The Role of 2,3-Diphophoglycerate

The Role of 2,3-Diphophoglycerate

     The major function of the hemoglobin molecule is the transport of oxygen to the tissues. The oxygen affinity of the hemoglobin molecule is associated with the spatial rearrangement of the molecule and is regulated by the concentration of phosphates, particularly 2,3-DPG in the erythrocyte. The manner in which 2,3-DPG binding to reduced hemoglobin (deoxyhemoglobin) affects oxygen affinity is complex. Basically, 2,3-DPG combines with the beta chains of deoxyhemoglobin and diminishes the molecule’s affinity for oxygen. 

    When the individual heme groups unload oxygen in the tissues, the beta chains are pulled apart. This permits the entrance of 2,3-DPG and the establishment of salt bridges between the individual chains. These activities result in a progressively lower affinity of the molecule for oxygen. With oxygen uptake in the lungs, the salt bonds are sequentially broken; the beta chains are pulled together, expelling 2,3-DPG; and the affinity of the hemoglobin molecule for oxygen progressively increases.

     In cases of tissue hypoxia, oxygen moves from hemoglobin into the tissues, and the amount of deoxyhemoglobin in the erythrocytes increases. This produces the binding of more 2,3-DPG, which further reduces the oxygen affinity of the hemoglobin molecule. If hypoxia persists, depletion of free 2,3-DPG leads to increased production of more 2,3-DPG and a persistently lowered affinity of the hemoglobin molecule for oxygen.

Figure: Hemoglobin molecular changes

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Alterations in Myeloid:Erythroid Ratio

     

     The M:E ratio is sensitive to hematologic factors that may impair red blood cell life span, inhibit overall production, or cause dramatic increases in a particular cell line. Each of these conditions reflects bone marrow dynamics through alterations of the M:E ratio. Many observations in the peripheral smear can be traced back to the pathophysiologic events at the level of bone marrow. A perfect example of this is the response of the bone marrow to anemia. As anemia develops and becomes more severe, the patient becomes symptomatic, and the kidney senses hypoxia secondary to a decreased hemoglobin level. Tissue hypoxia stimulates an increased release of erythropoietin (EPO), a red blood cell-stimulating hormone, from the kidney. EPO travels through the circulation and binds with a receptor on the youngest of bone marrow precursor cells, the pronormoblast. Bone marrow has the capacity to expand production 6-8 times in response to an anemic event. Consequently, the bone marrow delivers reticulocytes and nucleated red blood cells to the peripheral circulation prematurely if the kidney senses hypoxic stress. What is observed in the peripheral blood smear is polychromasia (stress reticulocytes, large polychromatophilic red blood cells) and nucleated red blood cells. Both of these cell types indicate that the bone marrow is regenerating in response to an event, a dynamic that represents the harmony between bone marrow and peripheral circulation.

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Bone Marrow & Myeloid:Erythroid Ratio

     

     The bone marrow is one of the largest organs of the body, encompassing 3% to 6% of body weight and weighing 1500 g in an adult. It is hard to conceptualize the bone marrow as an organ because it is not a solid organ that one can touch, measure, or weigh easily. Because bone marrow tissue is spread throughout the body, one can visualize it only in that context. It is
composed of yellow marrow, red marrow, and an intricate supply of nutrients and blood vessels. Within this structure are erythroid cells (red blood cells), myeloid cells (white blood cells), and megakaryocytes (platelets) in various stages of maturation, along with osteoclasts, stroma, and fatty tissue. Mature cells enter the peripheral circulation via the bone marrow sinuses, a central structure lined with endothelial cells that provide passage for mature cells from extravascular sites to the circulation. The cause and effect of hematologic disease are usually rooted in the bone marrow, the central factory for production of all adult hematopoietic cells. In the first 18 years of life, bone marrow is spread throughout all of the major bones of the skeleton, especially the long bones. As the body develops, the marrow is gradually replaced by fat until the prime locations for bone marrow in an adult become the iliac crest (located in the pelvic area) and the sternum (located in the chest area).
     In terms of cellularity, there is a unique ratio in the bone marrow termed the myeloid:erythroid (M:E) ratio. This numerical designation provides an approximation of the myeloid elements in the marrow and their precursor cells and the erythroid elements in the marrow and their precursor cells. The normal ratio of 3:1 to 4:1 reflects the relationship between production and life span of the various cell types. White blood cells have a much shorter life span than red blood cells (6 to 10 hours for neutrophils as opposed to 120 days for erythrocytes) and must be produced at a much higher rate for normal hematopoiesis.

