of Heme and Globin Synthesis


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- Substrate availability: iron (II) must be available for ferrochelatase.

- Feedback regulation: heme is a feedback inhibitor of ALA synthase.

- Subcellular localization: ALA synthase is in the mitochondria,

w ere e su s ra e, succ ny o , s pro uce .

ALA synthase is synthesized in the cytoplasm,

its transport across the mitochondrial membrane may be regulated.

- In erythropoietic cells, heme synthesis is coordinated with globin synthesis.If heme is available, globin synthesis proceeds. If heme is absent:

- Effects of drugs:

barbiturates and certain steroids can increase heme synthesis

- , .

62


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Defects in heme s nthesis

The porphyrias are classified depending on whether the enzyme

deficienc occursIn the erythropoietic cells of the bone marrow: ErythropoieticIn the liver: Hepatic

Either type may be hereditary or acquired.

The symptoms are caused by accumulation of intermediates

and deficiency of heme.

Accumulated intermediares are converted by nonenzymatic (light,

ox a ve e ec s s eps rom porp yr nogens o unuse u porp yr nswhich makes photosensitivity.

orp yr a re ers o e purp e co or cause y p gmen - e por-

phyrins in the urine.63

Defects in heme synthesis


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Pb poisoning3 1

4

25

Pb

6

Porpyrias of erithropoietic origin1: erithropoietic porphyria

2: hereditary protoporphyria

Porphyrias of liver origin

3: acute intermittent porphyria

4: porphyria cutanea tarda

5: hereditary coproporphyria6: variegate porphyria 64

Porphyrias


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1. individuals with an enzyme defect

prior to the synthesis of the

tetrapyrroles manifest abdominal

- ,

2. enzyme defects leading to the

accumulation of tetrapyrrole

intermediates show

,itches and burns (pruritus) when

exposed to visible light.

(Photosenstivity is a result of the

porphyrinogens to colored

porphyrins, which arephotosensitizing molecules that are

formation of superoxide radicals

from oxygen. These reactive oxygen

species can oxidatively damage

,of destructive enzymes from

lysosomes.)

65red urine, injured skin

Acute intermittent porphyria


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Gl cine + Succin l-CoA ALA PBG // ... Heme Hemo roteins

PBG

deaminase

ALAsynthase

ALA PBG Heme

no feedback inhibition!

//

.

activity is sufficient to produce heme for erythropoiesis. In the liver, however, if heme is

utilised or degraded by an elevated rate (e.g. certain drugs, hormones or ethanol are

metabolised b c tochrome P450 containin enz mes, the induce the level of this heme

containing enzyme) the decrease in the levels of heme induces ALA synthase. Under

these conditions the elevated levels of PBG cannot be further converted by PBG

deaminase. Both PBG (red urine) and ALA (neurotoxicity) are accumulated. Symtomps

abdomen syndrom, neurological abnormalities. Can be treated by infusion of high

concentration of heme. Barbiturates must be avoided beacuse they increase the level of

ALA synthase.

66

Summary of heme synthesis


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- It occurs in virtually all tissues but the highest rate is found in the

ver an one marrow.

- The first and the last three enzymes are located in the mitochondria.The middle 4 enzymes are located in the cytosol.

- Heme is s nthetized from 8 l cine and 8 succin l-CoA molecules.

- During synthesis the side chain modifications occur on the colorless.

- The last step oxidizes it to porphyrin (methylen to methene bridges)

.

- Porphyrins are produced by nonenzymatic (light, oxidative effects)

s eps rom porp yr nogens n porp yr as.

67


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68

Degradation of heme


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69

A HEME

Recycled!


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degradation Hemeoxigenaseslpeen, macrophages

Biliverdin

Biliverdin reductase

UDP

glkuronil

transzferz

BLOOD

LIVER

(albumin)Bilirubin

BILE Bilirubin

Bacterial flora

dconjugation, redcution

Saturation of methenyl

, INTESTINE

KIDNEY

70

feces urineStercobilin Urobilin

BilirubinThe high lipid solublity of bilirubin dictates


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The high lipid solublity of bilirubin dictates

- that it must be transported in the blood by a carrier(serum albumin)- that it is soluble in the lipid bilayers of cell membranes

- that it must be conjugated to a water-soluble substance for excretion

Bilirubin diglucuronide is excreted in the bile. It is subject to subsequent

rans orma ons o o er spec es y e n es na ora.

