Biochemistry of Blood PDF

# Biochemistry of Blood ## Contents - Haemoglobin structure - Types of normal Hb - Types of Abnormal Hb - Haem Biosynthesis - Haemoglobin Catabolism - Folic Acid - Cobalamin (Vitamin B12) - Vitamin K - Iron - Immunoglobulins ## Haemoglobin structure Haemoglobin = Haem + globin (Protein)....

# Biochemistry of Blood ## Contents - Haemoglobin structure - Types of normal Hb - Types of Abnormal Hb - Haem Biosynthesis - Haemoglobin Catabolism - Folic Acid - Cobalamin (Vitamin B12) - Vitamin K - Iron - Immunoglobulins ## Haemoglobin structure Haemoglobin = Haem + globin (Protein). Haem = Iron + Porphyrins. ### A. Porphyrins:- - Cyclic compounds derived from Porphin nucleus made of 4 pyrrole rings linked by 4 methenyl bridges (-CH=) labelled α, β, γ, δ. - In Porphyrins found in nature, side chains are substituted for H atoms. ### Types of Porphyrins:- 1. Type I isomer: The substituted groups attached to the 4 pyrrole rings are symmetrically arranged (AP – AP – AP – AP). 2. Types III isomer: The substituted groups attached to the 4th pyrrole ring are arranged in the reverse order (AP – AP – AP-PA). - Biologically important Porphyrins in Haem and Cytochromes are Type III isomer. ### B. Iron:- - Present in Ferrous state (Fe+2). - Linked to 4 Nitrogen atoms of 4 Pyrrole. - There is 2 additional bonds located on each side (perpendicular) to Haem plane called 5th and 6th coordination Bonds. - 5th bond is linked to N of Imidazole ring of proximal Histidine. - 6th bond is linked to Oxygen in HbO2 and empty in Deoxy Hb (Hb). - The 4 pyrrole rings are attached to side chains called Methyl, Vinyl, Methyl, Vinyl, Methyl, Propionyl, Propionyl, Methyl. (MV – MV – MP – PM). - Transport of O2 is based on a physical interaction between O2 and Iron of Haem to provide reversible association. ### C. Globin:- (Protein part = Apo Protein) - Simple protein (Histone) with high content of Histidine and Lysine. - Composed of 4 polypeptide chains: 2α chains and 2β chains. - α chain → 141 amino acid and folded into 7 right handed α helices. - β chain → 146 amino acid and folded into 8 right handed α helices termed from A → H starting from NH2 terminal. - Haem: Globin = 4:1 - Haem: Polypeptide chain = 4:4 - Each Haem molecule is linked to one polypeptide chain. - Myoglobin – Haemoglobin family of proteins has produced away in which Fe+2 can be bound to proteins to produce an O2 binding site. - Hb protects Fe+2 which binds O₂ from irreversible oxidation by providing an environment in which the first step in oxidation (binding of oxygen) is permitted, but the final step (oxidation) is blocked. ## Types Normal Hb ### I-Adult Hb (HbA):- #### a. Major Adult Hb (Hb A1) (α2β2):- - Contain 2 Alpha chains and 2 Beta chains. - Constitutes 95-97% of total Hb. #### b. Minor Adult Hb (Hb A₂) (α2 δ2):- - Contain 2 Alpha chains and 2 Delta chains. - Constitutes 2-4% of total Hb. - In 6 chain there is more than 1 amino acid different from β-chain. - β-Chain position 16 Glycine → δ-Chain position 16 Arginine. ### II-Glycosylated Hb (Hb A₁C):- - Modified form of Hb similar to HbA, but with glucose linked to 8-amino group of lysine at NH₂ terminal ends. - The reaction is non – enzymatic and depends on glucose concentration. - Present in normal value 5% of total Hb. - This percentage increases in Pre-Diabetic and Diabetic patients to 8-14%. - Hb A1C gives idea about blood glucose level during the last 3 months so it is useful in the assessment of Diabetic control. ### III- Fetal Hb (HbF) (α2γ2):- - Present normally in newborn and early fetal life. - At age of 7 months, 90% of HbF is replaced by HbA1. - Consist