Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 11. Iron kinetics and laboratory assessment

Kathryn Doig*

OUTLINE

Iron Chemistry

Iron Kinetics

Systemic Body Iron Regulation

Iron Transport

Cellular Iron Absorption and Disposition

Iron Recycling

Dietary Iron, Bioavailability, and Demand

Laboratory Assessment of Body Iron Status

Serum Iron (SI)

Total Iron-Binding Capacity (TIBC)

Percent Transferrin Saturation

Prussian Blue Staining

Ferritin

Soluble Transferrin Receptor (sTfR)

Hemoglobin Content of Reticulocytes

Soluble Transferrin Receptor/Log Ferritin

Thomas Plot

Zinc Protoporphyrin

Objectives

After completion of this chapter, the reader will be able to:

1. Describe the essential metabolic processes in which iron participates.

2. State whether body iron is regulated by excretion or absorption.

3. Describe the compartments in which body iron is distributed, including the relative amounts in each site.

4. Trace a molecule of iron from its absorption through the enterocyte to transport into mitochondria and then recycling via macrophages, including the names of all proteins with which it interacts and that control its kinetics.

5. Name the ionic form and number of molecules of iron that bind to one molecule of apotransferrin.

6. Explain how hepcidin regulates body iron levels.

7. Explain how individual cells absorb iron.

8. Explain how individual cells regulate the amount of iron they absorb.

9. Describe the role of each of the following in the kinetics of iron:

a.Divalent metal transporter 1 (DMT1)

b.Ferroportin

c.Transferrin (Tf)

d.Transferrin receptor (TfR)

e.Hepcidin

10. For the proteins listed in objective 9, distinguish those that are involved in the regulation of iron within individual cells versus those involved in systemic body iron regulation.

11. Recognize the names of proteins involved in hepatocyte iron sensing and regulation of hepcidin production.

12. List factors that increase and decrease the bioavailability of iron.

13. Name foods high in iron, both heme-containing and ionic.

14. For each of the following assays, describe the principle of the assay and the iron compartment assessed:

a.Total serum iron (SI)

b.Total iron-binding capacity (TIBC)

c.Percent transferrin saturation

d.Serum ferritin

e.Soluble transferrin receptor (sTfR)

f.Measures of the hemoglobin content of reticulocytes

g.Prussian blue staining of tissues and cells

h.Zinc protoporphyrin (ZPP)

15. Plot given patient values on a Thomas plot and interpret the patient’s iron status.

16. Calculate the percent transferrin saturation when given total serum iron and TIBC.

17. When given reference intervals, interpret the results of each of the assays in objective 14 plus a Thomas plot and sTfR/log ferritin and recognize results consistent with decreased, normal, and increased iron status.

18. Identify instances in which sTfR, hemoglobin content of reticulocytes, sTfR/log ferritin, and Thomas plots may be needed to improve diagnosis of iron deficiency.

CASE STUDY

After studying the material in this chapter, the reader will be able to respond to the following case study:

In 1995, Garry, Koehler, and Simon assessed changes in stored iron in 16 female and 20 male regular blood donors aged 64 to 71.1 They measured hemoglobin, hematocrit, serum ferritin concentration, and % transferrin saturation in specimens from the donors, who gave an average of 15 units (approximately 485 mL/unit) of blood over 3.5 years. The investigators collected comparable data from nondonors. Of the donors, 10 women and 6 men took a dietary supplement providing approximately 20 mg of iron per day. In addition, mean dietary iron intake was 18 mg/day for the women and 20 mg/day for the men. Over the period of the study, mean iron stores in women donors decreased from 12.53 to 1.14 mg/kg of body weight. Mean iron stores in male donors declined from 12.45 to 1.92 mg/kg. Nondonors’ iron stores remained unchanged. Based on hemoglobin and hematocrit results, no donors became anemic. As iron stores decreased, the calculated iron absorption rose to 3.55 mg/day for the women and 4.10 mg/day for the men—more than double the normal rate for both women and men.

1. Why did the donors’ iron stores decrease?

2. Why did the donors’ iron absorption rate rise? Explain using the names of all proteins involved.

3. Name the laboratory test(s) performed in the study used to evaluate directly the iron storage compartment?

4. What is the diagnostic value of the % transferrin saturation? What iron compartment does it assess?

Among the metals that are required for metabolic processes, none is more important than iron. It is critical to energy production in all cells, being at the center of the cytochromes of mitochondria. Oxygen needed for energy production is carried attached to iron by the hemoglobin molecule in red blood cells. Iron is so critical to the body that there is no mechanism for active excretion, just minimal daily loss with exfoliated skin and hair and intestinal epithelia. Iron is even recycled to conserve as much as possible in the body. To insure against times when iron may be scarce in the diet, the body stores iron as well.

