HEMA Chapter 7 Hgb Metabolism

Hemoglobin — basic composition

A globular tetramer of two pairs of unlike globin polypeptide chains and four heme groups; each globin chain contains one heme.

Hemoglobin — major functions

Transports O2 from lungs to tissues, CO2 from tissues to lungs, contributes to acid-base balance by binding/releasing H+, and transports nitric oxide.

Heme

Protoporphyrin IX with a central divalent ferrous iron atom (Fe2+).

Ferrous vs ferric iron in hemoglobin

Fe2+ in heme reversibly binds O2; oxidation to Fe3+ produces methemoglobin, whose iron cannot bind O2.

Hemoglobin oxygen capacity

Each of four heme iron atoms can reversibly bind one O2 molecule; one tetramer can carry up to four O2 molecules.

Hemoglobin concentration inside RBCs

Approximately 34 g/dL.

Hemoglobin molecular weight

Approximately 64,000 Daltons.

Globin chain lengths

Alpha and zeta: 141 amino acids; beta, gamma, delta, and epsilon: 146 amino acids; theta is listed as unknown.

Globin chain secondary structure

Each globin chain has eight helices separated by seven nonhelical segments.

Heme pocket

Heme is suspended in a pocket between the E and F helices of a globin chain.

F8 proximal histidine

The F8 proximal histidine forms the bond linking the globin chain to the heme iron.

E7 distal histidine

The E7 distal histidine is close to oxygen on the opposite side of the heme plane but is not directly linked to oxygen.

Primary structure of hemoglobin

The amino acid sequence of the globin polypeptide chains.

Secondary structure of hemoglobin

The arrangement of globin-chain segments into helices and nonhelical segments.

Tertiary structure of hemoglobin

The three-dimensional arrangement of the helices into the characteristic pretzel-like configuration.

Quaternary structure of hemoglobin

The complete tetrameric hemoglobin molecule formed by association of four globin chains.

Adult Hb A

Hb A is alpha2 beta2 and is the predominant adult hemoglobin.

Hb A subunit interactions

Strong alpha1-beta1 and alpha2-beta2 bonds stabilize dimers; alpha1-beta2 and alpha2-beta1 contacts are important in oxygenated/deoxygenated conformational changes.

Hb A1c

A glycated form of Hb A produced by nonenzymatic attachment of glucose to the N-terminal valine of the beta chain.

Normal Hb A1c in the chapter

Approximately 4%–6% of circulating Hb A is Hb A1c.

Hb A1c and glucose

In uncontrolled diabetes, Hb A1c increases in proportion to mean blood glucose over the preceding 2–3 months.

Heme biosynthesis location

Occurs in mitochondria and cytoplasm of bone marrow erythroid precursors from pronormoblast through circulating polychromatic erythrocyte.

Hemoglobin synthesis in mature RBCs

Mature RBCs cannot synthesize hemoglobin because they lose ribosomes and mitochondria.

First step of heme synthesis

In mitochondria, glycine and succinyl-CoA condense to form ALA, catalyzed by aminolevulinate synthase (ALA synthase).

ALA dehydratase

In the cytoplasm, ALA dehydratase converts ALA to porphobilinogen (PBG).

Cytoplasmic heme pathway

PBG undergoes transformations through hydroxymethylbilane, uroporphyrinogen III, and coproporphyrinogen III.

Final steps of heme synthesis

Protoporphyrinogen IX is converted to protoporphyrin IX in mitochondria; ferrochelatase adds Fe2+ to form heme.

Ferrochelatase

Heme synthase; the enzyme that adds Fe2+ to protoporphyrin IX to form heme.

Iron delivery to erythroid cells

Transferrin carries Fe3+ to erythroid precursors; iron is released in the endosome and transported to mitochondria, where it is reduced to Fe2+ for heme synthesis.

Hemoglobin assembly

Each globin chain binds heme, then forms a heterodimer with a nonidentical globin chain; two heterodimers combine to form the tetramer.

Alpha and zeta globin genes

Located on the short arm of chromosome 16.

Epsilon, gamma, delta, beta globin genes

Located as a cluster on the short arm of chromosome 11.

Globin gene copy numbers

There are two copies per diploid cell for most globin genes; alpha and gamma have four copies per diploid cell because there are two copies per chromatid.

Globin transcription vs translation

Transcription to mRNA occurs in the nucleus; translation of globin mRNA occurs on cytoplasmic ribosomes.

Balanced alpha and beta production

Although alpha genes produce more mRNA, alpha mRNA is translated less efficiently, so alpha and beta chains are produced in approximately equal amounts.

