Section XI - Drugs Acting on the Blood and the Blood-Forming docx

Adequate iron stores, as reflected by an iron saturation of transferrin of at least 30% and a plasma ferritin greater than 400 g/l, must be maintained, usually by repeated injections of

Section XI Drugs Acting on the Blood and the Blood-Forming Organs

Overview

The short life span of mature blood cells requires their continuous replacement, a process termed hematopoiesis New cell production must be responsive to both basal needs and situations of

increased demand For example, red blood cell production can vary over more than a fivefold range

in response to anemia or hypoxia White blood cell production increases dramatically in response to

a systemic infection, and platelet production can increase severalfold when platelet destruction results in thrombocytopenia

The regulation of hematopoiesis is complex and involves cell–cell interactions within the

microenvironment of the bone marrow as well as both hematopoietic and lymphopoietic growth factors A number of these hormonelike glycoproteins now have been identified and characterized, and, using recombinant DNA technology, their genes have been cloned and the proteins produced in quantities sufficient for use as therapeutic agents Clinical applications now are being developed, ranging from treatment of primary hematological diseases to uses as adjunctive agents in the

treatment of severe infections and in the management of patients who are undergoing chemotherapy

or marrow transplantation

Hematopoiesis also requires adequate supplies of minerals, both iron and copper, and a number of vitamins, including folic acid, vitamin B12, pyridoxine, ascorbic acid, and riboflavin Deficiencies of these minerals and vitamins generally result in characteristic anemias and, less frequently, a general failure of hematopoiesis Therapeutic correction of a specific deficiency state depends on the

accurate diagnosis of the anemic state and knowledge as to the correct dose, the use of these agents

in various combinations, and the expected response

This chapter deals with the growth factors, vitamins, minerals, and drugs that affect the blood and blood-forming organs

Hematopoietic Growth Factors

irradiated mice Their work led to the concept of colony-forming stem cells It also led to the

subsequent proof that stem cells present in human bone marrow are pluripotent—that is, they give rise to granulocytes, monocytes, lymphocytes, megakaryocytes, and erythrocytes

The role of growth factors in hematopoiesis was elucidated by Bradley, Metcalf, and others using bone marrow culture techniques (Bradley and Metcalf, 1966) Individual growth factors were isolated (Metcalf, 1985; Moore, 1991), and the target cells of these factors characterized The

pluripotent stem cell gives rise to committed progenitors, which can be identified as single forming units, and to cells that are increasingly differentiated.

colony-The existence of a circulating growth factor that controls erythropoiesis was first suggested by experiments carried out by Paul Carnot in 1906 (Carnot and Deflandre, 1906) He observed an increase in the red cell count in rabbits injected with serum obtained from anemic animals and

postulated the existence of a factor that he called hemapoietine However, it was not until the 1950s

that Reissmann (1950), Erslev (1953), and Jacobsen and coworkers (1957) defined the origin and

actions of the hormone, now called erythropoietin Subsequently, extensive studies of

erythropoietin were carried out in patients with anemia and polycythemia, culminating in 1977 with the purification of erythropoietin from urine by Miyake and colleagues The gene that encodes the protein was subsequently cloned and expressed at a high level in a mammalian cell system (Jacobs

et al , 1985; Lin et al. , 1985 ), producing a recombinant hormone that is indistinguishable from human urinary erythropoietin Similarly, complementary DNA and genomic clones for granulocyte, macrophage, and, most recently, megakaryocyte colony-stimulating factors have been isolated and sufficient quantities of biologically active growth factors produced for clinical investigation

(Kawasaki et al. , 1985 ; Lee et al. , 1985 ; Wong et al. , 1985 ; Yang et al. , 1986 ; Lok et al. , 1994 ; de Sauvage et al. , 1994 )

Growth Factor Physiology

Steady-state hematopoiesis involves the production of more than 200 billion (2 x 1011) blood cells each day This production is under delicate control, and, with increased demand, the rate can

increase severalfold The hematopoietic organ also is unique in that several mature cell types are derived from a much smaller number of pluripotent stem cells that are formed in early embryonic life These stem cells are capable of both maintaining their own number and differentiating under the influence of cellular and humoral factors [stem cell factor (SCF), Flt3 ligand (FL), interleukin-3 (IL-3), and granulocyte/macrophage colony-stimulating factor (GM-CSF)] to produce a variety of hematopoietic and lymphopoietic cells

Stem cell differentiation can be described as a series of steps that produce so-called burst-forming units (BFU) and colony-forming units (CFU) for each of the major cell lines (Quesenberry and Levitt, 1979) Although these early progenitors (BFU and CFU) are not morphologically

recognizable as precursors of a specific cell type, they are capable of further proliferation and differentiation, increasing their number by some 30-fold Subsequently, colonies of

morphologically distinct cells form under the control of an overlapping set of additional growth factors (G-CSF, M-CSF, erythropoietin, and thrombopoietin) Proliferation and maturation of the CFU for each cell line can further amplify the resulting mature cell product by another 30-fold or more, resulting in greater than 1000 mature cells produced for each committed stem cell (Lajtha et

al.