The largest percentage of body iron, nearly 65% of it, is held within hemoglobin in red blood cells of various stages () while about 25% of body iron is in storage, mostly within macrophages and hepatocytes.Table 11-12 The remaining 10% is divided among the muscles, the plasma, the cytochromes of cells, and various iron-containing enzymes within cells. A more functional approach to thinking about body iron distribution conceives of the iron as distributed in three compartments (Table 11-1). The functional compartment contains all iron that is functioning within cells. Though most of this is the iron in hemoglobin, the iron in myoglobin (in muscles) and cytochromes (in all cells) is part of the functional compartment. The storage compartment is the iron that is not currently functioning but is available when needed. The major sources of this stored iron are the macrophages and hepatocytes, but every cell, except mature red blood cells, stores some iron. The third compartment is the transport compartment of iron that is in transit from one body site to another in the plasma.

TABLE 11-1

Iron Compartments in Normal Humans

Compartment

Form and Anatomical Site

Percent of Total Body Iron

Typical Iron Content (g)

Functional

Hemoglobin iron in the blood

∼68

2.400

Myoglobin iron in muscles

∼10

0.360

Peroxidase, catalase, cytochromes, riboflavin enzymes in all cells

∼3

0.120

Storage

Ferritin and hemosiderin mostly in macrophages and hepatocytes; small amounts in all cells except mature red blood cells

∼18

0.667

Transport

Transferrin in plasma

< 1

0.001

Although the reactivity of iron ions makes them central to energy production processes, it also makes them dangerous to the stability of cells. Thus the body regulates iron carefully at the level of the whole body and also within individual cells, maintaining levels that are necessary for critical metabolic processes, while avoiding the dangers of excess iron accumulation. The conditions that develop when this balance is perturbed are described in Chapter 20. The routine tests used to assess body iron status are discussed here.

Iron chemistry

The metabolic functions of iron depend on its ability to change its valence state from reduced ferrous (Fe+2) iron to the oxidized ferric (Fe+3) state. Thus it is involved in oxidation and reduction reactions such as the electron transport within mitochondrial cytochromes. In cells, ferrous iron can react with peroxide via the Fenton reaction, forming highly reactive oxygen molecules.

Image

The resulting hydroxyl radical (OH•), also known as a free radical, is especially reactive as a short-lived but potent oxidizing agent, able to damage proteins, lipids, and nucleic acids. As will be described in the section on iron kinetics, there are various mechanisms within cells to reduce the potential for this type of damage.

Iron kinetics

Systemic body iron regulation

provides an overview of systemic body iron regulation that can be a reference throughout the section on iron Figure 11-1 kinetics. The total amount of iron available to all body cells, systemic body iron, is regulated by absorption into the body because there is no mechanism for excretion. Ferrous iron in the lumen of the small intestine is carried across the luminal side of the enterocyte by divalent metal transporter 1 (DMT1) (Figure 11-2). Once iron has been absorbed into enterocytes, it requires another transporter, ferroportin, to carry it across the basolaminal enterocyte membrane into the bloodstream, thus truly absorbing it into the body. Ferroportin is the only known protein that exports iron across cell membranes. When the body has adequate stores of iron, the hepatocytes sense that and will increase production of hepcidin, a protein able to bind to ferroportin, leading to its inactivation. As a result, iron absorption into the body decreases. When the body iron begins to drop, the liver senses that change and decreases hepcidin production. As a result, ferroportin is once again active and able to transport iron into the blood. Thus homeostasis of iron is maintained by modest fluctuations in liver hepcidin production in response to body iron status. The regulation of systemic body iron is summarized in Figure 11-3.

Image 

FIGURE 11-1 Overview of the iron cycle regulating systemic body iron. Iron is absorbed through the enterocyte of the duodenum and into the plasma via the portal circulation. There it binds to apotransferrin for transport to cells, such as the developing red blood cells. In red blood cells, the iron is used in hemoglobin that circulates with the cell until it becomes aged and is ingested by a macrophage. There the iron is removed from the hemoglobin and can be recycled into the plasma for use by other cells. The level of stored and circulating body iron is detected by the hepatocyte, which is able to produce a protein, hepcidin, when iron levels get too high. Hepcidin will inactivate the absorption and recycling of iron by acting on enterocytes, macrophages, and hepatocytes. When body iron decreases, hepcidin will also decrease so that absorption and recycling are again activated. Source: (From Doig K: Iron: the body’s most precious metal. Denver, 2013, Colorado Association for Continuing Medical Laboratory Education, Inc., p. 1.)