Hb A2

Hb A2 is alpha2 delta2 and is less than 3.5% of adult hemoglobin in the chapter.

Hb F

Hb F is alpha2 gamma2 and is the predominant fetal and newborn hemoglobin; healthy adults have about 1%–2%.

Embryonic hemoglobins

Gower-1 = zeta2 epsilon2; Gower-2 = alpha2 epsilon2; Portland = zeta2 gamma2.

Hb F at birth

The chapter gives approximately 60%–90% Hb F at birth, with Hb A about 10%–40%.

Adult hemoglobin distribution

From about two years through adulthood: Hb A >95%, Hb A2 <3.5%, and Hb F 1%–2%.

Hemoglobin switching

The gamma-to-beta switch begins in late fetal life and continues through adulthood, replacing most Hb F with Hb A.

KLF1

Krüppel-like factor 1; a key transcription factor involved in beta-globin activation and gamma-to-beta switching.

Other globin transcription factors

The chapter identifies GATA1, Ikaros, TAL1, p45-NF-E2, and LDB1 as additional regulators.

Locus control region (LCR)

An enhancer region more than 20 kb upstream that participates in globin gene activation through an active chromosome hub with promoters and transcription factors.

Translation-level globin regulation by heme

Heme promotes activation of a translation initiation factor and inactivates its repressor; low heme allows the repressor to block globin translation.

Rate-limiting step of heme synthesis

The initial glycine + succinyl-CoA reaction forming ALA, catalyzed by ALA synthase.

Heme negative feedback

Heme inhibits ALA synthase transcription and also inhibits ALA dehydratase and PBG deaminase; increased heme demand induces more ALA synthase.

Balanced globin and heme production

Balance is necessary because excess globin chains, protoporphyrin IX, or iron can accumulate and damage the cell.

Systemic regulation of erythropoiesis

Tissue hypoxia is detected by renal peritubular cells, which increase EPO; EPO increases RBC production and synthesis of RBC components including hemoglobin.

Hemoglobin reference interval — men

13.5–18.0 g/dL in the general reference interval given by the chapter.

Hemoglobin reference interval — women

12.0–15.0 g/dL in the general reference interval given by the chapter.

Hemoglobin reference interval — newborns

16.5–21.5 g/dL in the general reference interval given by the chapter.

High-altitude hemoglobin

People living at high altitude generally have slightly higher hemoglobin as compensation for oxygen-thin air.

Hemoglobin-oxygen dissociation curve

Plots percent hemoglobin oxygen saturation against PO2.

Why the oxygen dissociation curve is sigmoid

Cooperativity among hemoglobin subunits causes each bound O2 molecule to increase the oxygen affinity of remaining heme sites.

P50

The PO2 required for 50% oxygen saturation of hemoglobin.

Normal P50

Approximately 27 mm Hg.

Left shift

Indicates increased oxygen affinity and decreased P50; 50% saturation occurs below about 27 mm Hg.

Right shift

Indicates decreased oxygen affinity and increased P50; 50% saturation occurs above about 27 mm Hg.

Left-shift causes

Decreased 2,3-BPG, decreased H+/raised pH, decreased PCO2, decreased temperature, Hb F, or high-affinity hemoglobin variants.

Right-shift causes

Increased 2,3-BPG, increased H+/lowered pH, increased PCO2, increased temperature, or low-affinity hemoglobin variants.

Bohr effect

The effect of pH/H+ on hemoglobin oxygen affinity; lower pH shifts the curve right and promotes O2 release.

2,3-BPG

Binds between beta-globin chains of deoxygenated hemoglobin, stabilizes the tense T form, shifts the curve right, and promotes oxygen release.

T state

The tense, deoxygenated hemoglobin conformation with low oxygen affinity; stabilized by 2,3-BPG.

R state

The relaxed, oxygenated hemoglobin conformation with higher oxygen affinity.

T-to-R transition

Oxygen binding changes hemoglobin conformation, disrupts salt bridges, releases 2,3-BPG, and causes about a 15-degree rotation of the alpha1-beta1 and alpha2-beta2 dimers.

Oxygen loading vs unloading

High PO2 in lungs favors high affinity and oxygen loading; lower PO2 in tissues favors lower affinity and oxygen release.

Oxygen bound per gram hemoglobin

Approximately 1.34 mL O2 per gram of hemoglobin.

Arterial oxygen saturation reference interval

Approximately 96%–100%.

Myoglobin

A 17,000-Dalton monomeric oxygen-binding heme protein in cardiac and skeletal muscle with greater oxygen affinity than hemoglobin.