, 1969)

Hematopoietic and lymphopoietic growth factors are produced by a number of marrow cells and peripheral tissues The growth factors are glycoproteins and are active at very low concentrations, usually on more than one committed cell lineage Most show synergistic interactions with other factors, as well as "networking," wherein stimulation of a cell lineage by one growth factor induces the production of additional growth factors Finally, growth factors generally exert actions at

several points in the processes of cell proliferation and differentiation and in mature cell function (Metcalf, 1985) Some of the overlapping effects of the more important hematopoietic growth factors are illustrated in Figure 54–1 and listed in Table 54–1

Figure 54–1 Sites of Action of Hematopoietic Growth Factors in the Differentiation and Maturation of Marrow Cell Lines A self-sustaining pool of marrow stem cells differentiates under the influence of specific hematopoietic growth factors to form a variety of hematopoietic and lymphopoietic cells Stem cell factor (SCF), ligand (FL), interleukin-3 (IL-3), and granulocyte/macrophage colony-stimulating factor (GM-CSF), together with cell–cell interactions in the marrow, stimulate stem cells to form a series of burst-forming units (BFU) and colony-forming units (CFU): CFU-GEMM, CFU-GM, CFU-Meg, BFU-E, and CFU-E (GEMM, granulocyte, erythrocyte, monocyte, and megakaryocyte; GM, granulocyte and macrophage; Meg, megakaryocyte; E, erythrocyte) After considerable proliferation, further differentiation is stimulated by synergistic interactions with growth factors for each of the major cell lines—granulocyte colony-stimulating factor (G-CSF), monocyte/macrophage-stimulating factor (M-CSF), thrombopoietin, and erythropoietin Each of these factors also influences the proliferation, maturation, and, in some cases, the function of the derivative

cell line (seeTable 54–1).

Erythropoietin

While erythropoietin is not the sole growth factor responsible for erythropoiesis, it is the most

important regulator of the proliferation of committed progenitors (BFU-E and CFU-E) In its

absence, severe anemia is invariably present Erythropoiesis is controlled by a highly responsive feedback system in which a sensor in the kidney can detect changes in oxygen delivery to increase the secretion of erythropoietin, which then stimulates a rapid expansion of erythroid progenitors

Erythropoietin is produced primarily by peritubular interstitial cells of the kidney under the control

of a single gene on human chromosome 7 The gene product is a protein containing 193 amino acids, of which the first 27 are cleaved during secretion (Jacobs et al. , 1985 ; Lin et al. , 1985 ) The final hormonal peptide is heavily glycosylated and has a molecular weight of approximately 30,000 daltons Once released, erythropoietin travels to the marrow, where it binds to a receptor on the surface of committed erythroid progenitors and is internalized With anemia or hypoxemia, renal synthesis rapidly increases by 100-fold or more, serum erythropoietin levels rise, and marrow progenitor cell survival, proliferation, and maturation are dramatically stimulated This finely tuned feedback loop can be disrupted at any point—by kidney disease, marrow damage, or a deficiency in iron or an essential vitamin With an infection or an inflammatory state, erythropoietin secretion, iron delivery, and progenitor proliferation are all suppressed by inflammatory cytokines

Recombinant human erythropoietin (epoetin alfa), produced using a mammalian cell line (Chinese hamster ovary cells), is virtually identical to endogenous hormone Small differences in the

carbohydrate portion of the molecule do not appear to affect the kinetics, potency, or

immunoreactivity Currently available preparations of epoetin alfa include EPOGEN and PROCRIT, supplied in single-use vials of from 2000 to 10,000 U/ml for intravenous or subcutaneous

administration When injected intravenously, epoetin alfa is cleared from plasma with a half-life of

10 hours However, the effect on marrow progenitors is sufficiently sustained that it need not be given more often than three times a week to achieve an adequate response No significant allergic reactions have been associated with the intravenous or subcutaneous administration of epoetin alfa, and antibodies have not been detected, even after prolonged administration

Therapeutic Uses

Recombinant erythropoietin therapy can be highly effective in a number of anemias, especially those associated with a poor erythropoietic response As first shown by Eschbach and coworkers in

1987, there is a clear dose-response relationship between the epoetin alfa dose and the rise in

hematocrit in anephric patients, with eradication of their anemia at higher doses Epoetin alfa also has been shown to be effective in the treatment of anemias associated with surgery, AIDS, cancer chemotherapy, prematurity, and certain chronic inflammatory illnesses