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Iron Distribution

Iron-containing compounds in the body are one of two types:

1. Functional compounds that serve in metabolic (hemoglobin, myoglobin, iron-responsive element-binding protein) or enzymatic (cytochromes, cytochrome oxygenase, catalase, peroxidase) functions
2. Compounds that serve as transport (transferrin, transferrin receptor) or storage forms (ferritin and hemosiderin) for iron. 

     A poorly understood iron compartment is the intracellular “labile pool.” Iron leaves the plasma and enters the intracellular fluid compartment for a brief time before it is incorporated into cellular components (heme or enzymes) or storage compounds. This labile pool is believed to be the chelatable iron pool. The total iron concentration in the body is 40–50 mg of iron/kg of body weight. Men have higher amounts than women.

    Iron is found primarily in erythrocytes, macrophages, hepatocytes, and enterocytes (absorptive cells at the luminal [apical] surface of the duodenum). Hemoglobin constitutes the major fraction of body iron (functional iron) with a concentration of 1 gm iron/kg of erythrocytes, or about 1 mg iron/mL erythrocytes. Iron in hemoglobin remains in the erythrocyte until the cell is removed from the circulation. Hemoglobin released from the erythrocyte is then degraded in the macrophages of the spleen and liver, releasing iron. Approximately 85% of this iron from degraded hemoglobin is promptly recycled from the macrophage to the plasma where it is bound to the transport protein, transferrin, and delivered to developing normoblasts in the bone marrow for heme synthesis. The macrophages recycle 10 to 20 times more iron than is absorbed in the gut. This iron recycling provides most of the marrow’s daily iron requirement for erythropoiesis.

      Iron in hepatocytes and intestinal enterocytes is stored and utilized as needed to maintain iron homeostasis. The hepatocytes store iron that can be released and utilized when the amount of iron in the plasma is not sufficient to support erythropoiesis. Enterocytes that absorb dietary iron can either export it to the plasma or store it. Iron stored in enterocytes is lost when the cells are sloughed into the intestine. 

 

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Erythrocyte

Erythrocyte (Mature Erythrocyte)

• Bone marrow : 0 %
• Peripheral blood : Predominant cell type
• Size : 7-8 μm
• No nucleus
• No nucleoli
• No chromatin 
• No N/C ratio 
• Cytoplasm: Salmon with central pallor of about one-third of the diameter of the cell

NOTE: The mature erythrocyte has lost the blue-gray color and is salmon colored as hemoglobinization is complete. 

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Multiple Myeloma

     Multiple myeloma (myelomatosis) is a neoplastic disease characterized by plasma cell  accumulation in the bone marrow, the presence of monoclonal protein in the serum and/or urine and, in symptomatic patients, related tissue damage.

Download Multiple Myeloma lecture (ppt)

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Reticulocyte

Reticulocyte ( Polychromatic Erythrocyte / Diffusely Basophilic Erythrocyte )

• Size = 7-10 μm
• No nucleus (so there is no N/C ratio)
• No nucleoli
• No chromatin
• Cytoplasm: Color is slightly more blue/purple than the mature erythrocyte because there are RNA remnants.
• Bone marrow: 1 %
• Peripheral blood: 0.5 - 2.0 % 

NOTE:  When stained with supravital stain (e.g., new methylene blue), polychromatic erythrocytes appear as reticulocytes (contain precipitated ribosomal material)

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Orthochromic Normoblast

Orthochromic Normoblast ( Orthochromic Erythroblast / Metarubricyte )

• 1 - 4% of nucleated cells in BM
• Peripheral blood: 0 %
• Size = 10 -15 μm
• Low N/C ratio (1:2)
• Round nucleus
• No nucleoli
• Fully condensed chromatin 
• Cytoplasm: more pink or salmon than blue 

NOTE: The gray-blue color of the cytoplasm is becoming salmon as more hemoglobin is produced. 

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Polychromatophilic Normoblast

Polychromatophilic Normoblast ( Polychromatic Normoblast / Polychromatic Erythroblast / Rubricyte )

• 13-30% of nucleated cells in BM.
• Peripheral Blood : 0 % 
• Size = 12 - 15 μm
• Low N/C ratio (4:1)
• Eccentric nucleus
• No nucleoli 
• Chromatin irregular and coarsely clumped
• Cytoplasm: Gray-blue as a result of hemoglobinization 

NOTE: The blue color of the cytoplasm is becoming gray-blue as hemoglobin is produced. 