Myoglobin vs hemoglobin curves

Myoglobin has a hyperbolic, high-affinity curve; hemoglobin has a sigmoid curve due to cooperative subunit binding.

Hb F oxygen affinity

Hb F has a P50 of 19–21 mm Hg, causing a left shift and greater oxygen affinity than Hb A.

Why Hb F binds oxygen more strongly

Hb F has weakened 2,3-BPG binding due to an amino acid difference at a critical 2,3-BPG binding site.

Fetal advantage of Hb F

High oxygen affinity allows Hb F to withdraw oxygen more effectively from maternal circulation.

Why newborn RBC/Hb are high

Hb F delivers oxygen less readily to tissues, so fetal/newborn erythropoiesis produces more RBCs; RBC count, Hb, and hematocrit are higher than adult values and decline toward adult levels by about 6 months.

CO2 transport in tissues

CO2 enters RBCs and combines with water to form H2CO3, catalyzed by carbonic anhydrase; H2CO3 dissociates into H+ and HCO3−.

Bohr effect during CO2 transport

H+ binds oxyhemoglobin, promoting O2 release so O2 can diffuse into tissues.

Chloride shift

As HCO3− leaves the RBC for plasma, Cl− enters the RBC to maintain electroneutrality.

CO2 transport by hemoglobin

The chapter states about 85% of tissue CO2 is transported by hemoglobin as H+; some remains in cytoplasm and the remainder binds globin as a carbamino group.

CO2 transport in lungs

O2 binds deoxygenated hemoglobin; H+ is released, combines with HCO3− to form H2CO3, which becomes CO2 and H2O; CO2 diffuses out and is exhaled.

Nitric oxide transport

Hemoglobin binds, inactivates, and transports nitric oxide, a regulator of vascular tone.

S-nitrosohemoglobin

A form produced when nitric oxide binds to cysteine in the beta-globin chain.

Nitric oxide and hypoxic vasodilation

The chapter describes a proposed mechanism in which hemoglobin releases NO in hypoxic microvascular areas, promoting vasodilation; the theory is not universally accepted.

Dyshemoglobins

Dysfunctional hemoglobins unable to transport oxygen; the chapter discusses methemoglobin, sulfhemoglobin, and carboxyhemoglobin.

Methemoglobin

Hemoglobin with heme iron oxidized to Fe3+; ferric iron cannot bind oxygen.

Normal methemoglobin level

Reduction systems normally limit methemoglobin to about 1% of total hemoglobin despite continuous small-scale formation.

Methemoglobin reduction pathway

Predominantly NADH-cytochrome b5 reductase 3 (NADH-methemoglobin reductase).

Methemoglobinemia severity

Less than 25% is generally asymptomatic; above 30% may cause cyanosis and hypoxia symptoms; above 50% can lead to coma and death.

Acquired methemoglobinemia

Toxic methemoglobinemia from exogenous oxidants such as nitrites, primaquine, dapsone, or benzocaine.

Methylene blue treatment

At methemoglobin levels of about 30% or more, IV methylene blue is described as reducing ferric iron to ferrous iron through an NADPH-dependent pathway.

Hereditary methemoglobinemia — CYB5R3

CYB5R3 mutations reduce the ability to reduce methemoglobin; cytochrome b5 reductase deficiency is autosomal recessive.

Hb M

Inherited abnormal hemoglobins caused by alpha-, beta-, or gamma-globin mutations that favor the ferric state; the chapter describes Hb M as autosomal dominant.

Methemoglobin spectral measurement

Methemoglobin has an absorption peak around 630 nm; high levels produce chocolate-brown blood that does not return to normal red after oxygen exposure.

Sulfhemoglobin

An irreversible abnormal hemoglobin formed by adding sulfur to the pyrrole ring of heme; greenish pigment, ineffective for O2 transport, and persists for the life of the RBC.

Sulfhemoglobin causes

Can result from sulfanilamides, phenacetin, nitrites, phenylhydrazine, or sulfur-containing industrial/environmental exposure.

Sulfhemoglobin treatment

Avoid the offending agent; sulfhemoglobin cannot be converted back to normal Hb A and persists for the life of the cell.

Sulfhemoglobin vs methemoglobin

Sulfhemoglobin has a similar spectral peak to methemoglobin, but its spectral curve does not shift when cyanide is added.

Carboxyhemoglobin

Hemoglobin formed by combination of carbon monoxide with heme iron.

CO affinity for hemoglobin

Carbon monoxide has approximately 240 times the affinity for hemoglobin compared with oxygen.

Carboxyhemoglobin effect

CO binding shifts the O2 dissociation curve left, increasing remaining oxygen affinity and severely impairing oxygen release to tissues.