Hematology - 04 Hemoglobin and Iron

HEMATOLOGY HEMOGLOBIN AND IRON OUTLINE • Hemoglobin o Hemoglobin o Hemoglobin Structure o Hemoglobin Function o Hemoglobin Derivatives o Degradation of hemoglobin o Hemoglobinopathy o Hemoglobin Measurement • Iron Metabolism o Iron o Absorption o Transport o Utilization o Storage o Elimination o Laboratory Tests for Iron Metabolism HEMOGLOBIN GLOBIN CHAINS HEMOGLOBIN • • • • • • Known as Hgb or Hb The oxygen-binding protein in RBC (MW - 68,000 Dalton) Main cytoplasmic component of RBC o 1/3 of RBC weight o 34 g/dL per RBC Production: o 65% - nucleated stages of erythropoiesis o 35% - reticulocyte stage o RBC cannot produce Hgb because it lacks the necessary organelles Main Function: o Transport oxygen (lungs to peripheral tissues) ▪ 1 gram of Hgb = 1.34 mL of O2 o Transport CO2 (peripheral tissues to lungs) ▪ CO2 - metabolic waste that is excreted from lung in the form of bicarbonate o Participate in acid-base balance Reference ranges: o Female: 12-15 g/dL (120-150 g/L) o Male: 14-18 g/dL (140-180 g/L) o NewBorn: 16.5-21.5 g/dL (165-215 g/L) • • • • • HEMOGLOBIN STRUCTURE • Hemoglobin Molecule o Composed of: ▪ 4 Globin Chain - 2 identical pair ▪ 4 Protoporphyrin IX ▪ 4 Ferrous Iron • 1 gram of Hgb can carry 3.47 mg Iron ▪ 1 2-3 DPG/BPG • May or may not be present o Can carry 4 oxygen molecules due to 4 heme molecule ▪ Heme - functional component that binds 1 oxygen per molecule There are 39 different amino acids between Gamma & Beta Epsilon & Zeta - found in embryonic stage o Epsilon - analogous to Beta, Delta, & Gamma chains o Zeta - analogous to Alpha chains Globin Synthesis o Occurs in ribosomes from pronormoblast stage to reticulocyte stage o Occurs through transcription of DNA and translation of mRNA o Chromosome 16: α and ζ chains o Chromosome 11: ß, δ, γ, and ε chains Adult Hgb o Primarily composed of Alpha and Beta chains After birth, globin chains is primarily composed of Alpha and non-Alpha chains HEME • • • • • HEMOGLOBIN ASSEMBLY aka Ferroprotoporphyrin IX Belongs to the class of pigments known as porphyrins o Responsible for the red color of blood Each contains one protoporphyrin and ferrous iron o Protoporphyrin - aka protoporphyrin IX/ring Binds and carries oxygen 1 heme can bind one O2 • • HEME SYNTHESIS • Primary structure o Contain sequence o Will form helical & non-helical structure Secondary structure o Perform foldings to be arranged into a configuration Tertiary structure o Pretzel-like configuration o Where heme is bound o Will pair with different globin chain to for heterodimer and bind with another heterodimer to form quaternary structure HEMOGLOBIN MOLECULE • • Ferrochelatase - aka as heme synthase o Important enzyme needed for the insertion of iron in the center of protoporphyrin 4 porphyrin rings o Gives negative feedback to ALA synthase Porphyria - group of disorders caused by deficiency of one or more enzyme in heme synthesis HEMOGLOBIN FUNCTION FUNCTIONAL UPTAKE AND RELEASE OF OXYGEN o Heterodimer - with 2 different molecules ▪ Will bind to another with same composition • • • • • • Uptake and release of oxygen changes the state of Hgb 2,3-BPG - essential for R & S state o Its action is to stabilize salt bridges of the Hgb o Straightens a globin dimer o Inhibits binding of oxygen o Increases with high altitude ▪ More Hgb with stabilized producing T-state Hgb ▪ More Hgb cannot bind oxygen Tensed state (T) o Deoxygenated o Low oxygen affinity o Due to inclusion of 2,3-BPG Relaxed state (R) o Oxygenated o Has specific tilting of one globin pair at 15° o Tilting occurs due to lack of 2,3-BPG Carbon dioxide and hydrogen can stabilize salt bridges o Prevent binding of oxygen Diseases o Pulmonary insufficiency, congestive heart failure, severe anemia • DYSHEMOGLOBINS • • OXYHEMOGLOBIN DISSOCIATION CURVE • • • • • Graphical representation of hemoglobin affinity to oxygen Hemoglobin will transport oxygen to areas with low oxygen tension (capillaries) High O2 tension (lungs) = high oxygen affinity Shift to the Left: o ↑ O2 affinity/tension o ↑ pH o ↓ pCO2 o ↓ 2,3-BPG o ↓ Temperature Shift to the Right: (in areas with low oxygen tension) o ↓ O2 affinity o ↓ pH o ↑ pCO2 o ↑ 2,3-BPG o ↑ Temperature • • HEMOGLOBIN DERIVATIVES PHYSIOLOGIC FORMS OF HEMOGLOBIN • Oxyhemoglobin (HbO2) o Hemoglobin bound to oxygen o Bright red in color o Found in arterial blood o Hemoglobin Conformation: Relaxed (R) state ▪ Absence of 2,3-BPG Deoxyhemoglobin o Hemoglobin not bound to oxygen o Dark red in color o Found in venous blood o Hemoglobin Conformation: Tensed (T) state ▪ Presence of 2,3-BPG Dysfunctional hemoglobin variants Methemoglobin (Hi) o aka Hemiglobin, Ferrihemoglobin o Hemoglobin with ferric iron (Fe3+) – Hemin o Not able to bind oxygen o Color of blood: Chocolate-brown ▪ Chocolate-brown color seen after shaking o Seen in: ▪ Exposure of Hgb to oxidants • Oxidants oxidize Ferrous iron into ferric iron ▪ Methemoglobin reductase deficiency • Inherited disorder characterized by deficiency of the enzymes responsible maintaining or preventing formation of methemoglobin • Methemoglobin reductase is important in one of the shunts of RBC metabolism (methemoglobin reductase pathway) ▪ HbM • Hemoglobinopathy • Abnormal Hgb o >30% methemoglobin causes hypoxia o Reversible ▪ By administering reducing agents (Vit.C) Sulfhemoglobin (SHb) o aka Irreversible hemoglobin o Mixture of oxidizes, and partially denatured hemoglobin o Color of Blood: Mauve-Lavender, Greenish, Bluish o Causes: ▪ Prolonged constipation ▪ Exposure to certain drugs (sulfonamides [trimethoprim sulfamethoxazole], phenacetin, acetanilide, phenazopyridine) ▪ Enterogenous cyanosis ▪ Bacteremia (Clostridium perfringens, C. welchii) ▪ In vitro: addition of H2S to hemoglobin forming greenish pigment Carboxyhemoglobin (HbCO) o Hemoglobin with Fe2+ bound to CO ▪ Cannot bind oxygen due to a CO in place of O2 o Carbon monoxide (CO): ▪ Silent killer (CO is colorless and odorless) ▪ Seen in automobile exhaust, industrial pollutants (coal, gas, charcoal burning, tobacco smoke) ▪ 200-240x more affinity to hemoglobin than O2 ▪ 10,000x slower release from hgb molecule o Toxic levels: ▪ 10-15% - headache, dizziness ▪ >50% - coma and convultions o Color of blood: Cherry-Red ▪ Cherry-red is seen after shaking o Reversible ▪ By exposing patient with oxygen HEMOGLOBIN ONTOGENY HEMOGLOBIN Portland Gower I Gower II Hemoglobin F (HbF) Hemoglobin A1 Hemoglobin A2 • • • • GLOBIN PROPORTION CHAINS IN NEWBORN 2 zeta, 2 gamma 0 2 epsilon, 2 zeta 0 2 alpha, 2 epsilon 0 2 alpha, 80% 2 gamma 2 alpha, 2 beta 20% 2 alpha, 2 delta 0.5% INTRAVASCULAR CATABOLISM PROPORTION IN ADULTS (>1 Y.O.) 0 0 0 • • Alternate pathway Occurs in blood vessels • • Haptoglobin - transporter of free hgb Lack of haptoglobin cause globin chains to be filtered by the kidneys o Reabsorbed and converted to hemosiderin (insoluble form of stored iron) which excreted through the urine o If renal uptake of renal tubular cells are reached, hemoglobin in urine