Anemia of Chronic Renal Failure

Patients with the anemia of chronic renal disease are ideal candidates for epoetin alfa therapy The response in predialysis, peritoneal dialysis, and hemodialysis patients is dependent on severity of the renal failure, the erythropoietin dose and route of administration, and iron availability (Eschbach

et al , 1989; Kaufman et al. , 1998 ; Besarab et al. , 1999 ) The subcutaneous route of administration

is preferred over the intravenous, since absorption is slower and the amount of drug required is reduced by 20% to 40% Iron supply is especially critical Adequate iron stores, as reflected by an iron saturation of transferrin of at least 30% and a plasma ferritin greater than 400 g/l, must be maintained, usually by repeated injections of iron dextran (see"Therapy with Parenteral Iron").The patient must be closely monitored during therapy, and the dose of epoetin alfa must be adjusted

to obtain a gradual rise in the hematocrit, over a 2- to 4-month period, until a final hematocrit of

33% to 36% is reached Treatment to hematocrit levels greater than 36% is not recommended A study of patients treated to hematocrits above 40% showed a higher incidence of myocardial

infarction and death (Besarab et al. , 1998 ) Furthermore, the drug should never be used to replace emergency transfusion in patients who need immediate correction of a life-threatening anemia

It is currently recommended that the patient be started on a dose of 80 to 120 U/kg of epoetin alfa, given subcutaneously, three times a week It can be given on a once-a-week schedule, but

considerably more drug is required for an equivalent effect If the response is poor, the dose should

be progressively increased The final maintenance dose of epoetin alfa can vary from as little as 10 U/kg to more than 300 U/kg, with an average close to 75 U/kg, three times a week, in most patients Children under the age of 5 years generally require a higher dose Resistance to therapy is

commonly seen in the patient who develops an inflammatory illness or becomes iron deficient, so that close monitoring of general health and iron status is essential Less common causes of

resistance include occult blood loss, folic acid deficiency, carnitine deficiency, inadequate dialysis, aluminum toxicity, and osteitis fibrosa cystica secondary to hyperparathyroidism

The most common side effect of epoetin alfa therapy is aggravation of hypertension, seen in 20% to 30% of patients and most often associated with a too-rapid rise in hematocrit Blood pressure

control usually can be attained by either increasing antihypertensive therapy or ultrafiltration in dialysis patients or by reducing the epoetin alfa dose to slow the hematocrit response An increased tendency to vascular access thrombosis in dialysis patients also has been reported, but this remains controversial

Anemia in AIDS Patients

Epoetin alfa therapy has been approved for the treatment of HIV-infected patients, especially those

on zidovudine therapy (Fischl et al. , 1990 ) Excellent responses to doses of 100 to 300 U/kg, given subcutaneously three times a week, generally are seen in patients with zidovudine-induced anemia

In the face of advanced disease, marrow damage, and elevated serum erythropoietin levels (greater than 500 IU/L), therapy is less effective

Cancer-Related Anemias

Epoetin alfa therapy, 150 U/kg three times a week or 450 to 600 U/kg once a week, can reduce the transfusion requirement in cancer patients undergoing chemotherapy It also has been used to treat patients with multiple myeloma, with improvement in both their anemia and sense of well-being Here again, a baseline serum erythropoietin level may help to predict the response

Surgery and Autologous Blood Donation

Epoetin alfa has been used perioperatively to treat anemia and reduce the need for transfusion Patients undergoing elective orthopedic and cardiac procedures have been treated with 150 to 300 U/kg of epoetin alfa once daily for the 10 days preceding surgery, on the day of surgery, and for 4 days after surgery As an alternative, 600 U/kg can be given on days –21, –14, and –7 prior to surgery, with an additional dose on the day of surgery This can correct a moderately severe

preoperative anemia, hematocrit 30% to 36%, and reduce the need for transfusion Epoetin alfa also has been used to improve autologous blood donation (Goodnough et al. , 1989 ) However, as a routine, the potential benefit is small while the expense is considerable Patients treated for 3 to 4 weeks with epoetin alfa (300 to 600 U/kg twice a week), are able to donate only 1 or 2 more units than untreated patients, and most of the time this goes unused Still, the ability to stimulate

erythropoiesis for blood storage can be invaluable in the patient with multiple alloantibodies to homologous red blood cells.

Other Uses

Epoetin alfa has been designated an orphan drug by the United States Food and Drug

Administration (FDA) for the treatment of the anemia of prematurity and patients with

myelodysplasia In the latter case, even very high doses of more than 1000 U/kg 2 to 3 times a week have had limited success The possible use of very high dose therapy in other hematological

disorders, such as sickle cell anemia, is still under study Highly competitive athletes have used epoetin alfa to increase their hemoglobin levels ("blood doping") and improve performance

Unfortunately, this misuse of the drug has been implicated in the deaths of several athletes, and it should be discouraged