of 2 Alpha chains and 2 Gamma chains. - In y chain, there is more than 1 amino acid different from β chain. - B-Chain position 21 Histidine → y-Chain position 21 Serine. - HbF has a great affinity to O₂ because y-Chains don't bind 2, 3 BPG. - 2, 3 Bis Phospho Glycerate (2, 3 BPG) is responsible for lowering Hb affinity to O2 allowing Hb to release O2 at the typical PO2 of tissues. ## Abnormal Types of Hb ### I-Sickle cell disease (HbS):- - Glutamic amino acid is replaced by Valine amino acid at 6th position of Beta chain due to Mutation in the structural gene (α2S2). #### Pathogenesis:- - The presence of Valine (non-polar amino acid) instead of Glutamic acid (polar amino acid) at 6th position of β-chain leads to formation of sticky patches on the surface of both Oxy HbS and Deoxy HbS. - Both Deoxy HbS and Deoxy HbA contain complementary patches (grooves) - When Blood is deoxygenated → sticky patches and complementary patches of Deoxy HbS bind together → polymerization of deoxy HbS → Formation of long fibrous precipitates → sickling of RBCs. #### Effects of sickling of RBCs:- 1. Sickle shaped RBCs are easily fragile → Anaemia (sickle cell anaemia). 2. Sickle shaped RBCs are easily trapped in small Blood vessels → Thrombus formation and damage to tissues and organs as brain, bone, spleen, #### Diagnosis:- 1. Reticulocytes count between 10-20%. 2. Sickle shaped RBCs by microscopic examination. 3. Electrophoresis: shows HbS, no HbA. - HbS moves slowly due to less ve charges on its molecule, so appears in electrophoresis nearer to cathode than normal Hb. ### II-Thalassemia:- - Hereditary hemolytic diseases in which synthesis of their α or β globin chain is defective due to mutation affecting the regulatory gene. #### Types:- #### A. α-Thalassemia:- - Decreased or absent synthesis of α chains of Hb with compensatory increase in the synthesis of other chains. - It is 2 Types: - Pure β-chain (HbH): where there is 4 β-Chains (HbH disease). - Pure γ chain (Hb Bart's): where there is 4 γ-Chains. #### B. β-Thalassemia:- - Decreased or absent synthesis of β-chains. - Synthesis of α chain is normal and will combine with:- - δ-Chains → giving Excess HbA2 (α2 δ2). - γ-Chains giving Excess HbF (α2γ2). - The abnormal Hb doesn't function as normal Hb having abnormal O2 dissociation curve. #### Diagnosis:- 1. Anaemia: called Cooley's anaemia or Mediterranean Sea anaemia. 2. Electrophoresis: shows HbF, HbH, HbA2. ### III- Met Haemoglobinemia:- - This is oxidized Hb, containing Fe+3 instead of Fe+2 normally present in Haem. - The ability to react as an O2 carrier is lost. - Normal Erythrocyte contains small amount of Met Hb formed by spontaneous oxidation of Hb. #### Excess met Hb in blood may be due to: - Excess production of Met Hb. - Diminished ability to convert it back to normal Hb. ## Types and causes of Met Haemoglobinemia ### A. Congenital Met Haemoglobinemia:- #### a) Haemoglobin M (Hb М): - Congenital condition due to mutation in globin biosynthesis. - Distal or Proximal Histidine is replaced by Tyrosine, so Heme iron is stabilized in the Ferric state, since it forms a tight ionic complex with Phenolate Anion of Tyrosine. - Treatment with reducing agents as methylene blue is ineffective. - Treatment is only conservative as Blood Transfusion. #### b) Deficiency of NADH-Cytochrome b5 Met Hb reductase system: - This system consists of: 1. NADH (generated by Glycolysis). 2. Met Hb reductase = Flavoprotein Cytochrome b5 reductase. 