will be present <1% 97% 2.5% Ontogeny - development from fetal stage to adult Embryonic Hgb - Portland, Gower I & II HbF - major Hgb in fetal stage & newborn F cells - RBCs with HbF GLYCOSYLATED HEMOGLOBIN (HbA1c) • • • • aka glycated hemoglobin, glycohemoglobin 4-6% of circulation HbA1 HbA1 w/ glucose attached to N-terminal valine of Beta-chain. Index of metabolic control of diabetes during the preceding 2 to 3 months DEGRADATION OF HEMOGLOBIN • Two Mechanisms: o Extravascular catabolism o Intravascular catabolism HEMOGLOBINOPATHY • • EXTRAVASCULAR CATABOLISM • • • • • Occur in spleen, liver, kidneys Hemoglobin are dissociated to iron, globin, protoporphyrin Iron - Transported to bone marrow forrecycling (transferrin) Globin - Catabolized into the liver Protoporphyrin o Catabolized to bilirubin to urobilinogen o Excreted by the kidneys Qualitative defect of globin chain Major Groups: o Alpha Hemoglobinopathy – 2nd most common o Beta Hemoglobinopathy – most common o Gamma Hemoglobinopathy o Delta Hemoglobinopathy ß-HEMOGLOBINOPATHIES • • • • • • Hemoglobin S (HbS): α2ß26GLU→VAL o Most common and most severe abnormal hemoglobin o Substitution of Glutamic acid at 6 position is to Valine o Homozygous state (Hb SS)– sickle cell anemia o Heterozygous state (Hb AS)– sickle cell trait (resistant to P. falcifarum) o Common among Africans or African descent o Causes sickling of RBC (sickle cells/trepanocytes) under conditions of reduced O2 Hemoglobin C (HbC): α2ß26GLU→LYS o 2nd most common abnormal hemoglobin o Common among west African or west African descent o Presence of hemoglobin C crystal (Washington monument) Hemoglobin E (HbE): α2ß226GLU→LYS o 3rd most common o Common among southeast Asian (thai) o Most common hemoglobinopathy in the Philippines Hemoglobin D (HbD): α2ß226GLU→GLN Hemoglobin G (HbG): α2ß268ASP→LYS Hemoglobin H (HbH): ß4 o 4 beta-globin chains o Denatured Hgb o Pitted golf ball appearance, Raspberry appearance • Hemoglobin Bart’s: γ4 o Cause hydropsphetalis resulting to still birth or premature birth which may lead to fetal death o Due to high affinity to oxygen causing failure of Hgb to release oxygen during oxygenation of tissues EXAMINATION OF PLASMA OR SERUM FOR FAIRLEY’S PIGMENT • HEMOGLOBIN MEASUREMENT CYANMETHEMOGLOBIN METHOD • • • • • DETERMINATION OF FETAL HEMOGLOBIN reference method for hemoglobin assay Principle: Drabkin’s Reagent: o Potassium ferricyanide - active ingredient o Potassium cyanide - active ingredient o Non-ionic detergent – improves RBC lysis o Dihydrogen potassium phosphate Absorbance of cyanmethemoglobin at 540 nm is directly proportional to the hemoglobin concentration Many instruments now use sodium lauryl sulfate (SLS) to convert hemoglobin to SLS-methemoglobin SHAKING • • • • Used to observed changes in color of blood Whole blood is shaken in the air for 15 mins. The color formed are as follows: o Bright red – HbO2 o Cherry red – HbCO o Chocolate brown – Hi o Mauve lavender – SHb Katayama’s Test – method for HbCO o Blood + Ammonium sulfide----- (+) Rose red (-) Greenish brown Principle: Alkali Denaturation Test HbF resists alkali denaturation while HbA does not Normal Value Adults: 0.5 – 8% 1 year old: 1% • Acid Elution Test Resists acid elution, thus appear as isolated darkly – staining cell among a background of pale staining ghost cells 1-5% of rbc contain residual HbF Ghost cells - dehemoglobinized cells HEMOGLOBIN ELECTROPHORESIS • • • Principle: o Hgb molecules in an alkaline solution have a net