The clinical determination of plasma bilirubin distinguishes between conjugated

.

- Direct and indirect bilirubin values are used in the differential diagnosis

ofhyperbilirubinemia.

71

Jaundice (also called icterus) refers to the yellow color of skin, nailbeds, and sclerae (whites of

the eyes) caused by deposition of bilirubin, secondary to increased bilirubin levels in the blood

h erbili rubinemia . Althou h not a disease, aundice is usuall a s m tom of an underl in

disorder


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disorder.

Types of jaundice: Jaundice can be classified into three major forms described below. However, inclinical practice, jaundice is often more complex than indicated in this simple classification. For

,

metabolism. a. Hemolytic jaundice: The liver has the capacity to conjugate and excrete over

3,000 mg of bilirubin per day, whereas the normal production of bilirubin is only 300 mg/day. This

excess capacity allows the liver to respond to increased heme degradation with a correspondingincrease in conjugation and secretion of bilirubin-diglucuronide. However, massive lysis of red blood

cells (for example, in patients with sickle cell anemia, pyruvatekinase or glucose 6-phosphate

dehydrogenase deficiency) may produce bilirubin faster than it can be conjugated. Unconjugated

,

excreted into the bile, the amount of urobilinogen entering the enterohepatic circulation is

increased, and urinary urobilinogen is increased.] b. Hepatocellular jaundice: Damage to livercells (for example,in patients with cirrhosis or hepatitis) can cause unconjugated bilirubin levels in

the blood to increase as a result of decreased conjugation. Urobilinogen is increased in the urine

because hepatic damage decreases the enterohepatic circulation of this compound, allowing more

to enter the blood, from which it is filtered into the urine. The urine thus darkens, whereas stools

, . .If conjugated bilirubin is not efficiently secreted from the liver into bile (intra-hepatic cholestasis), it

can diffuse (leak) into the blood, causing a conjugated hyperbilirubinemia.] The similar thing

hapens in case of neonatal jaundice.

c. Obstructive jaundice: In this instance, jaundice is not caused by overproduction of bilirubin or

decreased conjugation, but instead results from obstruction of the bile duct (extrahepatic

cholestasis). 72


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Normal Hemol tic aundice

73


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Normal Physiological (neonatal) jaundice

In neonates, benign jaundice tends to develop because of two factors:

- the breakdown of fetal hemoglobin as it is replaced with adult hemoglobin

- immature hepatic metabolic pathways which are unable to conjugate and so excrete bilirubin as quickly as an adult.

Infants with neonatal jaundice are treated with colored light called phototherapy.

Phototherapy works through a process ofisomerization that changes the bilirubin into water-soluble isomers

that can be passed without getting stuck in the liver.Wikipedia

74


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Normal Biliary obstruction

75


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76

Functions of HemoglobinLung Circulation Tissues


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Lung Circulation Tissues

Hb.4O2O2O2inhaled

respiratory

chain

TCAexhaled

cycle

2 2

carbonic anhydrase

2 + 2

carbonic anhydrase

+

2 3H2CO3

.

Hb.carbamateH+ + HCO3

-H+ + HCO3

-

77

Quaternary structure of hemoglobin


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Hemoglobin

Quaternary structure: 4 subunits!

Four heme, four Fe2+, four O2

The 4 monomer are kept together by

secondar bonds:

Salt bridges

Hydrogen bonds

78


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HgA1: 22

79

Structure of one subunit of hemoglobin


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Tertiary structure of globin chain

-helix A: Ser3- Gly18

helix B: His20-Ser35

helix C: Phe36-Tyr42

helix D: His50-Gly51

helix E: Ser52-Ala71

helix F: Leu80-Ala88

-

helix H: Thr118-Ser138

(Name of the loop between two helices is

composed from names of the two helices: eg.