Myeloid Growth Factors

The myeloid growth factors are glycoproteins that stimulate the proliferation and differentiation of one or more myeloid cell lines They also enhance the function of mature granulocytes and

monocytes Recombinant forms of several of the growth factors have now been produced, including GM-CSF (Lee et al. , 1985 ), G-CSF (Wong et al. , 1985 ), IL-3 (Yang et al. , 1986 ), M-CSF or CSF-1 (Kawasaki et al. , 1985 ), SCF (Huang et al. , 1990 ), and, most recently, thrombopoietin (Lok et al. , 1994; de Sauvage et al. , 1994 ; Kaushansky et al. , 1994 ; Table 54–1)

The myeloid growth factors are produced naturally by a number of different cells including

fibroblasts, endothelial cells, macrophages, and T cells (Figure 54–2) They are active at extremely low concentrations GM-CSF is capable of stimulating the proliferation, differentiation, and

function of a number of the myeloid cell lineages (Figure 54–1) It acts synergistically with other growth factors, including erythropoietin, at the level of the BFU GM-CSF stimulates the CFU-GEMM (granulocyte/erythrocyte/macrophage/megakaryocyte), CFU-GM, CFU-M, CFU-E, and CFU-Meg (megakaryocyte) to increase cell production It also enhances the migration,

phagocytosis, superoxide production, and antibody-dependent cell media toxicity of neutrophils, monocytes, and eosinophils

Figure 54–2 Cytokine–Cell Interactions Macrophages, T cells, B cells, and

marrow stem cells interact via several cytokines [IL (interleukin)-1, IL-2, IL-3,

IL-4, IFN (interferon)- , GM-CSF, and G-CSF] in response to a bacterial or a

foreign antigen challenge SeeTable 54–1 for the functional activities of these

various cytokines

The activity of G-CSF is more focused Its principal action is to stimulate the proliferation,

differentiation, and function of the granulocyte lineage It acts primarily on the CFU-G, although it can also play a synergistic role with IL-3 and GM-CSF in stimulating other cell lines G-CSF enhances phagocytic and cytotoxic activities of neutrophils Unlike GM-CSF, G-CSF has little effect on monocytes, macrophages, and eosinophils At the same time, G-CSF reduces

inflammation by inhibiting IL-1, tumor necrosis factor, and interferon gamma

Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF)

Recombinant human GM-CSF (sargramostim ) is a 127–amino acid glycoprotein produced in yeast

Except for the substitution of a leucine in position 23 and variable levels of glycosylation, it is identical to endogenous GM-CSF While sargramostim, like natural GM-CSF, has a wide range of effects on cells in culture, its primary therapeutic effect is the stimulation of myelopoiesis The initial clinical application of sargramostim was in patients undergoing autologous bone marrow transplantation By shortening the duration of neutropenia, transplant morbidity was significantly reduced without a change in long-term survival or risk of inducing an early relapse of the malignant process (Brandt et al. , 1988 ; Rabinowe et al. , 1993 ) The role of GM-CSF therapy in allogeneic transplantation is less clear The effect of the growth factor on neutrophil recovery is less

pronounced in patients receiving prophylactic treatment for graft-versus-host disease (GVHD), and studies have failed to show a significant effect on transplant mortality, long-term survival, the appearance of GVHD, or disease relapse However, it may improve survival in transplant patients who exhibit early graft failure (Nemunaitis et al. , 1990 ) It also has been used to mobilize CD34-positive progenitor cells for peripheral blood stem cell collection for transplantation following myeloablative chemotherapy Sargramostim has been used to shorten the period of neutropenia and reduce morbidity in patients receiving intensive chemotherapy (Gerhartz et al. , 1993 ) It also will stimulate myelopoiesis in some patients with cyclic neutropenia, myelodysplasia, aplastic anemia,

or AIDS-associated neutropenia (Groopman et al. , 1987 ; Vadhan-Raj et al. , 1987 )

Sargramostim (LEUKINE) is administered by subcutaneous injection or slow intravenous infusion at

a dose of 125 to 500 g/m2 per day Plasma levels of GM-CSF rise rapidly after subcutaneous injection and then decline, with a half-life of 2 to 3 hours When given intravenously, infusions should be maintained over 3 to 6 hours With the initiation of therapy, there is a transient decrease

in the absolute leukocyte count secondary to margination and sequestration in the lungs This is followed by a dose-dependent, biphasic increase in leukocyte counts over the next 7 to 10 days

Once the drug is discontinued, the leukocyte count returns to baseline within 2 to 10 days When GM-CSF is given in lower doses, the response is primarily neutrophilic, while at larger doses, monocytosis and eosinophilia are observed Following bone marrow transplantation or intensive chemotherapy, sargramostim is given daily during the period of maximum neutropenia until a sustained rise in the granulocyte count is observed Frequent blood counts are essential to avoid an excessive rise in the granulocyte count The dose may be increased if the patient fails to respond after 7 to 14 days of therapy However, higher doses are associated with more pronounced side effects, including bone pain, malaise, flulike symptoms, fever, diarrhea, dyspnea, and rash Patients can be extremely sensitive to GM-CSF, demonstrating an acute reaction to the first dose,

characterized by flushing, hypotension, nausea, vomiting, and dyspnea, with a fall in arterial oxygen saturation due to sequestration of granulocytes in the pulmonary circulation With prolonged

administration, a few patients may develop a capillary leak syndrome, with peripheral edema and both pleural and pericardial effusions