negative charge and move toward the anode in an electrophoretic system Note: Electrophoretic mobility greater than HbA at pH 8.6 are known as fast hemoglobin – Hb Bart’s, Hb H, Hb I. o Slowest = Hgb C Mediums Cellulose Acetate Agar pH 8.4 - 8.6 Screening procedure Buffer: Tris EDTA Boric Acid Stain: Ponceau S SPECTROSCOPIC METHOD • Schumm’s test o Principle: Plasma or serum is layered with ether and mixed with saturated yellow ammonium sulfide o Result: Positive: narrow band is seen in green filter paper at 558nm Citrate Agar pH 6.0 - 6.2 Confirmatory test qualitative, simple direct vision, spectroscope is used SPECTROPHOTOMETRIC METHOD • • • • HbCO – 555 nm Hi – 630 nm SHb – 630 nm Sahli’s Acid Hematin Method 1. Place N/10 HCl into Hb tube up to 2 grams. 2. Blood sample in Sahli’s Hb piptette up to 20 uL. 3. Add blood sample to acid solution. 4. Mix with a stirrer. 5. Allow to stand for 10 minutes. 6. Add distilled water drop by drop till the color of the solution matches to brown glass standard. 7. Take the reading of the lower meniscus from the graduated tube in grams. o Uses comparator block o Empty tube in the middle Note: If S band is present, Solubility Test or Sickling test (Dithionate tube test, Sodium Metabisulfite test) must be performed • Factors: 1. The direction and rate of migration is dependent on Isoelectric point of Hgb, and pH of buffer 2. Migration is also dependent on Ionic strength of buffer SPECIFIC GRAVITY METHOD • • • • • ENTEROCYTES aka Copper sulfate method o only reagent used is copper sulfate S.G. 1.053 = 12.5 g/dL of Hgb Used for screening of blood donors >12.5 g/dL Hgb – drop of blood sinks <12.5 g/dL Hgb – drop of blood floats GASOMETRIC METHOD (OXYGEN CAPACITY METHOD) • • • Principle: Hgb will combine and liberate a fixed quantity of Oxygen. The blood is hemolyzed with saponin and the gas is collected and measured in a van slyke apparatus Oxygen combining capacity of blood. 1.34ml O2 /g of Hgb Formula MECHANISM • • MEASUREMENT OF HEMOGLOBIN BREAKDOWN • • • • Estimation of Exhaled carbon monoxide Estimation of Hemoglobin carbon monoxide Estimation of fecal urobilinogen IRON METABOLISM IRON • • • • Cationic bivalent [Ferrous (Fe2+)], trivalent [Ferric (Fe3+)] 70% of total body iron is transported in ferrous state to heme portion of hemoglobin Hepcidin – master regulator hormone of systemic iron metabolism Main processes (AT-USE) o Absorption o Transport o Utilization o Storage o Elimination ABSORPTION • • • • • Normal Daily Diet: 15mg of iron Only 1-2 mg are absorbed Duodenum and upper jejunum - site of maximum Fe absorption Fe3+ should be reduced to ferrous state (vitamin C or ferric reductase) o Enzyme found in enterocytes in intestines that convert ferric iron to ferrous iron Two forms of Absorbed Iron: o Heme Iron – meat, readily absorbable ▪ Consumable meat containing myoglobin ▪ Organic ferrous state - incorporated with hemoglobin/myoglobin ▪ In Ferrous state (Fe2+) o Non-Heme Iron – inorganic Fe ▪ Acquired from green leafy vegetables ▪ Usually on Ferric state (Fe3+) • • • • • • • • • • • • Heme iron are transported the enterocyte by the heme carrier protein 1 (heme transporter) Once heme iron is inside the enterocyte, the heme will be degraded by Heme oxygenase into three parts: ferrous iron, CO, Bilirubin IX Non-heme iron is first converted into ferrous state by the action of duodenum-specific cytochrome B-like protein (DCYTB) o To maximize the conversion of ferric to ferrous by DCYTB, they are chelated by ascorbic