Image 

FIGURE 11-2 Absorption of ionic iron in the small intestine. Ferric iron in the intestinal lumen is reduced prior to transport across the luminal membrane of the enterocyte by divalent metal transporter 1 (DMT1). It is carried across the opposite membrane into the blood by ferroportin. It is reoxidized by hephaestin (not shown) as it exits for transport in the blood. Source: (From Doig K: Iron: the body’s most precious metal. Denver, 2013, Colorado Association for Continuing Medical Laboratory Education, Inc., p. 6.)

Image 

FIGURE 11-3 Summary of body iron regulation. When body iron stores drop sufficiently low that plasma iron also drops, the liver’s iron-sensing system is activated and hepcidin production is decreased. As a result, ferroportin in the enterocyte and macrophage membranes will transport iron into the circulation. Plasma iron will rise and body stores will be restored. The hepatocyte iron-sensing system recognizes this and produces hepcidin. Hepcidin inactivates ferroportin in the enterocyte and macrophage membranes so that less iron is absorbed and recirculated. The resulting drop in iron stores and plasma iron is detected by the liver, repeating the cycle. Source: (From Doig K: Iron: the body’s most precious metal. Denver, 2013, Colorado Association for Continuing Medical Laboratory Education, Inc., p. 18.)

The mechanism by which the hepatocytes are able to sense iron levels and produce hepcidin is highly complex, with multiple stimulatory pathways likely involved. Although this system is not yet fully elucidated, a number of critical molecules have been identified. One postulated system for the interaction of these molecules is modeled in . The proteins involved include, at least, the hemochromatosis receptor (HFE), transferrin receptor 2, hemojuvelin, bone morphogenic protein (BMP) and its receptor (BMPR), and sons of mothers against decapentaplegic (SMAD).Figure 11-43 Table 11-2 lists their functions. The importance of these various molecules to iron kinetics has been demonstrated via mutations, both natural in humans and induced in mice, that lead to either iron overload or iron deficiency. Testing for these mutations is increasing in molecular diagnostic laboratories. The diseases associated with the known human mutations are described in Chapter 20.

Image 

FIGURE 11-4 Hepatic iron-sensing systems leading to hepcidin production. One system of iron sensing by hepatocytes involves the release of the hemochromatosis receptor (HFE) from transferrin receptor 1(TfR1) when the latter binds transferrin (Tf). The freed HFE then binds to transferrin receptor 2 (TfR2), initiating a membrane signal that phosphorylates sons of mothers against decapentaplegic (SMAD) proteins that move to the nucleus and upregulate hepcidin gene expression. A second system activated when body iron is high in secretion of bone morphogenic protein 6 (BMP6) by liver macrophages. BMP6 binds with its receptor, BMPR, and coreceptor, hemojuvelin (HJV), to phosphorylate SMAD and ultimately increase hepcidin production. Matriptase 2 can inactivate HJV and is an important mechanism to reduce hepcidin production when body iron is low. Source: (From Camaschella C, Silvestri L: Molecular mechanisms regulating hepcidin revealed by hepcidin disorders. The Scientific World 11: 1357-1366, 2011 and Doig K: Iron: the body’s most precious metal. Denver, 2013, Colorado Association for Continuing Medical Laboratory Education, Inc., p. 17.)

TABLE 11-2

Functions and Locations of Proteins Involved in Body Iron Sensing and Hepcidin Production

Protein

Location

Function

Hemochromatosis protein (HFE)

Hepatocyte membrane

A protein that is bound to transferrin receptor 1 (TfR1) until released by the binding of transferrin to TfR1

Transferrin receptor 2 (TfR2)

Hepatocyte membrane

A hepatocyte transferrin receptor that is able to bind freed HFE to initiate an internal cell signal for hepcidin production

Bone morphogenic protein (BMP)

Secreted product of macrophages

The ligand secreted by macrophages that initiates signal transduction when it binds to its receptor in a cell membrane

Bone morphogenic protein receptor (BMPR)

Hepatocyte (and other cells) membrane

A common membrane receptor initiating signal transduction within a cell when its ligand (BMP) binds

Hemojuvelin (HJV)

Hepatocyte membrane

A coreceptor acting with BMPR for signal transduction, leading to hepcidin production

Sons of mothers against decapentaplegic (SMAD)

Hepatocyte (and other cells) cytoplasm

A second messenger of signal transduction activated by BMPR-HJV complex, able to migrate to the nucleus and upregulate hepcidin gene expression

Iron transport

Iron exported from the enterocyte into the blood is ferrous and must be converted to the ferric form for transport in the blood. Hephaestin, a protein on the basolaminal enterocyte membrane, is able to oxidize iron as it exits the enterocyte. Once oxidized, the iron is ready for plasma transport, carried by a specific protein, apotransferrin (ApoTf). Once iron binds, the molecule is known as transferrin (Tf). Apotransferrin binds two molecules of ferric iron.