3. Cytochrome b5. - This system reduces heme Fe³ back to the Fe+2 state. - Treatment with reducing agents is Effective. ### B. Acquired (Toxic) Met Haemoglobinemia:- - Arises following the ingestion of large amounts of drugs as Phenacetin or Sulphonamides, excess Nitrites or certain oxidizing agents in diet. - Treatment with injection of reducing agents like Vitamin C or Glucose or Methylene blue is Effective in reversing Acquired Met Haemoglobinemia. #### Diagnosis of Methemoglobinemia:- 1. Cyanosis. 2. Examination of Blood color (Brownish). 3. Pulse Oximetry to test saturation of Blood Oxygen. 4. DNA sequencing of Globin chain. ## Haem Biosynthesis - Haem is Iron Protoporphyrin III synthesized in : - a. Bone marrow (85%) for synthesis of Hb. - b. Liver for synthesis of Cytochromes. - The initial (first) and last 3 enzymatic steps → Mitochondrial Enzymes. - Intermediate steps → Cytoplasmic Enzymes. ### 1) Synthesis of 5-Amino Levulinic Acid (δ-ALA): - Glycine + Succinyl CoA → ALA Synthase PLP → a-amino B-keto → ALA Synthase COASH → Adipic Acid → δ-Amino Levulinic Acid (δ-ALA) CO2 ### 2) Synthesis of Porphobilinogen (PBG): - 2 δ-Amino Levulinic Acid → ALA Dehydratase → 2 H2O → Porphobilinogen (PBG) First precursor Pyrrole ### 3) Synthesis of Uroporphyrinogen: - 4 Porphobilinogen → Uroporphyrinogen I Synthase → 4 NH2 → Uroporphyrinogen I Open chain linear tetra pyrrole ### 4 Porphobilinogen → Uroporphyrinogen III Co-Synthase ### 4) Synthesis of Coproporphyrinogen: - Uroporphyrinogen III → Decarboxylase → 4 CO2 → Coproporphyrinogen III ### 5) Synthesis of Protoporphyrinogen III, Protoporphyrin and Haem: - Coproporphyrinogen III → Coproporphyrinogen Oxidase → 02 → 2 CO2 → Protoporphyrinogen III - Protoporphyrinogen III → Protoporphyrinogen Oxidase → 6 H - Protoporphyrinogen → Heme Synthase or Ferrochelatase → Protoporphyrin III - Protoporphyrin III → Haem ## Fate of RBCs and Hb catabolism - RBCs exit from arterioles in splenic pulp and enter splenic sinus. - Old RBCs are destroyed by Macrophages in spleen, liver and bone marrow. ### Hemoglobin - Heme → Biliverdin & Bilirubin → Form Bile pigments → Exerted in Bile ✓ 0.2-0.8 mg/dL in Blood - Iron → Reutilized for Hb synthesis - Globin → Hydrolyzed into amino acids which is Reutilized in protein synthesis ## Hemoglobin Catabolism:- - Heme Catabolism occurs in RES by Heme Oxygenase + O2 + NADPH. - Heme Oxygenase → Biliverdin Reductase → Heme → Biliverdin 02 (Green) NADPH+H → Bilirubin (Yellow) NADPH+H Fe+3 RES ## Folic Acid (Folacin = Pteroyl Glutamic Acid) ### Chemistry: - Pteridine Nucleus (Bicyclic Nitrogenous compounds) + P-Amino Benzoic Acid (PABA) + Glutamic Acid. - Pteroic Acid + Glutamic Acid. - Animal cells Can't: - Synthesize PABA. - Attach Glutamate to Pteroic Acid. - Bacteria and plants can → so Animal cells require folic Acid in their Diet. ### Requirements: - 200 µg/day. ### Sources: - Leafy vegetables, yeast, cauliflower, liver. ## Functions: - Formation of the important coenzyme Tetra hydro Folic Acid (FH₁ = THF). - Folic Acid → Di Hydro Folate → Folic Acid → Tetra Hydro Folate Reductase NADPH+H+ Reductase NADPH+H+ NADP+ NADP+ - FH4 is the coenzyme for one Carbon metabolism → It can carry one carbon moiety as: - ✓ Methyl (-CH3) - Methylene (-CH2-) - Methenyl (-CH=) - Formyl (-CHO) - / Formimino (-CH = NH) ### Sources of the one carbon Group:- 1. Beta carbon of Serine (Major source). 2. Glycine. 3. Formate (Intermediate of Tryptophan Metabolism through Kynurenine pathway). ### Functions of one carbon Group:- 1. Synthesis of some amino acids as Glycine, Serine, Methionine, and Histidine. 2. Purine Biosynthesis (Formation of C2 and C8 of Purine ring). ✓ It is coenzyme for Formyl Transferase. 