acids, citrates, other organic acids and amino acids o The chelation of ferric iron in the intestinal lumen will result in better absorption of ferric iron Once converted into ferrous state, it can now enter the enterocyte through the DMT1 Ferrous iron from heme iron can be transported out from enterocyte to the circulation o The exit is facilitated by heme oxygenase Ferrous iron from nonheme iron will be exported by DMT1 Both heme and nonheme irons are exported out of the cell into circulation through the Ferroportin 1 o Ferroportin 1 is facilitated by the action of Hephaestin which will reoxidize the ferrous iron to ferric iron o Only ferric state of iron can be bound to transferrin o Transferrin is the plasma Fe transporter; transport Fe through the circulation to the cells requiring iron (liver, erythroid marrow) Once transferrin has reached the cells needing iron, the cells will consume transferrin with iron by process of endocytosis (engulfing iron) Once plasma transferrin with iron goes to the liver, it will activate the action of hepcidin Hepcidin is a negative regulator of instestinal absorption of iron o It will bind to ferroportin causing destruction of ferroportin molecule o This will prevent exit/transport of iron, Body only absorbs iron it needs The iron trapped inside are stored in the form of ferritin (storage form of iron) Ferritin has two fates: excreted or absorbed If the body needs iron, ferritin can reenter the circulation Unused iron will be excreted by shedding of epithelial cells through defecation IMPORTANT SUBSTANCES • • • • • • • • • • Heme Carrier Protein 1 – internalize heme iron to the enterocyte o Found in duodenal enterocyte membrane Heme oxygenase – degrades heme (enterocytes) o Result to Fe2+, CO Bilirubin IX Ascorbic acid, citrates, amino acids, other organic acids - chelates nonheme iron for efficient conversion Duodenum-Specific Cytochrome B-like Protein (DCYTB) o Converts non-heme iron (Fe3+) to Fe2+ Divalent Metal Transporter 1 (DMT1) o Transport Fe2+ across the enterocyte to the portal circulation Ferroportin – a basolateral protein that mediates transport of Fe2+ out of the enterocyte. Hephaestin – a copper-containing iron oxidase that facilitates egress of Fe2+ by reoxidation to ferric state o Ferric state - can be bound to transferrin (transport protein of iron) o Transferrin - transfer iron to cells Hepcidin – an antimicrobial peptide (liver) that binds to ferroportin and degrades it. o Negative regulator of the intestinal iron absorption Ferritin – soluble form of stored iron o If needed, it will be absorbed o If not needed. It will be excreted by desquamation (shedding of skin through defecation) Enterocytes - cells lining the intestine TRANSPORT • • • Transferrin – transport protein of Iron o Only 1/3 of transferrin iron-binding sites are occupied by Fe3+ o 2/3 do not carry iron (serum UIBC) ▪ UIBC - Unbound Iron binding Capacity o Total Iron Binding Capacity (TIBC) – total iron-binding sites of transferrin Transports iron from the enterocytes to transferrin receptors of the marrow normoblasts. Transferrin receptors – glycoprotein dimer located virtually on all cells except RBC o Provides transferrin-bound iron access into normoblasts. o Can bind two transferrin molecule • • • • ELIMINATION • • • • • Mitochondrial Iron – incorporated rapidly into protoporphyrin to form heme o Ferrochelatase - enzyme that bind ferrous iron into the rings of protoporphyrin Iron from senescent RBCs is turned over to macrophage