Cellular iron absorption and disposition

Individual cells regulate the amount of iron they absorb to minimize the adverse effects of free radicals. This is accomplished by relying on an iron-specific carrier to move it into the cell by a process calledreceptor-mediated endocytosis (Figure 11-5). Cell membranes possess a receptor for transferrin, transferrin receptor 1 (TfR1). TfR1 has the highest affinity for diferric Tf at the physiologic pH of the plasma and extracellular fluid. When the TfR1 molecules bind Tf, they move and cluster together in the membrane. Once a critical mass accumulates, the membrane begins to invaginate, progressing until the invagination pinches off a vesicle inside the cytoplasm called an endosome. Hydrogen ions are pumped into the vesicle. The resulting drop in pH changes the affinity of transferrin for iron, so the iron releases. Simultaneously, the affinity of TfR1 for apotransferrin at that pH increases so the apotransferrin remains bound to the receptor. The iron is exported from the vesicle into the cytoplasm of the cell by divalent metal transporter 1 (DMT1). Cytoplasmic trafficking is still not fully understood. However, some of the iron finds its way to storage. Other molecules of iron are transferred into the mitochondria,4 where they are incorporated into cytochromes, or in the case of red blood cells, into heme for the production of hemoglobin. Meanwhile, the endosome returns to the cell membrane, where the endosome membrane fuses with the cell membrane, opening the endosome and essentially reversing its formation. At the pH of the extracellular fluid, TfR1 has a very low affinity for apotransferrin, so the apotransferrin releases into the plasma, available to bind iron once again. The TfR1 is also available again to carry new molecules of Tf-bound iron into the cell.

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FIGURE 11-5 Cellular iron regulation. A critical mass of transferrin receptor 1 (TfR) with bound transferrin (Tf) will initiate an invagination of the membrane that ultimately fuses to form an endosome. Hydrogen ion (H+) inside the endosome releases the iron from Tf, and once reduced, it is transported into the cytosol by divalent mental transporter 1 (DMT1). In the cytosol, iron may be stored as ferritin or transferred to the mitochondria, where it is transported across the membrane by mitoferrin (not shown). The TfR with apotransferrin is returned to the cell membrane, where the ApoTf releases and the TfR is available to bind more Tf for iron transport into the cell. Source: (From  http://walz.med.harvard.edu/Research/Iron_Transport/Tfr-Tf-Figure-1.gif)

Cells are able to store iron so they have a reserve if supplies of new iron decline. Although all cells store iron, those cells that are central to regulating systemic body iron, macrophages and hepatocytes, typically contain the most. Ferric iron is stored in a cage-like protein called apoferritin. Once iron binds, it is known as ferritin. One ferritin molecule can bind more than 4000 iron ions.5 Ferritin iron can be mobilized for use during times of iron need by lysosomal degradation of the protein.6 Partially degraded ferritin is known as hemosiderin and is considered to be less metabolically available than ferritin, though greater understanding of ferritin chemistry may revise this view.

In order to regulate the amount of iron inside the cell and avoid free radicals, cells are able to control the amount of TfR1 on their surface. The process depends on an elegant system of iron-sensitive cytoplasmic proteins that are able to affect the posttranscriptional function of the mRNA for TfR1.7 The result is that when iron stores inside the cell are sufficient, production of TfR1 declines. Conversely, when iron stores inside the cell are low, TfR1 production increases. This is useful diagnostically to detect iron deficiency because a truncated form of the TfR1 is sloughed from cells and is measurable in serum assoluble transferrin receptor (sTfR).8 The serum sTfR levels reflect the number of TfRs expressed on cells. So increases in sTfR can indicate increases in membrane TfR that result from low intracellular iron as seen in iron deficiency anemia.

Red blood cells deserve special mention in regard to cellular iron regulation. Because production of hemoglobin requires them to acquire far more iron than other cells, special mechanisms exist that allow them to circumvent the usual limitations on iron accumulation. The complete understanding of these processes has yet to be developed, but hypotheses suggest that the iron may bypass the cytoplasmic iron-sensing system, moving directly into the mitochondria from the endosome.9

Iron recycling

When cells die, their iron is recycled. Multiple mechanisms salvage iron from dying cells. The largest percentage of recycled iron comes from red blood cells. Senescent (aging) red blood cells are ingested by macrophages in the spleen. The hemoglobin is degraded, with the iron being held by the macrophages as ferritin. Like enterocytes, macrophages possess ferroportin in their membranes.10 This allows macrophages to be iron exporters so that the salvaged iron can be used by other cells. The exported iron is bound to plasma apotransferrin, just as if it were newly absorbed from the intestine.

