Hemoglobin

Heme = Fe++ + Porphyrin Ring + Globin

Hemoglobin Function

Conjugated protein for transport of O2 & CO2

&

Buffer (O2 Dissociation Curve)

Basic Structure

How many? subunits:

How many? ironcontaining tetrapyrrole heme rings How many? polypeptide globin chains

? identical alpha chains ? identical non-alpha chains

Basic Structure

Each chain arranged in -helix w/ 8 individual helical segments (labeled A H)

Each globin molecule has both hydrophobic & hydrophilic areas

Basic Structure

Fe-containing heme ring w/in hydrophobic region of globin Heme Pocket

Hydrophobic nature protects Fe residue from oxidation Each Fe at center of heme residue held in place & kept active (Fe++) by 2 histidine residues

Hemoglobin Chemistry

O2 binds to heme (Iron)

97% carried this way 20% bound 80% free in plasma

CO2 binds to globin

CO2 causes acidity in plasma, effects the pH balance of the blood Blood can carry both at one time different binding

Basic Structure

Each globin (141 aa) All non- chains (146 aa) W/ considerable structural homology among non-alpha chains Full saturation: 1 gm Hgb holds 1.34 mL of O2

Hgb F

Normal Hgb:

Hgb A1

95 - 98% (adult) 15 - 40% at birth predominates at 6 12 mons

< 2% (adult) 1 - 0.8% (3 y/o) <2% present at 1 year of age 10% still present at 4 mons 65-90% of total HgB at birth 90 - 95% - fetal life

Hgb A2

2 - 3% (adult) 0 - 1.8%- cord blood 5 - 6 mons- adult levels

Hgb Gower 1 & 2; Portland Hgb - Embryonic

Hgb derivatives (Heme changes)

Oxyhgb (bright red) vs Reduced hgb (purple red)

Maintained

by methemoglobin- cytochrom C

reductase 575 nm

Hgb derivatives (Heme changes)

Carboxyhgb (cherry pink)

200

X affinity vs O2 Collect in citrate Test: ammonia + blood (25% COHgb) CO poisoning occur at levels of 20-30% 540 nm

Heavy smokers: Smokers: 1-10%

Hgb derivatives (Heme changes)

Methgb (0.5% normal in blood)

Sulfhgb (0- 2.2% normal in blood)

Fe++ Fe+++ brown No O2 binding cyanotic Induced by oxidizing agents (nitrates, nitrites, quinones, chlorates) G-6-PD deficiency 525 nm

Sulfa drugs (sulfonamides); aromatic amines trinitrotoluene Irreversible binding RES

Myoglobins Porphyrins

Iron Metabolism

Body Iron Distribution and Storage

Utilization Duodenum (average, 1 - 2 mg per day) Dietary iron Utilization

Plasma transferrin (3 mg) Muscle (myoglobin) (300 mg) Circulating erythrocytes (hemoglobin) (1,800 mg)

Bone marrow (300 mg)

Storage iron

Liver (1,000 mg)

Sloughed mucosal cells Desquamation/Menstruation Other blood loss (average, 1 - 2 mg per day) Iron loss

Reticuloendothelial macrophages (600 mg)

Major Iron Compartments

Metabolic: Hgb Myoglobin

1800 - 2500 mg 300 - 500 mg

Storage: Iron storage 0 - 1000 mg Transit: Serum iron 3 mg Total 3000 - 4000 mg

Red cell indices

Hemoglobin (gm\dl): 13.5- 17 M 12- 15 F Hematocrit (Vol%): Volume of RBCs / Total blood 39- 49 % M 33- 43% F RBCs ct (106/ul): 4.3- 5.9 M 3.5- 5 F

Iron Deficiency Anemia (IDA)

IDA - Pathogenesis:

Decreased Iron stores Decreased Hb Synthesis Delayed maturation of erythroblasts (cytoplasmic) Decreased cytoplasm, more division (microcytes) Decreased hb content (hypochromia) Anemia.