AB, CD)

The hem group is found in the apolar

polar groups facing the surface.80

Structure of heme


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COOHCOOH

N

NN

N

Fe

Heme is the prosthetic group for hemoglobin (myoglobin)

Heme consists of one ferrous (Fe2+) iron ion coordinated in the center of he

tetrapyrrole ring of proto porphyrin IX.

Fe-protoporphyrin IX81

Tertiary structure of hemoglobin


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Haemoglobin is a globular verytightly stuffed compaque

molecule. H dro hobe inside

proximal His

hydriphyl side chains outside.

Heme is inside of the hydrophobe

ocket. Isolated fee heme unableto keep Fe in 2+ state, only

pocked inside the globin chain.

If Fe is oxidized to Fe3+ ferri

(methemoglobin) it cannot bind

O2. Iron has 6 coordinative

(covalent ) bindings:

- .

5.: His-F8 of globin (proximal His)

This makes the bond between

oxygen

bindin site

6.: O2

82

His-E7 helps to bound O2(distal His)

(no O2 is show here)

Polimorfism of globinsI. II. III.


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I.Embrionyc haemoglobins

2 2

Hb Gower 2 22

or an 22

II.Foetal Haemoglobins

Hb F 2 2

III.Adult haemoglobins

HbA1 (98%)

HbA2 2% 22

83

Expression of hemoglobin genes

during development is related tothe changing oxygen uptake

Comparison of Mb and Hb


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Myoglobin

One ol e tide chain

Hemoglobin

Four ol e tide chains

One heme, one Fe, one O2 Tertiary structure only

Four heme, four Fe, four O2 Tertiary and quaternary structure

O2 storage in muscle Regulated affinity to O2 binding

O2 transport in RBC84

Ox en-bindin to Hb


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100Oxygen-binding curve for Hb

- sigmoidal / cooperative

- low affinity in the veins

- hi h affinit in the arteries80

ion

- p50 25 mmHg

40s

atura

20% venous

pressure

arterial

pressure

00 20 40 60 80 100

pO2

(mmHg)

85

O -bindin to Mb


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- hyperbolic / non-cooperative

- high affinity for O2,

higher than that of Hb

80

ion

- p50 1 mmHgHbMb

40s

atura

20%

00 20 40 60 80 100

pO2

(mmHg)

86

O2-binding causes conformational changes


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87

The uaternar structure chan es

I d Hb i i t f th l f th


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In deoxyHb iron is out of the plane of theheme. Followin oxi en bindin iron moves

into the plane. The movement of iron is

followed by the movement of the protein

.

Upon oxygen binding:

1

1

twists relative to 2

2 heme-heme distance reduces

centra cav ty constr cts

In short, the deoxy state relaxes andswitches to the oxy state. These changes

transmits the structural chan es to the other

heme groups and INCREASES their O2

binding. This is cooperativity.88

v y


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60

80

turation

0

20%s

pO2

(mmHg)

In other words the oxygen binding to the next subunits requires less energy

89

because part of the salt bridges are already broken. That is why the affinity

becomes larger. This explains the sigmoid saturation curve.

u y


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O2 affinity is decreased by:

1. 2,3BPG (2,3-bisphospho-glycerate)

-produced by the shunt of the glycolysis in RBC

-releases O2

2. Low pH- Bohr effect,-metabolicall active tissues CO and H+

-releases O2

. -metabolycally active warm tissues

90

. m noac sequence

-Foetal Hb binds O2 with more affinity than adult Hb

Control: 1. 2,3-bisphospho-glycerate


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2,3 BPG binds to the positively charged

beta chains.

-helps the relase of oxygen at tissues

-reduces O2 affinity

At high altitude the level of BPG increases

facilitating the release of oxygen at tissues.

91

At low external oxygen more 2,3 BPG binds to the

increased amount of deoxiHb, 2,3BPG will not

inhibit its own production (BPG mutase). IncreasedBPG synthesis.

Glucose S nthesis of 2 3-BPG

Glucose 6 P


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Glucose-6-P

Biphosphoglycerate mutase

, -

2,3-biphosphoglycerate

-

ADP

ATP

-

P ruvate

,

At hi h altitude the level of BPG increases facilitatin the

Lactate

release of oxygen at tissues. At lowexternal oxygen more 2,3

BPG binds to the increased amount of deoxiHgb, 2,3BPG will

not inhibit BPG mutase. Increased BPG synthesis.

