M.Prasad NaiduMSc Medical Biochemistry,

Ph.D.Research Scholar

Hemoglobin is the red blood pigment exclusively found in erythrocytes.

Normal range:Concentration of Hb in blood in

Males is 14 – 16 g/dlFemales is 13 – 15 g/dl

Structure:Tetrameric allosteric protein

Mol wt 64,450 Conjugated protein globin +heme Globin is the apoprotein part Heme is the non-protein part (Prosthetic part) Chromo protein Example of quaternary structure of protein Globular protein Consist of 4 poly peptide chain 2 of one type

and 2 of another type ( α2 and β2) held together by noncovalent bonds.

αhelical conformation constitutes 75% globin molecules.

4 of helices are terminated by proline residues. Each polypeptide chain contains heme group

and oxygen binding cite. Heme consist of protoporphyrin ring and Fe

atom Fe atom in heme can forms 6 bonds. 4with N of protoporphyrin 5th one imidazole N of proximal histidine. In OxyHb , 6th binds to O2

Function:1.Delivery of O2 from the lungs to the tissues

2. Transport of CO2 and protons (H+) from tissue to

lungs for expiration.

Effect of 2,3,BPG on O2affinity of Hb

levels are higher in younger children compared to elders.2,3 BPG is produced in the erythrocytes from an intermediate (1,3 Bisphosphoglycerate) of glycolysis.

This short pathway is refered as Rapaport Leubering cycle.

Significance of Rapaport leubering cycle

It prevents accumulation of ATP not needed by the erythrocyteIt supplies 2,3 BPG in oxygen transport which is required for the Hb function.

2,3 BPG regulates the binding and release of Oxygen from hemoglobin.

2,3 BPG accounts for about 16% of the non carbonate buffer value of erythrocyte fluid.

HEMEIron containing compound belonging to the class of protoporphyrin compounds

Protoporphyrin is composed of 4 pyrrole rings which are linked by methene (=CH) bridges to from tetrapyrrole (porphyrin)

4 methyl , two vinyl and two propionate side chain gps are attached to the porphyrin rings.

These can be arranged in 15 different ways One of these isomers called protoporphyrin IX is biologically active.

Iron (Fe2+) held in the center of the protoporphyrin molecule by co-ordination bonds with the four nitrogen of the protoporphyrin ring.

Iron has 6 valency 4 bonds are formed b/n iron and nitrogen

atoms of the porphyrin ring system 5th bond is formed between nitrogen atom of

histidine residue of the globin polypeptide chain, known as proximal histidine.

6th bond is formed with oxygen

Heme Synthesis

Takes place in all cells (occurs to the greatest

extent in the bone marrow and liver)

Occurs both in mitochondria and cytosol.

Stages of Heme Synthesis

1.Biosynthesis of - aminolevulinic acid (ALA)

from the precursor glycine and succinyl CoA

2.Formation of porphobilinogen (PBG) from δ

amino levulinic and

3. conversion of the porphobilinogen to the

cyclic tetrapyrrol porphyrin ring and heme.

Biosynthesis of δ ALA

1st step is biosynthesis of Heme

Condensation step (succinyl CoA + glycine)

Rate controlling step in heme synthesis

Occurs in mitochondria

Formation of PBG

Occurs in cytosol

2 mol of δ ALA condense to form one mol of

porphobilinogen ad 2H2O.

Enzyme is ALA dehydratase , Zn containing enzyme.

Enzyme is inhibited by lead and ALA is excreted in urine

(lead poisoning).

Conversion of porphobilirogen to porphyrins and heme 4 BPG condense head to tail to form a linear tetrapyrrole. Enzyme is PBG deaminase.PBG cylized spontaneouusly to form UPGI and UPG III , Enzyme is UPG III cosynthase

UPG III is converted to CPG III by decarboxylation of all acetate(A) side chain to methyl (M), enzyme is UPG decarboxylase.

CPG III enter mitochondria , it is converted to PPG III by CPG oxidase enzyme.