Haptoglobin and hemopexin are plasma proteins able to salvage free hemoglobin or heme, respectively, preventing them from urinary loss at the glomerulus and returning the iron to the liver. Like macrophages, hepatocytes are important to iron salvage. They also possess ferroportin so that the salvaged iron can be exported to transferrin and ultimately to other body cells.11 These salvage pathways are described in greater detail in Chapter 23.

Dietary iron, bioavailability, and demand

Under normal circumstances, the only source of iron for the body is from the diet. Foods containing high levels of iron include red meats, legumes, and dark green leafy vegetables.12 Although some foods may be high in iron, that iron may not be readily absorbed and thus is not bioavailable. Iron can be absorbed as either ionic iron or nonionic iron in the form of heme. Ionic iron must be in the ferrous (Fe+2) form for absorption into the enterocyte via the luminal membrane carrier, divalent metal transporter (DMT1). However, most dietary iron is ferric, especially from plant sources. As a result, it is not readily absorbed. Furthermore, other dietary compounds can bind iron and inhibit its absorption. These include oxalates, phytates, phosphates, and calcium.12 Release from these binders and reduction to the ferrous form are enhanced by gastric acid, acidic foods (e.g., citrus), and an enterocyte luminal membrane protein, duodenal cytochrome B (DcytB). Thus, although the U.S. diet contains on the order of 10 to 20 mg of iron/day, only 1 to 2 mg is absorbed.12 This amount is adequate for most men, but menstruating women, pregnant and lactating women, and growing children usually need additional iron supplementation to meet their increased need for iron. Chapter 20 discusses this further.

Heme with its bound iron is more readily absorbed than ionic iron.13 Thus meat, with heme in both myoglobin of muscle and hemoglobin of blood, is the most bioavailable source of dietary iron. The means by which heme is absorbed by enterocytes is not entirely clear. Although one carrier has been identified, it is actually more efficient at carrying folic acid.1415 So the primary heme carrier protein is still being sought.

Laboratory assessment of body iron status

Disease occurs when body iron levels are either too low or too high (Chapter 20). The tests used to assess body iron status are able to detect both conditions. They include the traditional or classic iron studies: serum iron (SI), total iron-binding capacity (TIBC), percent transferrin saturation, and Prussian blue staining of tissues. More recently, ferritin assays have been included among the routine tests. For special circumstances when the results of routine assays are equivocal or too invasive, newer assays include the soluble transferrin receptor (sTfR) and hemoglobin content of reticulocytes. The results of these measured parameters can be combined to calculate an sTfR/log ferritin ratio or graph a Thomas plot. Finally, zinc protoporphyrin is another assay with special application in sideroblastic anemia. Diagnostically, the tests can be organized to assess each of the iron compartments as indicated in Table 11-3.

TABLE 11-3

Assessment of Body Iron Status

Laboratory Assay

Typical Adult Male Reference Interval

Diagnostic Use and Compartment Assessed

Serum iron level

50–160 μg/dL

Indicator of available transport iron

Serum transferrin level (TIBC)

250–400 μg/dL

Indirect indicator of iron stores

Transferrin saturation

20%–55%

Indirect indicator of iron stores with transport iron

Serum ferritin level

40–400 ng/mL

Indicator of iron stores

Bone marrow or liver biopsy with Prussian blue staining

Normal iron stores visualized

Visual qualitative assessment of tissue iron stores

Soluble transferrin receptor (sTfR) level

1.15–2.75 mg/L

Indicator of functional iron available in cells

sTfR/log ferritin index

0.63–1.8

Indicator of functional iron available in cells

RBC zinc protoporphyrin level

< 80 μg/dL of RBCs

Indicator of functional iron available in red blood cells

Hemoglobin content of reticulocytes

27–34 pg/cell

Indicator of functional iron available in developing red blood cells

RBC, Red blood cell; TIBC, total iron-binding capacity.

Serum iron (si)

Serum iron can be measured colorimetrically using any of several reagents such as ferrozine. The iron is first released from transferrin by acid, and then the reagent is allowed to react with the freed iron, forming a colored complex that can be detected spectrophotometrically. Reference intervals are reported separately for men, women, and children, and will vary from laboratory to laboratory and from method to method. The serum iron level has limited utility on its own because of its high within-day and between-day variability; it also increases after recent ingestion of iron-containing foods and supplements. To avoid the apparent diurnal variation, the standard practice has been to collect the specimen fasting and early in the morning when levels are expected to be highest. However, this practice has recently been questioned.16 A diurnal variation in hepcidin has been detected that may explain some of the serum iron variability and may still support the early-morning phlebotomy practice.17 A typical reference interval is provided in Table 11-3.