Granulocyte Colony-Stimulating Factor (G-CSF)

Recombinant human G-CSF (filgrastim, NEUPOGEN) is a 175–amino acid glycoprotein produced in

Escherichia coli Unlike natural G-CSF, it is not glycosylated and carries an extra N-terminal

methionine The principal action of filgrastim is the stimulation of CFU-G to increase neutrophil production (Figure 54–1) It also enhances the phagocytic and cytotoxic functions of neutrophils

Filgrastim has been shown to be effective in the treatment of severe neutropenia following

autologous bone marrow transplantation and high-dose chemotherapy (Lieschke and Burgess, 1992) Like GM-CSF, filgrastim shortens the period of severe neutropenia and reduces morbidity secondary to bacterial and fungal infections When used as a part of an intensive chemotherapy regimen, it can decrease the frequency of both hospitalization for febrile neutropenia and

interruptions in the chemotherapy protocol G-CSF also has proven to be effective in the treatment

of severe congenital neutropenias In patients with cyclic neutropenia, G-CSF therapy, while not eliminating the neutropenic cycle, will increase the level of neutrophils and shorten the length of the cycle sufficiently to prevent recurrent bacterial infections (Hammond et al. , 1989 ) Filgrastim therapy can improve neutrophil counts in some patients with myelodysplasia or marrow damage (moderately severe aplastic anemia or tumor infiltration of the marrow) The neutropenia of AIDS patients receiving zidovudine also can be partially or completely reversed Filgrastim is now

routinely used in the patient undergoing peripheral blood stem cell (PBSC) collection and a stem cell transplant It encourages the release of CD34+ progenitor cells from the marrow, reducing the number of collections necessary for transplant Moreover, filgrastim-mobilized PBSCs appear more capable of rapid engraftment PBSC-transplanted patients require fewer days of platelet and red blood cell transfusions and a shorter duration of hospitalization than do patients receiving

autologous bone marrow transplants

Filgrastim is administered by subcutaneous injection or intravenous infusion over at least 30

minutes at a dose of 1 to 20 g/kg per day A usual starting dose in a patient receiving

myelosuppressive chemotherapy is 5 g/kg per day The distribution and clearance rate from

plasma (half-life of 3.5 hours) are similar for both routes of administration A continuous 24-hour intravenous infusion can be used to produce a steady-state serum concentration of the growth factor

As with GM-CSF therapy, filgrastim given daily following bone marrow transplantation or

intensive chemotherapy will increase granulocyte production and shorten the period of severe neutropenia Frequent blood counts should be obtained to determine the effectiveness of the

treatment The dosage may need to be adjusted according to the granulocyte response, and the duration of therapy will depend on the specific application In marrow transplantation and intensive

chemotherapy patients, continuous daily administration for 14 to 21 days or longer may be

necessary to correct the neutropenia With less intensive chemotherapy, fewer than 7 days of

treatment may be needed In AIDS patients on zidovudine or patients with cyclic neutropenia, chronic G-CSF therapy often will be required

Adverse reactions to filgrastim include mild to moderate bone pain in those patients receiving high doses over a protracted period, local skin reactions following subcutaneous injection, and, rarely, a cutaneous necrotizing vasculitis Patients with a history of hypersensitivity to proteins produced by

E coli should not receive the drug Marked granulocytosis, with counts greater than 100,000/ l, can

occur in patients receiving filgrastim over a prolonged period of time However, this is not

associated with any reported clinical morbidity or mortality and rapidly resolves once therapy is discontinued Mild to moderate splenomegaly has been observed in patients on long-term therapy

The therapeutic roles of other growth factors still need to be defined M-CSF may play a role in stimulating monocyte and macrophage production, though with significant side effects, including splenomegaly and thrombocytopenia Because of their primary effect on primitive marrow

precursors, IL-3 and FL may be used in combination with GM-CSF and G-CSF Administration of IL-3 followed by GM-CSF has been shown to give a greater neutrophil response than GM-CSF alone (Ganser et al. , 1992 ) This combination also may be more effective in promoting the release

of marrow CD34+ stem cells in patients undergoing stem cell pheresis SCF, IL-1, IL-6, IL-9, and IL-11 need to be studied alone and in combination with each other, as well as with both GM-CSF and G-CSF The combination of IL-3 followed by GM-CSF also needs to be studied in protocols that include the reinfusion of harvested stem cells for their growth-promoting activity

