Haemoglobin – Applied Physiology

Dr Meera Narendran   BHMS,MD(Hom)

Haemoglobin is the red pignent of blood consist of 2 parts.
Globin A specific simple protein constituting 96% of Hb
Haem (Non specific prosthetic group) Iron containing pigment. It is a protoporphyrin compound. consist of 4 pyrrole groups joined together. The porphyrin can combine with metals forming metalloporphynin compound. Haem is a metalloporphynin where the metal is iron. The iron content of the haemglobin is about 0.34% and about 3gram of iron is present as hemoglobin in the total amount of blood of an adult Iron remains in ferrous (fe++) from. Globin helps haem to keep the iron in ferrous state and to combine loosly and reversily with molecule oxygen.

Molecular Weight Molecular weight of hemoglobin is about 68,000 . It contains 4 atoms of iron and 8 atoms of Sulphur. The hemoglobin molecule is probably an aggregate of 4 unitary molecule ,each having one atom of iorn and 2 atom of sulphur and with a molecular weight of 17,000. chemical formulas->C712 H1130 O245 N214 S2 Fe4

1. The most characteristic property of hemoglobin is the ease with which it contains with Oxygen and dissociates from it 100ml of H2O absorb 1/3 of O2 at body temperature,under atmospheric pressure . But 100ml of blood under the same conditions will take up 20ml of Oxygen (60times)due to the presence of Hb. (About 1,200ml of Oxygen can be carried by the total amount of blood of an adult man )[One gm Hb combines of normal temparature Pressure (NTP) with 1.34 ml of Oxygen .] This corresponds to 2 atoms of O2 for each atom of Fe. The compound oxyhemoglobin gives off its full Oxygen contains when placed in vaccum.
2) Oxyhaemoglobin holds its Oxygen loosely which can be easily displaced by many other gases forming more stable compounds (Eg:-Co, No, H2O will form carboxyhemoglobin ,Nitric oxide Hb, Sulphaemoglibin respectively).
3) The globin part of Hb directly combine with Co2 to form carbamino compounds.
4) Crystallisation . Hemoglobin can be easily crystallised . The form of the crystals, their solublity & easy of crystallisation are charateristic of the species from which Hb is obtained . Most bloods including human blood ,form rhombic prism or needles. The distinction of Hb lies globin part of the molecule and not in the heam part . It is Known that the aminoacid compesition of the various globins (derived from Hb of different species) varies considerably in respect to their cystine content .

5) Isoelectric pH of hemoglobin (reduced Hb) is 6.8 ,that of Oxyhaemoglobine is 6.6.
6) Spectroscopic appearance Reduced Hb gives one broad between the Fravuhofer’s lines D and E (corresponding to the wavelength– Lamda 559) That of oxyhemoglobin consist of 2 bands between D & E. The band nearer D is called Alpha band (Lamda -579) The band nearer E is called Beta band (Lamda 542)

In man probably there are at least 2 varieties
—–Foetal hemaglobin (Hbf)
—–Adult heamoglobin (HbA) Foetal heamoglobin differs chemically & Spectroscopically from the adult Hb . This is Due to some difference in the globin fraction . This properties helps to compenasate the relative anoxia of foetal blood. At low O2 Pressure foetal haemoglobin can take up larger volumes of O2 than adult haemoglobin.Foetal haemoglobin is 70 % saturated at 20mm of O2 pressure whereas adult haemoglobin is only 20% saturated at this pressure . A small quantity of foetal Hb persist for some weeks or month after birth.

Normal Values of Hb
In Males — 14.5 -15.5gm%
Females à 13.5-14.5gm%
New born à 2o-23gm%

Methods of estimation of Hb
1) Tallovist;s Method A drop of blood from patients finger is soaked in a piece of filter paper and compared againts a standard colour scale betore the blood on the filter paper dries up. This method doesnot give accurate results.
2) Haldane’s haemoglobinometer (Haldares modification of Gower;s method)
3) Gower;s haemoglobinometer Here the standard used is a solution of pirocarmine gelatin.
4) Sabli’s Method Here Hcl is used ,which convert Hb into acid haematin
5) Von-fleischl’s haemometer Set of coloured glasse
6) Colourimetric with Colouremeter
7) Method of Van-slyke and Stadie
8) Spectomeric method. It is the modern method and is based as the measurememt of abosrphon of lightat at certain wavelength of cyanohaemoglobin formed by treating the Hb with ferricyanide and then with xyanide
Derivative of Haemoglobin