Total iron-binding capacity (TIBC)

The amount of iron in plasma or serum will be limited by the amount of transferrin that is available to carry it. To assess this, transferrin is maximally saturated by addition of excess ferric iron to the specimen. Any unbound iron is removed by precipitation with magnesium carbonate powder. Then the basic iron method as described above is performed on the absorbed serum, beginning with the release of the iron from transferrin. The amount of iron detected represents all the binding sites available on transferrin—that is, the total iron-binding capacity (TIBC). It is expressed as an iron value, although it is actually an itransferrin saturated with iron is calculated ndirect measure of transferrin. A typical reference interval is provided in Table 11-3.

Percent transferrin saturation

Since the TIBC represents the total number of sites for iron binding and the SI represents the number bound with iron, the degree to which the available sites are occupied by iron can be calculated. The percent of transferrin saturated with iron is calculated as:

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It is important that both the SI and TIBC be expressed in the same units, but it does not matter which units are used in the calculation. A typical reference interval is provided in Table 11-3. A convenient rule of thumb evident from the table is that about one third (1⁄3) of transferrin is typically saturated with iron.

Prussian blue staining

Prussian blue is actually a chemical compound with the formula Fe7(CN)18. 18 The compound forms during the staining process, which uses acidic potassium ferrocyanide as the reagent/stain. The ferric iron in the tissue reacts with the reagent, forming the Prussian blue compound that is readily seen microscopically as dark blue dots. Tissues can be graded or scored semiquantitatively by the amount of stain that is observed. Prussian blue stain is considered the gold standard for assessment of body iron. Staining is conducted routinely when bone marrow or liver biopsies are taken for other purposes. Although ferric iron reacts with the reagent, ferritin is not detected, likely due to the intact protein cage. However, hemosiderin stains readily.

Ferritin

As mentioned above, until the development of serum ferritin assays, the only way to truly assess body iron stores was to take a sample of bone marrow and stain it with Prussian blue. Such an invasive procedure prevented regular assessment of body iron. The development of the serum immunoassay for ferritin provided a convenient assessment of body iron stores. Though ferritin is an intracellular protein, it is secreted by macrophages into plasma for reasons that are not yet understood.19 The level of serum ferritin has been shown to correlate highly with stored iron as indicated by Prussian blue stains of bone marrow.20 Typical reference intervals are provided in Table 11-3.

There is a significant drawback in the interpretation of serum ferritin results. Ferritin is an acute phase protein or acute phase reactant (APR).21 The APRs are proteins that are produced, mostly by the liver, during the acute (i.e., initial) phase of inflammation, especially during infections. They include cytokines that are nonspecific, but also other proteins with the apparent intent to suppress bacteria. Since bacteria need iron, the body’s production of ferritin during the acute phase seems to be an attempt to sequester the iron away from the bacteria. Thus increases in ferritin can be induced without an increase in the amount of systemic body iron. These rises may not be outside the reference interval but still high enough to elevate a patient’s ferritin above what it would otherwise be. Ferritin values between 20 and 100 ng/mL are most equivocal, making it difficult to recognize true iron deficiency when an inflammatory condition is also present.22 Therefore, the predictive value of a ferritin result within the reference interval is weak. However, only a decreased level of stored body iron can lower ferritin levels below the reference interval, so the predictive value of a low ferritin result is high for iron deficiency.

Soluble transferrin receptor (stfr)

As described above, cells regulate the amount of TfR on their membrane based on the amount of intracellular iron. When the latter drops, the cell expresses more TfR on the membrane. A truncated form of the receptor is shed into the plasma and can be detected with immunoassay.8 Thus increases in the sTfR reflect either increases in the amounts of TfR on individual cells, as in iron deficiency, or an increase in the number of cells each with a normal number of TfRs. The latter occurs during instances of rapid erythropoiesis, such as a response to hemolytic anemia. Typical reference intervals are provided in Table 11-3.

Hemoglobin content of reticulocytes

Chapter 15 describes how some hematology instruments are able to report a value for the amount of hemoglobin in reticulocytes; it is analogous to the mean cell hemoglobin (MCH), but just for reticulocytes. Because, under normal conditions, the number of circulating reticulocytes represents the status of erythropoiesis in the prior 24-hour period, the amount of hemoglobin in reticulocytes provides a near real-time assessment of iron available for hemoglobin production.23 The hemoglobin content of reticulocytes will drop when iron for erythropoiesis is restricted. A representative adult reference interval is provided in Table 11-3. Separate reference intervals may be provided for children and infants.