Thrombopoietin

The cloning and expression of a recombinant human thrombopoietin, a cytokine that selectively stimulates megakaryocytopoiesis, is another major milestone in the development of hematopoietic growth factors as therapeutic agents (Lok et al. , 1994 ; de Sauvage et al. , 1994 ; Kaushansky et al. , 1994) If future clinical trials live up to the early promise of the demonstrated ability of this new cytokine to increase rapidly the platelet count in animals (Harker, 1999), the combined use of thrombopoietin with G-CSF or GM-CSF together with erythropoietin will have a great impact in the treatment of primary hematological diseases and the anemia, neutropenia, and thrombocytopenia associated with high-dose chemotherapy In a study of a small number of patients with

gynecological cancers receiving carboplatin (Vadhan-Raj et al. , 2000 ), recombinant human

thrombopoietin (rHuTPO) therapy reduced the duration of severe thrombocytopenia as well as the need for platelet transfusions Larger, randomized, controlled trials are now under way to define fully the clinical merits and safety of rHuTPO The optimal dose and schedule of administration in various clinical settings also need to be worked out Both rHuTPO and pegylated recombinant human megakaryocyte growth and development factor (PEG-rHyMGDF) give delayed platelet responses Following a single bolus injection, platelet counts show a detectable increase by day 4 and a peak response by 12 to 14 days The platelet count then returns to normal over the next 4 weeks The peak platelet response follows a log-linear dose response Platelet activation and

aggregation are not affected, and patients are not at increased risk of thromboembolic disease, unless the platelet count is allowed to rise to very high levels These kinetics need to be taken into account when planning therapy in a chemotherapy patient

Drugs Effective in Iron Deficiency and Other Hypochromic Anemias

Iron and Iron Salts

Iron deficiency is the most common cause of nutritional anemia in human beings It can result from inadequate iron intake, malabsorption, blood loss, or an increased requirement, as with pregnancy When severe, it results in a characteristic microcytic, hypochromic anemia However, the impact of iron deficiency is not limited to the erythron (Dallman, 1982) Iron also is an essential component

of myoglobin; heme enzymes such as the cytochromes, catalase, and peroxidase; and the

metalloflavoprotein enzymes, including xanthine oxidase and the mitochondrial enzyme

-glycerophosphate oxidase Iron deficiency can affect metabolism in muscle independently of the effect of anemia on oxygen delivery This may well reflect a reduction in the activity of iron-

dependent mitochondrial enzymes Iron deficiency also has been associated with behavioral and learning problems in children and with abnormalities in catecholamine metabolism and, possibly, heat production (Pollit and Leibel, 1982; Martinez-Torres et al. , 1984 ) Awareness of the ubiquitous role of iron has stimulated considerable interest in the early and accurate detection of iron

deficiency and in its prevention

History

Iron has been used in the treatment of illness since the Middle Ages and the Renaissance However,

it was not until the sixteenth century that iron deficiency was recognized as the cause of "green sickness," or chlorosis, in adolescent women Sydenham subsequently proposed iron as a preferred therapy over bleedings and purgings, and in 1832, the French physician Pierre Blaud recognized the need to use adequate doses of iron to successfully treat chlorosis Blaud's nephew later distributed the "veritable pills of Blaud" throughout the world The treatment of anemia with iron followed the principles enunciated by Sydenham and Blaud until the end of the nineteenth century At that time the teachings of Bunge, Quincke, von Noorden, and others cast doubt on their treatment of

chlorosis The dose of iron employed was reduced, and the resulting lack of efficacy brought

discredit on the therapy It was not until the third and fourth decades of the twentieth century that the lessons taught by the earlier physicians were relearned

The modern understanding of iron metabolism began in 1937 with the work of McCance and

Widdowson on iron absorption and excretion and Heilmeyer and Plotner's measurement of iron in

plasma Then in 1947, Laurell described a plasma iron transport protein that he called transferrin

Hahn and coworkers (1943) were the first to use radioactive isotopes to quantitate iron absorption and define the role of the intestinal mucosa to regulate this function In the next decade, Huff and associates (1950) initiated isotopic studies of internal iron metabolism The subsequent

development of practical clinical measurements of serum iron, transferrin saturation, plasma

ferritin, and red cell protoporphyrin permitted the definition and detection of the body's iron store status and iron-deficient erythropoiesis

Iron and the Environment

Iron exists in the environment largely as ferric oxide or hydroxide or as polymers In this state, its biological availability is limited unless it is solubilized by acid or chelating agents For example, to meet their needs, bacteria and some plants produce high-affinity chelating agents that extract iron from the surrounding environment Most mammals have little difficulty in acquiring iron; this is explained by an ample iron intake and perhaps also by a greater efficiency in absorbing iron

Human beings, however, appear to be an exception Although total dietary intake of elemental iron

in human beings usually exceeds requirements, the bioavailability of the iron in the diet is limited