3. Synthesis of Deoxy Thymidylic Acid (dTMP). ✓ It is coenzyme for Thymidylate synthase. ## Deficiency:- - Folate deficiency may be True (primary) or secondary to B12 deficiency. - Folate deficiency leads to:- 1. Macrocytic anaemia with Megaloblastic changes in Bone marrow. ↓↓↓ Purines and dTMP → Inhibition of DNA synthesis → slows down the maturation of RBCs → production of abnormally large (Macrocytic) RBCs with Fragile Membranes. 2. Glossitis and Gastrointestinal disturbances. ## Folic Acid Antagonists: - Substances used in treatment of malignant diseases (cancer) as Methotrexate (Amethopterin) and Aminopterin. • They prevent reduction of Folic Acid to Tetra hydro Folic Acid (THF= FH4), so they block synthesis of Nucleic Acids in Malignant cells. • Folic Acid antagonist (Trimethoprim) → Antibiotic. ## Cobalamin (Vitamin B12) ### Chemistry:- - It has two characteristic components:- 1. The central portion of the molecule (Corrin ring) Consists of 4 reduced and extensively substituted pyrrole rings. - Very similar to Porphyrin structure, but differs from Porphyrin in that: - ✓ 2 Pyrrole rings (A and D) are joined directly rather than through Methenyl bridge. - ✓ Co-ordinated to the four inner Nitrogen atoms of Corrin Ring is an atom of Cobalt. 2. 5. 6 di methyl Benzimidazole Riboside. - Cobalt is in a coordination state of six. - 5th position is attached to 5, 6 dimethyl Benzimidazole Riboside. - 6th position is coordinated to:- - + Cyanide (CN) → Cyano Cobalamin. - Hydroxyl (OH) → Hydroxy Cobalamin. - Methyl (CH3) → Methyl Cobalamin. - 5\ deoxy Adenosine → 5\ deoxy Adenosyl Cobalamin. ### Requirements:- - 2 µg / day. ### Sources:- - It is not present in vegetable foods. - Neither animals nor plants can synthesize it. - The only source in nature is through synthesis by microorganisms in animal Intestine. - Negligible (Small) amounts are provided by Intestinal flora. - The only sources of the vitamin are foods of animal origin as liver, kidney, meats, milk and eggs. ### Functions:- - In foods, Vitamin B12 occurs bound to protein, and to be utilized Vitamin B12 is first removed from protein by acid hydrolysis in the stomach. - Then, Vitamin B12 combines with the intrinsic factor (glycoprotein secreted by parietal cells of gastric mucosa) which carries Vitamin B12 to ileum for Absorption. - As (Cobalamin-Intrinsic factor) complex crosses ileal mucosa, intrinsic factor is released and the vitamin is transferred to plasma (Absorbed). - In Man, there are only 2 bio chemical reactions in which Vit. B12 participate 1) Homocysteine → Methionine Synthase → Methionine Methyl THF Methyl Cobalamin Cytoplasmic THE - Methyl Cobalamin ← Hydroxy Cobalamin ← Deoxy Adenosyl Cobalamin 2) L- Methyl Malonyl CoA → Methyl Malonyl CoA Mutase → 5\ deoxy Adenosyl Cobalamin Mitochondrial → Succinyl CoA ### Deficiency:- 1. B12↓↓↓→ Pernicious anaemia → Macrocytic Megaloblastic anaemia (thought to be due to the effect of B12 on folate Metabolism). 2. Neurologic disorders due interference with myelin sheath integrity with sensory and motor Losses (progressive demyelination of nervous tissue). ✓ Decreased B12 → ↓↓ Methionine → ↓↓ SAM → ↓↓ Methylation of Phosphatidyl Ethanolamine to Phosphatidyl Choline (Essential for Myelin Sheath formation). 