Clinical Features:

General features of Anemia

Pallor,

Weakness, Lethargy Breathlessness on exertion Palpitations heart failure pedal edema

Clinical Features:

Special features in IDA:

Angular

cheilitis, atrophic glossitis, Esophageal atrophy/web dysphagia, Koilonychia, brittle nails, gastric atrophy

Microcytic Hypochromic A (IDA)

DEC Hb, HCT, RBC MCV < 70 (IDA & THALASSEMIA) Low Retic count High RDW PBS: Microcytic Hypochromic RBCs

PBS: Response to Iron Tx

Dimorphic blood film

Other LAB Tests:

DEC SERUM Fe DEC SERUM FERRITIN

Most useful, cost effective test of Fe stores > 100 ng/ml r/o IDA Except in acute hepatitis or liver necrosis but not chronic liver dse (elevated- release of Fe stores) Falsely elevated- disseminated TB & Hodgkin's disease

INC TIBC (high specificity- near 100% but poor sensitivity- < 30%) BM: ASYNCHRONOUS RBC-POIESIS

Hemochromatosis: Overview

Def: Xss accumulation of Fe w/ organ damage Iron arithmetic:

Normal total body pool: 2-5 gm Hgb (2 gm) Storage: ferritin & hemosiderin (0.5 - 1 gm) Myoglobin (0.3 gm) Normal

liver storage: 0.5 gm Hemochromatosis:

Total body Fe > 50 gm At least 1/3 is stored in liver

Hemochromatosis: classification

Genetic (hereditary) vs secondary (acquired)

Secondary causes iron overload:
Parenteral

iron overload (transfusions, iron-dextran

injections) Ineffective erythropoiesis hemolytic anemias (most common secondary cause) Increased oral intake (Bantu siderosis) Congenital atransferrinemia Chronic liver disease (mostly alcoholic)

Genetic Hemochromatosis

Homozygous recessive HFE gene on chr 6p (near HLA gene locus) MC mutation in HFE: tyrosine substituted for cysteine at aa 282 (C282Y) Gene frequency: 6% northern European whites; homozygosity 0.45% (1 in 220) M: F = 6:1 Males show disease earlier (no menses)

Hereditary Hemochromatosis: pathogenesis

Genetic Hemochromatosis: Pathology

Deposition hemosiderin in multiple organs (brown granules of aggregated ferritin): Liver & pancreas (most severe) Myocardium Pituitary, adrenal, thyroid, parathyroid glands Joints (synovitis) and skin Testes (atrophy)

Hemochromatosis

Genetic dse Excess amounts of iron Arthritis, cirrhosis, DM, heart failure, HCC

Hemochromatosis: Clinical Dx

Signs:
Abdominal

pain Hepatomegaly Skin pigmentation (brown-gray) DM Cardiac dysfunction Arthritis Hypogonadism

Hemochromatosis: Clinical Dx

Diagnostic tools:
Markedly

elevated serum ferritin (best screening test) HFE gene molecular analysis Liver biopsy w/ quantitative Fe analysis

Normal: < 1,000 ug Fe/gm Hemochromatosis: > 10,000 ug Fe/gm

Evaluate for secondary causes (vs. genetic)

Anemia of Chronic Disease: ACD

ACD - ETIOLOGY: ANY CHRONIC DSE

Inflammatory:
Temporal

Non-inflammatory:
CHF COPD DM

arteritis (may be a presenting sign) Rheumatoid arthritis Cancer

ACD

Infections: TB, SBE,

Osteomyelitis Chronic UTI or PN Fungal

Chronic inflammatory disorders:

Malignancy:

Mets Leukemia Lymphoma Myeloma

RA SLE Sarcoid Collagen Vascular Dse Polymyalgia Rheumatica Chronic Hepatitis Decubitus ulcer

ACD
Fe SEQUESTRATION IN BM HISTIOCYTES Defect w/ defective Fe utilization & shorten RBC survival

Storage Iron IL-1 TNF IFN

BM Storage Iron (Blue)

Erythroid Precursors (dec responsiveness to EPO)

ACD - LAB DX:

INC. ESR INC. HAPTOGLOBIN Dec serum Fe Dec TIBC BM: Inc BM Fe stores w/ dec sideroblasts

ACD

Normal

IDA

Sample question #1
ACD is due to inadequate production of, or poor response to, which one of the following?
A. B. C. D. E. Iron Folate Erythropoietin Ferritin Hemosiderin AFP, Nov. 15, 2000