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Basophilic Normoblast

Basophilic Normoblast (Basophilic Erythroblast / Prorubricyte)

• 1-3% of nucleated cells in BM.
• Peripheral blood : 0 %
• Size = 16-18 μm
• Round to slightly oval nucleus
• Moderate N/C ratio (6:1) 
• Dark blue cytoplasm
• Indistinct nucleoli ( 0 - 1 nucleoli ) 
• Coarsening ( slightly condensed ) chromatin

 

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Pronormoblast

Pronormoblast (also known as Proerythroblast / Rubriblast) is the first microscopically recognizable cell in erythrocyte lineage.

• 1% of Nucleated Cells in BM.
• Peripheral Blood: 0%
• 1% of Nucleated Cells in BM.
• Size: 20-25 μm
• Nucleus: Round to slightly oval
• High N/C ratio (8:1)
• 1-3 faint nucleoli 
• Fine chromatin
• Cytoplasm: Dark blue; may have prominent Golgi

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Glycosylated Hemoglobin

     HbA_1C on chromatography is a minor component of normal adult hemoglobin (HbA) that has been modified posttranslationally (HbA₃ on starch block electrophoresis). A component usually has been added to the N terminus of the β-chain. The most important subgroup of HbA₁ is HbA_1C , which has glucose irreversibly attached. This hemoglobin is referred to as glycosylated hemoglobin. HbA_1C is produced throughout the erythrocyte’s life, its synthesis dependent on the concentration of blood glucose. Older erythrocytes typically contain more HbA_1C than younger erythrocytes having been exposed to plasma glucose for a longer period of time. However, if young cells are exposed to extremely high concentrations of glucose ( >400 mg/dL)  for several hours, the concentration of HbA_1C increases with both concentration and time of exposure.
     Measurement of is routinely used as an indicator of control of blood glucose levels in diabetics because it is proportional to the average blood glucose level over the previous two to three months. 

     Average levels of HbA_1C are 7.5% in diabetics and 3.5% in normal individuals.

 

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Structure of Hemoglobin

     Hemoglobin is the life-giving substance of every red blood cell, the oxygen-carrying component of the red blood cell. Each red blood cell is nothing more than a fluid-filled sac, with the fluid being hemoglobin. Every organ in the human body depends on oxygenation for growth and function, and this process is ultimately controlled by hemoglobin. In 4 months (120 days), red blood cells with normal hemoglobin content submit to the rigors of circulation. Red blood cells are stretched, twisted, pummeled, and squeezed as they make their way through the circulatory watershed.

The hemoglobin molecule consists of two primary structures:

1- Heme

This structure involves four iron atoms in the ferrous state (Fe²⁺ ), because iron in the ferric state (Fe³⁺ ) cannot bind oxygen, surrounded by protoporphyrin IX, or the porphyrin ring, a structure formed in the nucleated red blood cells. Protoporphyrin IX is the final product in the synthesis of the heme molecule. It results from the interaction of succinyl coenzyme A and delta-aminolevulinic acid in the mitochondria of the nucleated red blood cells. Several intermediate by-products are formed, including porphobilinogen, uroporphyrinogen, and coproporphyrin. When iron is incorporated, it combines with protoporphyrin to form the complete heme molecule. Defects in any of the intermediate products can impair hemoglobin function.
2- Globin

This structure consists of amino acids linked together to form a polypeptide chain, a bracelet of amino acids. The most predominant chains for adult hemoglobins are the alpha and beta chains. Alpha chains have 141 amino acids in a unique arrangement, and beta chains have 146 amino acids in a unique arrangement. The heme and globin portions of the hemoglobin molecule are linked together by chemical bonds.

2,3-Diphosphoglycerate (2,3-DPG)

2,3-DPG is a substance produced via the Embden-Meyerhof pathway during anaerobic glycolysis. This structure is intimately related to oxygen affinity of hemoglobin. As 2,3-DPG increased, the affinity of hemoglobin to oxygen is decreased. 

     Each hemoglobin molecule consists of four heme molecules with iron at the center and two pairs of globin chains. The heme structure sits lodged in the pocket of the globin chains. Hemoglobin begins to be synthesized at the polychromatic normoblast stage of red blood cell development. This synthesis is visualized by the change in cytoplasmic color from a deep blue to a lavender-tinged cytoplasmic color. Of hemoglobin, 65% is synthesized before the red blood cell nucleus is extruded, with an additional 35% synthesized by the reticulocyte stage. Normal mature red blood cells have a full complement of hemoglobin, which occupies a little less than one-half of the surface area of the red blood cell.

Figure: Hemoglobin molecule: note four heme molecules tucked inside globin chains. 

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