Metabolism of Iron

The body store of iron is divided between essential iron-containing compounds and excess iron, which is held in storage From a quantitative standpoint, hemoglobin dominates the essential

fraction (Table 54–2) This protein, with a molecular weight of 64,500 daltons, contains four atoms

of iron per molecule, amounting to 1.1 mg of iron per milliliter of red blood cells (20 mM) Other forms of essential iron include myoglobin and a variety of heme and nonheme iron-dependent enzymes Ferritin is a protein-iron storage complex, which exists as individual molecules or in an aggregated form Apoferritin has a molecular weight of about 450,000 daltons and is composed of

24 polypeptide subunits; these form an outer shell within which resides a storage cavity for

polynuclear hydrous ferric oxide phosphate Over 30% of the weight of ferritin may be iron (4000

atoms of iron per ferritin molecule) Aggregated ferritin, referred to as hemosiderin and visible by

light microscopy, constitutes about one-third of normal stores, a fraction that increases as stores enlarge The two predominant sites of iron storage are the reticuloendothelial system and the

hepatocytes, although some storage also occurs in muscle (Bothwell et al. , 1979 )

Internal exchange of iron is accomplished by the plasma protein transferrin (Aisen and Brown, 1977) This 1-glycoprotein has a molecular weight of about 76,000 daltons and two binding sites for ferric iron Iron is delivered from transferrin to intracellular sites by means of specific transferrin receptors in the plasma membrane The iron–transferrin complex binds to the receptor, and the ternary complex is taken up by receptor-mediated endocytosis Iron subsequently dissociates in a pH-dependent fashion in an acidic, intracellular vesicular compartment (the endosomes), and the receptor returns the apotransferrin to the cell surface, where it is released into the extracellular environment (Klausner et al. , 1983 )

Human cells regulate their expression of transferrin receptors and intracellular ferritin in response to the iron supply When iron is plentiful, the synthesis of transferrin receptors is reduced and ferritin production is increased Conversely, with iron deficiency, cells express a greater number of

transferrin receptors and reduce ferritin concentrations to maximize uptake and prevent diversion of iron to stores Isolation of the genes for the human transferrin receptor and ferritin has permitted a better definition of the molecular basis of this regulation Apoferritin synthesis is regulated by a system of cytoplasmic binding proteins (IRP-1 and -2) and an iron-regulating element on mRNA (IRE) When iron is in short supply, IRP binds to mRNA IRE and inhibits the translation of

apoferritin Conversely, when iron is abundant, binding is blocked and apoferritin synthesis

increases (Klausner et al. , 1993 )

The flow of iron through the plasma amounts to a total of 30 to 40 mg per day in the adult (about 0.46 mg/kg of body weight) (Finch and Huebers, 1982) The major internal circulation of iron involves the erythron and the reticuloendothelial cell (Figure 54–3) About 80% of the iron in plasma goes to the erythroid marrow to be packaged into new erythrocytes; these normally circulate for about 120 days before being catabolized by the reticuloendothelium At that time a portion of the iron is immediately returned to the plasma bound to transferrin, while another portion is

incorporated into the ferritin stores of the reticuloendothelial cell and is returned to the circulation more gradually Isotopic studies indicate some degree of iron wastage in this process, wherein defective cells or unused portions of their iron are transferred to the reticuloendothelial cell during maturation, bypassing the circulating blood When there are abnormalities in maturation of red cells, the predominant portion of iron assimilated by the erythroid marrow may be rapidly localized

in the reticuloendothelial cell as defective red cell precursors are broken down; this is termed

ineffective erythropoiesis With red cell aplasia, the rate of turnover of iron in plasma may be

reduced by one-half or more, with all the iron now going to the hepatocyte for storage

Figure 54–3 Pathways of Iron Metabolism in Human Beings (Excretion Omitted)

The most remarkable feature of iron metabolism is the degree to which the body store is conserved

Only 10% of the total is lost per year by normal men, i.e., about 1 mg per day Two-thirds of this

iron is excreted from the gastrointestinal tract as extravasated red cells, iron in bile, and iron in exfoliated mucosal cells The other third is accounted for by small amounts of iron in desquamated skin and in the urine Physiological losses of iron in men vary over a narrow range, from 0.5 mg in the iron-deficient individual to 1.5 to 2 mg per day when excessive iron is consumed Additional losses of iron occur in women due to menstruation While the average loss in menstruating women

is about 0.5 mg per day, 10% of normal menstruating women lose over 2 mg per day Pregnancy imposes a requirement for iron of even greater magnitude (Table 54–3) Other causes of iron loss include the donation of blood, the use of antiinflammatory drugs that cause bleeding from the gastric mucosa, and gastrointestinal disease with associated bleeding Much rarer are the

hemosiderinuria that follows intravascular hemolysis and pulmonary siderosis, wherein iron is deposited in the lungs and becomes unavailable to the rest of the body