Heme Synthesis

Porphyrin Synthesis & Structure

Heme Synthesis

Glycine & Succinyl Co-A -amino levulinic acid (-ALA)

The

rate-limiting step in heme synthesisintra-mitochondrial ALA- synthase

-ALA travels to cytoplasm; converted to PBG, a monopyrrole

Heme Synthesis

PBG (monopyrrole) Protoporphyrin IX (biologically active tetrapyrrole) Fe inserted into tetrapyrrole ring in mitochondria
Heme synthesis:
Stimulated

by Fe Repressed when Fe is inadequate (IDA)

Sideroblastic Anemia

SIDEROBLASTIC A. MECHANISM:
Defective prdn of heme under- hgb erythroid precursors w/ microcytosis REFRACTORY A. (RESISTANT TO TX) INEFFECTIVE ERYTHROPOIESIS (ANEMIA W/ HYPERPLASTIC BM)

SIDEROBLASTIC A - ETIOLOGY & Mechanism:

HEREDITARY (X LINKED/ AR)

HEPATOSPLEENOMEGALY; THROMBOEMBOLISM

SIDEROBLASTIC A - ETIOLOGY & Mechanism:

ACQUIRED: IDIOPATHIC

NEUTROPENIA

W/ PELGER HUET CELLS PRONE TO Fe OVERLOAD; TE 10% DEVELOP AML

SIDEROBLASTIC A - ETIOLOGY & Mechanism:

ACQUIRED: DRUG INDUCED

ALCOHOL- FOLATE DEF. + INH- VIT. B6 METABOLISM

MALNUtrition

CHLORAMPHENICOL-

MITOCHONDRIAL

INHIBITION LEAD- HEME PATHWAY Zinc, cycloserine, plavix

DISEASE ASSO. (THYROID; CA; LYMPHOMA; MM; RA)

Sideroblastic A Lab Dx:

PBS: Papenheimer Bodies; Basophilic stippling in Pb poisoning; Dimorphic (macrocytic + intensely microcytic RBCs) in patient w/ acquired sideroblastic a; anisopoikilocytosis; Target cells

Sideroblastic A Lab Dx:

Serum Fe: Inc Stigmata of a myelodysplastic syndrome BM: Ringed sideroblasts on BM Fe stain; inc hemosiderin

Porphyria

Porphyria cutanea tarda

Etiology:
Uroporphyrinogen Anti

decarboxylase def

HC Ab photosensitivity

S/S:
Cutaneous

Porphyria cutanea tarda

Features:
Portal

Lab tests:
Inc

inflam w/ cirrhosis HCC Inc hepatic Fe Inc urinary uroporphyrin

urine uroporphyrin Inc aminotransferase

Acute intermittent porphyria

AD w/ incomplete penetrance (other family members w/ the condition maybe asymptomatic) Drugs & alcohol induced (P450 enzyme inducers) Acute attack: urine turns dark on standing (high ALA & PBG levels). Levels remain moderately raised between attacks

Variegate porphyria
AD disorder Features: Cutaneous fragility & photosensitivity Acute neurological attacks common

Hereditary coproporphyria
AR Uroporphyrinogen synthetase defect expressed in RBCs w/ inc porphyrin levels in stool Acute neurological attacks + cutaneous manifestations

Globin Synthesis

Location of the Globin Genes

Duplication of genes for: Globin

Globin (G & A) * G & A differ from one another only at position 136 (glycine & alanine respectively)

Globin chain switch

Globin chain synthesis

cluster - chromosome 16 z 2 1 z2e2 z22 2e2 22 Gower 1 Portland Gower II F Fetal <1% Embryonic

22
2b2 e G A b

A2
Adult A

1.5-3.5%
>95%

b cluster - chromosome 11

Hemoglobin Catabolism

Ferritin

Reduced

Normal

Increased

Iron deficiency

HB electrophoresis

Anemia of Chronic Disease

Bone marrow Ringed sideroblasts

Further investigations to find the cause are

necessary

If HB electrophoresis is normal then do alpha gene mapping

Hypochromia
Microcytic Note: Anisocyosis: RDW poikilocytes

WAKAS!!!