3. Homocystinuria and Methyl Malonic Aciduria. ## Vitamin K ### Requirements:- - 70-140 mg/day. ### Sources:- - Vitamin K₁ → vegetable oils, green Leafy vegetables as spinach, cabbage, cauliflower and peas. - Vitamin K2 → synthesized by intestinal flora & found in animal tissue. - Good sources Vitamin K include tomatoes, cheese, egg yolk and liver. ### Functions:- - Vitamin K is required for the conversion of several blood clotting factors (II, VII, IX, X) (1972) to the active state. - All these factors are proteins synthesized in liver in an inactive form. ### Mechanism of Vitamin K dependent activation of Prothrombin:- - Prothrombin is synthesized in the liver in an inactive precursor form called Pre Prothrombin. - Conversion of pre Prothrombin to active Prothrombin requires vitamin K dependent carboxylation of glutamic Acid to γ-carboxy glutamic Acid. - γ-Carboxy glutamic Acids are good chelators and allow active Prothrombin to bind (chelate) calcium. - Prothrombin-Calcium complex binds to the phospholipid membrane, where Proteolytic conversion to thrombin can occur. #### To summarize:- 1. Vitamin K is an essential cofactor for carboxylase enzyme that forms γ-Carboxy glutamic Acid from glutamic acid in the specific protein molecule. 2. Vitamin K probably acts as a cofactor necessary for oxidative phosphorylation (Coenzyme Q which is essential component of oxidative phosphorylation, is similar to vitamin K in structure). - ☑ N.B: Vitamin K is an antidote to Dicumarol anticoagulant. ### Deficiency:- - Manifested by bleeding tendency from minor wounds. - Diagnosed by prolonged blood coagulation time. ## Iron ### Daily Requirements:- #### Infant & Children #### Adolescents #### Adults #### Above 50 years #### Male 15 mg/day 18 mg/day 10 mg/day 10 mg/day #### Female 15 mg/day 18 mg/day 18 mg/day 10 mg/day - Increased demands for iron in females are due to pregnancy, Menstruation and Lactation. ### Sources:- - Organ meats as liver, heart, kidney, whole wheat, spinach and molasses. - Milk is a poor source of iron, ### Absorption:- - Of Dietary iron, 10% is absorbed in the duodenum. #### Factors that help Iron absorption are:- 1. Acidity: low PH helps to liberate iron from organic combinations. 2. Reducing Agents: Ferric (Fe¹³) Vitamin C & Glutathione ►Ferrous (Fe+2). 3. Proteins with sulfur containing amino acids change Fe+3 → Fe+2. 4. Type of iron compounds in diet, Heme iron is absorbed directly. 5. Iron deficiency enhances iron absorption. #### Factors which inhibit Iron Absorption are:- 1. Achlorhydria (No HCl) or Hypochlorhydria (Low HCl). 2. Phytates. 3. Oxalates. ### Mechanism of Iron Absorption - Iron is Transported (Fe+2) into enterocytes by divalent metal transporter (DMT1) at the Apical (luminal) side of the cell. - Heme → Heme Transporter (HT) into Enterocytes. Apical protein - Heme Oxygenase → In Enterocytes, Heme Porphyrin → Remove Fe and adds it to intracellular Fe+2 pool in cytoplasm. - Some Fe+2 is stored as ferritin in Enterocytes. - Remaining Fe+2 is transported to plasma by the Transporter Ferro Protein (FP-1). ### Distribution:- - Total body content of iron is 5 gram, - Haemoglobin contains 3 gram (60%). - 5% of total iron is held by a Porphyrin (Heme) combined with proteins as Myoglobin, Cytochromes, Cytochrome Oxidase, Catalase and Peroxidase. - Iron is stored as Ferritin, Transferrin and Hemosiderin. - Liver is the main storage organ for Iron. - Ferritin is the main storage form for Iron. ### Plasma level:- - 50 – 150 µg/dL. ### Excretion:- - Mainly by faeces either as unabsorbed iron or sloughed intestinal cells. ### Metabolism:- - Iron is released from mucosal cell and enters the portal blood as Fe+2. - In plasma, Fe+2 is converted by serum Ferroxidase (Ceruloplasmin) to Fe+3. - Fe+3 combine with Iron binding protein (Transferrin). - Protein bound Iron is 50-150 µg/dL, but Total Iron Binding Capacity (TIBC) is 300 – 360 µg/dL. - 35% of Transferrin is saturated with Iron. - Iron is taken rapidly by Bone marrow for Hb synthesis. - Remaining Iron is stored in the liver as Hemosiderin. ### Function:- 1. Important for compounds essential for cellular respiration as Hb and Tissue iron (Myoglobin, Cytochromes, Cytochrome Oxidase, Catalase, Peroxidase). 