1) Oxyhaemoglobin :—It is a compound of haemoglobin with oxygen . Iron remains in the ferrous (fe++) state in oxyhaemoglobin. It is not a stable compound . oxygen may beremoved when the blood is expesed to a vacuum. It has got 2 absorption bands between D & E.
2) Methaemoglobin :—It is also a compound of Hb with Oxygen . It can be produced after treating the blood with potassium ferricyanide . It is chocolate-brown in colour. it is a stable compound . Oxygen cannot be removed by the exposing the blood to a vacuum . Iron remains in the ferric state . It has got the absorption band between C&P
3) Carbohaemoglobin :–It is a compound of Hb with Co2. The compound is formed by Union of Co2 the globin portion.
4) Carboxhaemoglobin or Corbomonoxy- haemoglobin
Haemoglobin combined with Co insted of Oxygen . It is present in blood in coalgas poisoming. It has got two absorption bands between D&E. The ablity of human Hb at 380C for Co is 210 times greater than O2
5) Sulphaemoglobin It is formed by the combination of Hb with H2S . The compound is very stable and is Sometimes found in the blood after certain kinds of drug perisoning
6) Hb combined with No insted of Oxygen found in nitric oxide peisoning.

Derived Products
A Iron – containing
(i) Haematin The derivatives can exist in 2 forms – acid and alkaline and may be prepared from Hb by the action of acid or alkali . This is sometimes found in urine in old cases of haemarrhege. It is a ferric compound . Acid haematin has got an absorption band between C & D.
(ii) Haemin. Haemin is haematin hydrochloride . It is prepared by boiling oxyhemolobine Nacl and glacial acetic acid . It is a ferric compound.
(iii) Haemochromogen When alkaline haematin is reduced by ammonium sulphide , this derivative is obtaind . It is a ferrous Compound. Haem with ferrous iron is combined with denatured globin of all the haemoglobin derives, haemochromogen possess the most charactarestic specturm. It has a dishinct band between D & E , as wellas fainter band between E & b line . Due to this property this compound is offen used to identify doublful blood stains.
(iv) Cathaemoglobin It is a compound of haem containing ferric iron with denatured globin.
(v) Haem It is a ferrous Compound produced by the reduction of haemabin in alkaline solution.

B) Iron – free
(i) Haematoporphynin This derivative can exist in 2 form -acid &alkaline. It is prepared by mixing blood with sulphoricacid . When mixing with alkali , the alkaline variety is formed. Normal urine contains traces . It is found in the blood and Urine in sulphonic poisoning and in certain case of liver disease
ii) Haemopyrrole. When haematoporphynin is reduced , this coumpound is formed . It is probably a mixture of several pyrrole compounds.
(iii) Haematoidin This compound is produced by the breakedoun of Hb in the body . It is found as yellowish-red crystals in the region of old blood extravasation’
(iv) Bilirubin It is the chief pigment of bile is produced from Hb in the whole of the reticulo-endothelial system. From it are derived all the other bile pigments, the pigment of the stool, stercobilin, the pigment of unine such as urobilinogen,urobilin,urtilin and urochrome

Synthesis of Hb
Hb is synthesised by the cells of erythroid series in the red bone marrow . Hb is first seen at the state of internediate normoblast. four molecule of prophobinlinogen combine and after some forther changes ,eventvally protoprophynin is formed. Proto prophynin now in corporates with iron atom to become haem 4 heam molecules are now joined with one globin molecule to form one molecule of haemoglobin.

Factors required for synthesis of Hb
For the synthesis of globin , aminoacids are required . For the Synthesis of haem, the raw or building materials resired like glycine, sccintl (COA ete) are all avilable from the metabolic intermediates Iron has to be Supplied in the food. Traces of copper and cobalt are necesarry although clinically thier deficiency ading to failure of Hb synthesis is practically unknown. Pyridoxine deficiency also leads to depression of Hb synthesis.