Soluble transferrin receptor/log ferritin

Although ferritin and sTfR values alone can point to iron deficiency, the ratio of sTfR to ferritin or sTfR to log ferritin improves the identification of iron deficiency when values are equivocal.2425 Because the sTfR rises in iron deficiency and the ferritin (and its log) drops, these ratios are especially useful when one of the parameters has changed but is not outside the reference interval. A typical reference interval is provided in Table 11-3.

Thomas plot

Thomas and Thomas26 demonstrated that when the sTfR/log ferritin is plotted against the hemoglobin content of reticulocytes, a four-quadrant plot results that can improve the identification of iron deficiency (Figure 11-6).27 In instances where there is true iron deficiency, the sTfR will rise and the ferritin will drop so that the sTfR/log ferritin will be high and the hemoglobin content of reticulocytes will be low; patient results will plot to the lower right quadrant. In instances where the ferritin may be falsely elevated by inflammation, the sTfR/log ferritin will be normal despite reduced availability of iron for hemoglobin production—thus a low hemoglobin content in reticulocytes. In this instance, patient values will plot to the lower left quadrant called functional iron deficiency because the systemic body stores are adequate but not available for transport and use by cells. As iron deficiency develops, other cells are starved before erythrocytes; production of hemoglobin in reticulocytes remains at a normal level for as long as possible. However, the body’s other iron-starved cells will increase sTfR production and systemic iron stores of ferritin will be depleted, thus elevating the sTfR/log ferritin value. These early iron-deficient patients’ results will plot to the upper right quadrant called latent iron deficiency. By incorporating several different assessments of iron status, the use of the Thomas plot, as it is called, can improve the identification of iron deficiency in instances when other tests are equivocal. Chapter 20 will elucidate further the impact of various diseases on the parameters of the Thomas plot.

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FIGURE 11-6 Thomas plot. Plotting the ratio of soluble transferrin receptor to log ferritin (sTfR/log ferritin) against the hemoglobin content of reticulocytes produces a graph with four quadrants. Patients with values within the reference intervals for each assay will cluster in the upper left quadrant. Those with functional iron deficiency, like the anemia of chronic inflammation, will cluster at the lower left. Latent iron deficiency, before anemia develops, will cluster to the upper right with frank iron deficiency in the lower right quadrant. Source: (Modified from Doig K: Iron: the body’s most precious metal. Denver, 2013, Colorado Association for Continuing Medical Laboratory Education, Inc., p. 24.)

Zinc protoporphyrin

Zinc protoporphyrin (ZPP) accumulates in red blood cells when iron is not incorporated into heme and zinc binds instead to protoporphyrin IX. It is easily detected by fluorescence. Although ZPP will rise during iron-deficient erythropoiesis, the value of this test is greatest when the activity of the ferrochelatase is impaired, as in lead poisoning (Chapter 20).

Summary

• Iron is so critical for transport and use of oxygen that the body conserves and recycles it, and it does not have a mechanism for its active excretion. Free radical production by iron ions severely damages cells and thus demands regulation. The body adjusts its iron levels by intestinal absorption, depending on need.

• Iron is absorbed into enterocytes as ferrous iron by the divalent metal transporter 1 (DMT1) on the luminal side of the cells. Heme can also be absorbed. Iron is exported into the plasma via ferroportin, a protein carrier in the enterocyte basolaminal membrane.

• Iron is carried in the plasma in ferric form attached to apotransferrin. Each molecule of apotransferrin can bind two molecules of iron. Apotransferrin with bound iron is called transferrin.

• Individual cells absorb iron when diferric transferrin binds to transferrin receptor 1 (TfR1) on their surfaces. Bound receptors cluster and invaginate the membrane to form an endosome. Iron released by acid within the endosome is exported into the plasma and ultimately into the mitochondria for incorporation into cytochromes and heme. Alternatively, it can also be stored as ferritin in the cytosol. The iron-depleted endosome fuses with the cell membrane, releasing the apotransferrin and thus allowing the TfR1 on the cell membrane to bind more diferric transferrin.

• Macrophages ingest dying red blood cells. They salvage and store the iron derived from heme.

• Hepatocytes sense body iron status through the interaction of the hemochromatosis receptor, transferrin receptor 2, hemojuvelin, bone morphogenic protein, and SMAD.

• When the hepatocyte iron-sensing system detects that body iron levels are high, the hepatocyte secretes hepcidin. Hepcidin inactivates ferroportin in enterocyte, macrophage, and hepatocyte membranes, reducing the absorption of new iron and the release of stored iron. When the hepatocyte senses low body iron, hepcidin secretion is reduced, and ferroportin is active for intestinal iron absorption and macrophage and hepatocyte iron export into the plasma.

• Individual cells adjust the number of transferrin receptors on their surface to regulate the amount of iron they absorb; receptor numbers rise when the cell needs additional iron but decrease when the iron in the cell is adequate. Truncated soluble transferrin receptors are also shed into the plasma in proportion to their number on cells.