2. Remaining iron is present in the stored form as Ferritin, Transferrin and Hemosiderin and used in Hb Bio synthesis ### Iron deficiency:- #### Abnormal Levels - Due to intake, ↓↓ absorption or ↑↑ loss of iron. - Characterized by Microcytic Hypochromic anaemia, - Protein Bound iron and ↑↑ TIBC. - Serum Ferritin Less than 20 µg/ml. - Common in Infancy as Milk is deficient in Iron. - Partial Gastrectomy Complications include Iron deficiency Anaemia. ### Iron Toxicity (Hemosiderosis):- - Excessive accumulation of iron in tissues as heart, spleen, pancreas, liver, and under skin (Hemochromatosis) due to repeated blood Transfusion in Hemolytic anaemia. - May lead to Bronze Diabetes. - Characterized by || TIBC. - Serum Ferritin More than 50 µg/ml. ## Immunoglobulins - Ig molecule is Y shaped with 4 polypeptide chains: 2 identical Light chains (L) & 2 identical Heavy chains (H) linked by disulphide Bonds. - Both L and H chains have - Variable regions (VL & VH) at the amino terminal part. - Constant regions (CL & CH) at the carboxy terminal part. - The Variable regions have variable amino acid sequence and form the antigen binding site of Ig. - Each Ig has 2 identical Antigen binding sites. - Constant regions have constant amino acid sequence in all Igs of same class. - Igs are glycoproteins containing carbohydrate group attached to CH. - Immunoglobulins are Proteins secreted from Plasma cells on Antigen stimulation. They are γ-Globulins. ### Classes of Immunoglobulins - There are 5 classes of Ig that differ in are sequence of CH | Ig | Ig Existence | Heavy chain | Symbol | Subclasses | % | |---|---|---|---|---|---| | 1 | IgG | Gamma | Y | IgG1, IgG2, IgG3 | 75% | | | Serum Monomer | | | | | | 2 | IgA | Alpha | α | IgA1, IgA2 | 10-15% | | Secretory Dimer | | | | | | 3 | IgM | Mu | µ | IgM, IgM2 | 5-10% | | | Pentamer | | | | | | 4 | IgD | Delta | δ | | 0.2% | | | Monomer | | | | | | 5 | IgE | Epsilon | ε | | Very low concentration | | | Monomer | | | | | ## Functions of Immunoglobulins ### I- Immunoglobulin G (IgG):- 1. Has low M.W → can: - Traverse Blood vessels to tissues. - Pass through placenta to fetus providing passive immunity for the newborn in the first 6 month after delivery. 2. It has antibacterial, antiviral, antiprotozoal and antitoxins activity. 3. It fixes and activates complement (by classical pathway). 4. It increases in the secondary immune response. ### II-Immunoglobulin M (IgM):- 1. Produced in Primary immune response. 2. It fixes and activates complement (by classical pathway). 3. Gives agglutination reaction with antigen. ### III-Immunoglobulin A (IgA):- 1. Provides Immunity for the new born (found in colostrum and milk). 2. Responsible for Local Immunity at mucous membrane surface which are the main entry sites of pathogens. 3. Fixes and activates complement (by Alternative pathway). ### IV- Immunoglobulin D (IgD):- 1. Receptor for antigens on surface of B-Lymphocytes. 2. Antibody against Insulin and food toxin. ### V- Immunoglobulin E (IgE):- 1. Protects against Parasites by causing release of enzymes from Basophils. 2. Mediates allergy (Type I Hypersensitivity reaction). - ✓ IgE binds to receptors on Mast cells or Basophils by its Fc. - Antigen binds to this IgE → release of mediators as Histamine, Bradykinin and Serotonin. - ✓ Histamine and Bradykinin cause vasodilatation of small blood vessels of skin leading to Urticaria. - Serotonin causes vasoconstriction and bronchoconstriction leading to Bronchial Asthma.