Fate of haemoglobin
When RBC, become old they receptured mostly in spleen (as well as in the liver & bone marrow ) . The Hb is liberated from the ruptured RBC & phagocytozed by the phagocytes of RE system. Within the phagocytes the tetrapyrrole ring is opened up that is heam is converted into a compond where 4 pyrrole ring are lie side by side. The iron is still attached with the tetrapyrrole straight chain compound and probabily the globin also remains attached with it . Susequently both globin and iron are removed . The terapyrrole straight chain compound thus formed (free iron and globin) is called biliverdin. Biliverdin is Oxidized to form bilirubin . All these changes occur within the phagocyte of RE system. Bilirubin now comes out of the phagocyte and in plasma combine with albumin and is transported in the plasma as bilirubin- albumin complx. This complex is frequently called free bilirubin; The free bilirubin ultimately enters the liver and here the albumin is removed from the free bilirubin and the bilirubin is conjugated with glucornicacid a derivative of glucose) to form bilirubin gluccoronides, which is water soluble.

A small amount of bilirobin is conjugated with sulphate radicals to form bilirubin sulphate. The conjugated water soluble bilirabin is called “conjugated bilirubin”. Glucoronyl transferase activity can be increased by the drug phenobarbitone, Hyperbilirubinaemia and kernicterus in the neonates can thus be sucessfully treated by phenobarbitone. All conditions which produce execusion erythrocyte destruction eg: Malaria, mismatched blood transfusion, erythroblastosis faetalis, bites by some types of poisonous snakes thus leads to excessive free bilirubinn formation & jauadice clinically called haemdytic jauxdice, develops. But the urine doesnot contain free bilirutrin in heamolytic jaundice, as the compound cannot pass the renal filter. Instead the urine contains excessive urobilinogen. The normal bilirubin concentration in plasma is below 0.5-1.0mg/100l, which rises greatly in haemolytic jaundice.

Applied physiology
1) Hyperbilirubinaemia
Elevation in serum bilirubin level may result from insreased rate of production or decreased rate of disposal. Depending upon the nature of the bilirubin elevated ,the condition maybe grouped into conjugated or unconjugated hyperbilirubinaemia. Based on the cause it can be divided into congenital and aquired . Congenital (eg:Gilbert’s disease,crigler Najjar syndrome, Dubin -Johnsons syndrome, Rotor syndrome)Aquaried can be conjugated or non-conjugated The cojugated biliurbin is discharged into the biliary canaliculi are get mixed up with bile. This is the main colouring matter of bile . Via the bile , the conjugated biliubin ultimately enters the duodenum. In the intestine , when it comes in contact with the instestinalbacteria bilirubin glucoronide is hydrolysed by bacterial enzymes and non-conjugated called free bilirubin is formed . This free bilirutain reduced to form urobilinigen and stercobilinogens . Part of the urobilinogen and stercobilinogens are absorbed by blood , which then circulate in the blood and is excreated in the urine . Then rest is excreated in the stool. Golden yellow colour of stool is due to this pigments and if stool be kept exposed to the sun and air , the colour becomes blackish because of the oxidation of these pigments. The conjugation of biliubin in liver is catalyzed by the enzyme “glucoronyl transferase” Many drugs especially some steroids (for eg:-Methandrostenolone (Dianabol) a compound with androgenic activity ,used clinically for it’s Protein anabolic effect) compete with bilirubin for conjugating with glocoromides . unless bilirubin conjugate with glucornic acid it cannot be excerated via bile. so excessive use of this drugs often leads to accumulation of free bilirubinin plasma (jaundice )

2) Hemoglobinopathies The hemoglobinopathies are a group of conditions in which the structure /production of haemoglobin has been alttered in some way
Classification of haemoglobionopathies
(a) stractural haemoglobin variants
The sickle cell sundrome
(b) Defective haemoglobin synthesis
(c) persistence of fetal haemglobin
(i) Sickle cell syndrome Sickle -cell haemoglobin (HbS) is the major abnormal haemoglobin in this group . The 6 th amino acid of the beta chain is valine instread of the normal glutamic acid of HbA .The RBC in sickel cell anaemia are sickle shaped , When the Hbs is in the reduced state when the oxygen tension is low Hbs molecules tend to aggregated to form filament s or to tubular structures and preciptate from solution . They have shorter life span . The blood flow in the capillaries may be blocked by the cells causing severe pains and damage to the tissues Erythrocytes of people with sickle cell trait contain both HbA and Hbs . In those suffering from sickle cell disease only Hbs is present .

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