and reused. (after 120 days) STORAGE • • Two Forms: Ferritin o Soluble form of storage iron o Can be measured in serum o Apoferritin – protein component of ferritin without the iron Average daily loss of iron: 1-2 mg (sweat, urine, feces) Defecation: 1mg/day o Majority of eliminated iron Lactation & Menstruation: 1mg/day o Increased iron loss among women Perspiration, Skin exfoliation: minimal loss LABORATORY TESTS FOR IRON METABOLISM SERUM/FERRIC IRON • • • • • • Used for differential diagnosis of disorders of iron metabolism. Measures of Fe2+ bound to serum transferrin Exhibits diurnal variation o Highest in the morning o Lowest in the evening (25% lower) Sample: o Non-hemolyzed serum o Obtained in the morning (12 hr fasting) Reference range: 50-150 ug/dL (10-30 umol/L) Decreased: Iron deficiency anemia, inflammatory disorders, acute infection, after immunization, post-AMI TOTAL IRON BINDING CAPACITY (TIBC) • • • • UTILIZATION • Hemosiderin o Insoluble form of storage iron o Partially degraded or precipitated form of ferritin Both forms are found in liver, bone marrow, spleen Liver - majority of ferritin and hemosiderin is located When the body needs iron, stored iron returns to transferrin which will be transported to the cells requiring iron Indirectly measures the concentration of transferrin by measuring its ability to bind iron o Transferrin - 1/3 bound to iron; 2/3 UIBC Measures UIBC (chromogen spectrophotometry) and TIBC (immunoassay) o Eg. UIBC = 18; TIBC = 21 Transferrin ability = TIBC - UIBC = 21-18 = 3 Sample: o Non-hemolyzed serum o 12 hr fasting (regardless in morning/evening) Reference value: 250-540 ug/dL (47-70 umol/L) o Increased: Iron Deficiency Anemia o Decreased: Anemia of Chronic disease PERCENT SATURATION • • • Transferrin saturation Percentage of sites available for carrying iron Formula: • Reference Value: o Male: 16-60% o Female: 16-50% SERUM FERRITIN • • • • • • • Acute phase reactant (positive) o Protein level affected after inflammation Measure of body’s tissue iron stores Good indicator of iron storage status o Generally, first lab test that declines following iron deficiency Plasma ferritin = ferritin body stores Useful in diagnosing iron deficiency. Method: Radioimmunoassay Reference Value: o Men: 15-200 ug/L o Female: 12-150 ug/L o Increased: Thalassemia, Anemia of Chronic Disease, Sideroblastic Anemia o Decreased: Iron Deficiency Anemia FREE ERYTHROCYTE PROTOPORPHYRIN • • • • Intermediate product of hemoglobin synthesis in the RBC. Method: hematofluorometry Principle: Protoporphyrin without iron will bind to zinc to form fluorescent zinc protoporphyrin which is measured using hematofluorometer. o RBC produces slightly more protoporphyrin than necessary Reference Value: <80 ug/dL o Increased: heme synthesis disorder, iron deficiency anemia, lead poisoning (plumbism), sideroblastic anemia PRUSSIAN BLUE STAIN • • • • • • • Prussian blue - supravital stain o Stains iron blue; blue inclusions in cells Visual estimation of iron in macrophage, nucleated RBC, and reticulocytes. RBC & Retics in peripheral blood should not have visible iron o RBC lack transferrin receptors Bone Marrow: 20-80% nRBCs have visible iron (1-3 inclusions) Sideroblast – nRBC with iron (granules) o Found in bone marrow o Can be normal Siderosomes – granules found in sideroblast Siderocytes – reticulocytes in bone marrow with iron o Presence of siderocyte indicate abnormality o Normal iron in bone marrow should not be visible under light microscope