This enzyme catalyzed the conversion of two of the proprionate side chains into vinyl groups by oxidative decarboxylation.

This enzyme act only on type III coproporphyrinogen

Oxidation of protoporphyrinogen III to protoporphyrin is catalyzed by another mitochondrial enzyme, protoporphyrinogen oxidase.

Final step involves the incoporation ferrous iron into protoporphyrin in a reaction catalyzed by mitochondrial heme synthase or ferrochelatase

Regulations of Heme Synthesis

1.ALA synthase allosteric enzyme that catalyzed controlling step, feed back inhibition by Heme.

2.Iron atom itself may be the active regulatory species

3.Several substances like steroid hormone metabolites ethanol, Barbiturate etc induce the synthesis of hepatic ALA

Disorders of Heme biosynthesisPorphyrias

Breakdown of HemoglobinRBC life span is 120 days , after this they are degraded by RE system

Fate of Bilirubin

Fate of BilirlubinMetabolism & excretion of bilirubin occurs in

liver & intestine Uptake by liver parenchymal cells Conjugation in smooth endoplasmic reticulum

in liver Secretion of conjugated bilirubin Excretion of bilirubin in the form of stercobilin

and urobilinogen through feces and urine respectively

Normal concentration of serum bilirubin Total bilirubin 0.1 - 1.0 mg/dl Conjugated (direct) bilirubin 0.1 – 0.4 mg/dl Unconjugated (indirect)bilirubin 0.2 – 0.8

mg/dl

Bilirubin in blood morethan 1mg/dl is called clinical hyperbilirubinemia.

Hyper bilirubinemia : Increased bilirubin production Decreased hepatic uptake Decreased hepatic conjugation Decreased excreation of bilirubin into bile

Bilirubin accumulates in allthese conditions approximately 2.2 - 5 mg/dl.

The skin and sclere appears yellowish due to deposition of bilirubin in the tissue.

The condition is called jaundice or Icterus.

Jaundice: (Hyperbilirubimia)acquired or inherited Conjugated or Unconjugated

4 types Hemolytic or prehepatic Hepato cellular / Hepatic / intrahepatic Obstructive / Post hepatic Neonatal / Physiological jaundice

Acquired hyperbilirubimia Prehepatic or Hemolytic

eg: - sickle haemoglobin (sickle cell anemia)

- deficiency of enzyme glucose 6-phosphate dehydrogenase.

- Incompatible blood transfusion

Biochemical features Increased plasma unconjugated bilirubin Increased amount of urobilinogen in urine

and Feces. Absence of bilirubin in urine.

Intrahepatic / Hepatic / Hepatocellular JaundiceInfection (viral hepatitis) Toxic chemicals (alcohol)Drugscirrhosis

Uncojugated hyperbilirubinemia and presence of some conjugated hyperbilirubinemia

Biochemical Features Increased plasma concentrtaion of conjugated

and unconjugated bilirubin. Decreaed amount of urobilinogen in urine and

faeces Presence of bilirubin in the urine Raise level of alanine transaminase enzyme

Posthepatic or Obstructive Jaundiceoccurs due to obstruction in the passage of conjugated bilirubin from the liver cells to the intestine this condition is also called as cholestasis.

Causes Blockage of common bile duct by gallstones Carcinoma of the head of the pancreas carinoma of the duct

Biochemical Features:Increased conjugated bilirubin in plasmaAbsence of urobilinogen in faeces and urinePresence of bilirubin and bile salts in urineRaised ALP in plasma

Neonatal of physiologic jaundice Mild jaundice in the first few days after birth Results from an accelerated haemolysis and

immature liver enzyme system for conjugation Liver is deficient in enzyme UDP-glucuronyl

transferase. Increased unconjugated bilirubin is seen If it exceeds 20 – 25 mg/dl it penetrates the

BBB This results in hyperbilirubinaemic toxic

encephalopathy or kernicterus

Inherited hyperbilirubinaemia Gilbert’s syndrome Crigler Najjar syndrome Dubin Johnson syndrome Rotor syndrome