92

Control: 2. The Bohr effect

Metabolically active tissues are rich on CO and H+


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Metabolically active tissues are rich on CO2 and H+.

+ 2 2 .

Why?

NH3+

R CO 2

N

R

CO 2-

N-terminus of

+ +-

O

His 146 of Asp 94 of

These additional char es form additional salt brid es to further

cross-link the Hb quaternary structure and stabilize the tense deoxy

state. Hence, they lead to the release of O2.

93

Control: 2. The Bohr effect


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low level of CO2

high level of CO2

o ncrease an 2 concen ra ons

decrease the affinity of Hb for O2 94

Control: 3. Temperature


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Hb is a temperature controller. O2 binding to Hb is (usually) exothermic; oxygen release from Hb isendotermic, that is, heat is given out. This also means that when oxyHb arrives at muscle, heat is

required to liberate O2. Whilst this isnt generally a problem to humans, it is for animals from colder

.

needed to free oxygen. At the other extreme, in the heavily worked flight muscles of some birds,

efficient heat loss is essential to avoid overheating. Here O2

release requires 3 times as much heat as it

does in man.

Control: 4. Amino acid se uence


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HbA: 22

HbF: 22

One of the changes in the chain vs

e c a n s s er, w c es n

the central cavity.

deoxyHbF for BPG relative to

deoxyHbA

This increases the affinity of HbFfor O2.

96

Abnormal hemoglobinsPoint mutations in the core region


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Hemoglobin M

Change E7 or F8 His to Tyr,

therefore Fe2+ oxydizes to Fe3+,

therefore it cannot bind O2.97

Abnormal hemoglobinsMutations at subunit interfaces


98/103

Sickle cell anemia

Hemoglobin S: Glu6Val in chains

Wikipedia

98

Abnormal hemoglobinsMutations at subunit interfaces


99/103

mutation

Altered surface of deoxyHbS causes polymerization

Wikipedia

99

Abnormal hemoglobins


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Thalassemias

.the severity of the disease might vary.

Glucose is spontaneously covalently bound to Hb.

% of Hb glucosylated depends on blood sugar levels.

Significance in the early diagnosis of diabetes mellitus.

100

CONTENTS

I. IRON METABOLISM

1. Introduction


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.

3. Transport of iron and Storage of iron

4. Regulation of iron metabolism: hepcidin

II. HEME METABOLISM

1. Biosynthesis of heme, Porphyrias

2. Degradation of heme, Jaundice

III. HEMOGLOBIN, MYOGLOBIN

1. Structure of hemoglobin

2. Polymorphism of globins

. , , , ,

4. Abnormal hemoglobins: Sicle cell anemia, MetHb, HbA1c

101

Exam essa uestions


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. , ,

storage of iron. Regulation of iron uptake at body and cellular level.

2. Heme synthesis, Porpyrias.

3. Heme breakdown. Jaundice.

4. Hemoglobin: structure and function, Regulation of O2 binding .Globin

polymorphysm and abnormalities.

102

Example for Simple questions

Give a short definition to

1. Ferritin

2. Transferrin3. Transferrin receptor

1. List Heme containing proteins of human body!

2. What is the mechanism of iron uptake to

ll ?


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3. Transferrin receptorcells?

4. Hepcidin

5. Ferroportin

6. DMT1

3. What is effects of hepcidine?

4. List 3-5 intermediates of heme synthesis!

-7. Hemocromatosis

8. Porpyrins

9. Heme

.

6. Classification of porphyrias?

7. Types of jaundice and short explanation to

them!10. ALA synthase

11. Ferrochelatase12. Porhyrias

8. List the factors which affect O2 binding of Hg!

9. What is the composition of adult and foetalHg?

13. Hemoxigenase

14. UDP glucuronyl transferase

15. Bilirubin

16. Hemoglobin

17. Myoglobin

18. Coo erativit

10319. Bohr effect

20. Sicle cell anemia

21. H A1c

Documents

2.Iron Heme Hb