• Cells store iron as ferritin when they have an excess. Iron can be released from ferritin when needed by degradation of the protein by lysosomes. Partially degraded ferritin can be detected in cells as stainable hemosiderin. Ferritin is secreted into the plasma by macrophages in proportion to the amount of iron that is in storage. Ferritin is elevated in plasma by the acute phase response, unrelated to amounts of stored iron.

• Dietary iron is most bioavailable as heme from meat sources. Plant sources typically supply ferric iron that must be released from iron-binding compounds and reduced before absorption.

• Most body iron is found in hemoglobin or stored as ferritin. Less than 10% of all body iron is found in muscles, plasma, cytochromes, and iron-dependent enzymes throughout body cells.

• Laboratory tests for assessment of iron status include total serum iron, total iron-binding capacity, percent transferrin saturation, serum ferritin, soluble transferrin receptor, tissue staining for hemosiderin, zinc protoporphyrin, and the hemoglobin content of reticulocytes. Additional parameters derived from these, the Thomas plot and sTfR/log ferritin, are particularly useful for the recognition of iron deficiency when other test results are equivocal.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. Iron is transported in plasma via:

a. Hemosiderin

b. Ferritin

c. Transferrin

d. Hemoglobin

2. What is the major metabolically available storage form of iron in the body?

a. Hemosiderin

b. Ferritin

c. Transferrin

d. Hemoglobin

3. The total iron-binding capacity (TIBC) of the serum is an indirect measure of which iron-related protein?

a. Hemosiderin

b. Ferritin

c. Transferrin

d. Hemoglobin

4. For a patient with classic iron study values that are equivocal for iron deficiency, which of the following tests would be most helpful in determining whether iron deficiency is present or not?

a. Zinc protoporphyrin

b. Peripheral blood sideroblast assessment

c. Soluble transferrin receptor

d. Mean cell hemoglobin

5. What membrane-associated protein in enterocytes transports iron from the intestinal lumen into the enterocyte?

a. Transferrin

b. Ferroportin

c. DMT1

d. Ferrochelatase

6. Iron is transported out of macrophages, hepatocytes, and enterocytes by what membrane protein?

a. Transferrin

b. Ferroportin

c. DMT1

d. Ferrochelatase

7. Below are several of the many steps in the process from absorption and transport of iron to incorporation into heme. Place them in proper order.

i. Transferrin picks up ferric iron.

ii. Iron is transferred to the mitochondria.

iii. DMT1 transports ferrous iron into the enterocyte.

iv. Ferroportin transports iron from enterocyte to plasma.

v. The transferrin receptor transports iron into the cell.

a. v, iv, i, ii, iii

b. iii, ii, iv, i, v

c. ii, i, v, iii, iv

d. iii, iv, i, v, ii

8. What is the fate of the transferrin receptor when it has completed its role in the delivery of iron to a cell?

a. It is recycled to the plasma membrane and released into the plasma.

b. It is recycled to the plasma membrane, where it can bind its ligand again.

c. It is catabolized and the amino acids are returned to the metabolic pool.

d. It is retained in the endosome for the life span of the cell.

9. The transfer of iron from the enterocyte into the plasma is REGULATED by:

a. Transferrin

b. Ferroportin

c. Hephaestin

d. Hepcidin

10. What is the percent transferrin saturation for a patient with total serum iron of 63 μg/dL and TIBC of 420 μg/dL ?

a. 6.7%

b. 12%

c. 15%

d. 80%

11. Referring to Figure 11-6, into which quadrant of a Thomas plot would a patient’s results fall with the following test results:

Soluble transferrin receptor: increased above reference interval

Ferritin: decreased below reference interval

Hemoglobin content of reticulocytes: within the reference interval

a. Normal iron status

b. Latent iron deficiency

c. Functional iron deficiency

d. Iron deficiency

12. A physician is concerned that a patient is developing iron deficiency from chronic intestinal bleeding due to aspirin use for rheumatoid arthritis. The iron studies on the patient show the following results:

Laboratory Assay

Adult Reference Intervals

Patient Values

Serum ferritin level

12–400 ng/mL

25 ng /mL

Serum iron level

50–160 μg/dL

45 μg/dL

Total iron-binding capacity (TIBC)

250–400 μg/dL

405 μg/dL

Transferrin saturation

20%–55%

CALCULATE IT

How would these results be interpreted?

a. Latent iron deficiency

b. Functional iron deficiency

c. Iron deficiency

d. Equivocal for iron deficiency

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*The author extends appreciation to Mary Coleman, whose coverage of iron metabolism in the prior editions provided the foundation for this chapter.