The limited physiological losses of iron point to the primary importance of absorption as the

determinant of the body's iron content Unfortunately, the biochemical nature of the absorptive process is understood only in general terms After acidification and partial digestion of food in the stomach, its content of iron is presented to the intestinal mucosa as either inorganic iron or heme iron These fractions are taken up by the absorptive cells of the duodenum and upper small intestine, and the iron is transported either directly into the plasma or stored as mucosal ferritin Absorption appears to be regulated by two separate transporters: DCT1, which controls uptake from the

intestinal lumen, and a second transporter, which governs movement of mucosal cell iron across the basolateral membrane to bind to plasma protein Mucosal cell iron transport and the delivery of iron

to transferrin from reticuloendothelial stores are both determined by the HFE gene, a novel MHC

class 1 molecule localized to chromosome 6 (Peters et al. , 1993 ) Regulation is finely tuned to prevent iron overload in times of iron excess, while allowing for increased absorption and

mobilization of iron stores with iron deficiency Normal absorption is only about 1 mg per day in the adult man and 1.4 mg per day in the adult woman, and 3 to 4 mg of dietary iron is the most that can be absorbed under normal conditions Increased iron absorption is seen whenever iron stores are depleted or when erythropoiesis is increased and ineffective Patients with hereditary

hemochromatosis, secondary to a defective HFE gene, also demonstrate increased iron absorption,

as well as loss of the normal regulation of iron delivery to transferrin by reticuloendothelial cells The resulting increased saturation of transferrin opens the door to abnormal iron deposition in nonhematopoietic tissues

Iron Requirements and the Availability of Dietary Iron

Iron requirements are determined by obligatory physiological losses and the needs imposed by growth Thus, the adult man has a requirement of only 13 g/kg per day (about 1 mg), whereas the menstruating woman requires about 21 g/kg per day (about 1.4 mg) In the last two trimesters of pregnancy, requirements increase to about 80 g/kg per day (5 to 6 mg), and the infant has similar requirements due to its rapid growth These requirements (Table 54–4) must be considered in the context of the amount of dietary iron available for absorption

In developed countries, the normal adult diet contains about 6 mg of iron per 1000 calories,

providing an average daily intake for the adult male of between 12 and 20 mg and for the adult female of between 8 and 15 mg Foods high in iron (greater than 5 mg/100 g) include organ meats such as liver and heart, brewer's yeast, wheat germ, egg yolks, oysters, and certain dried beans and fruits; foods low in iron (less than 1 mg/100 g) include milk and milk products and most nongreen vegetables The content of iron in food is affected further by the manner of its preparation, since iron may be added from cooking in iron pots

Although the iron content of the diet is obviously important, of greater nutritional significance is the bioavailability of iron in food (Hallberg, 1981) Heme iron is far more available, and its absorption

is independent of the composition of the diet Heme iron, which constitutes only 6% of dietary iron, represents 30% of iron absorbed Nevertheless, it is the availability of the nonheme fraction that deserves the greatest attention, since it represents by far the largest amount of dietary iron that is ingested by the economically underprivileged In a vegetarian diet, nonheme iron is absorbed very poorly because of the inhibitory action of a variety of dietary components, particularly phosphates (Layrisse and Martinez-Torres, 1971) Two substances are known to facilitate the absorption of nonheme iron—ascorbic acid and meat Ascorbate forms complexes with and/or reduces ferric to ferrous iron While meat facilitates the absorption of iron by stimulating production of gastric acid,

it is possible that some other effect, not yet identified, also is involved Either of these substances can increase availability severalfold Thus, assessments of available dietary iron should include not only the amount of iron ingested but also an estimate of its availability based on the intake of

substances that enhance or inhibit its absorption and iron stores (Figure 54–4; Monsen et al. , 1978 )

Figure 54–4 Effect of Iron Status on the Absorption of Nonheme Iron in Food The percentages of iron absorbed from diets of low, medium, and high bioavailability in individuals with iron stores of 0, 250, 500, and 1000 mg are portrayed (After Monsen et al. , 1978 ©American Journal of Clinical Nutrition Courtesy of American Society for Clinical Nutrition With permission.)

A comparison of iron requirements with available dietary iron is made in Table 54–4 Obviously, pregnancy and infancy represent periods of negative balance The menstruating woman also is at risk, whereas iron balance in the adult man and nonmenstruating woman is reasonably secure The difference between dietary supply and requirements is reflected in the size of iron stores These will

be low or absent when iron balance is precarious and high when iron balance is favorable (seeTable

53–2) Thus, in the infant after the third month of life and in the pregnant woman after the first trimester, stores of iron are negligible Menstruating women have approximately one-third the stored iron found in the adult man, indicative of the extent to which the additional average daily loss

of about 0.5 mg of iron affects iron balance

Iron Deficiency

The prevalence of iron-deficiency anemia depends on the economic status of the population and on the methods used for evaluation In developing countries, as many as 20% to 40% of infants and pregnant women may be affected (WHO Joint Meeting, 1975), while studies in the United States suggest that the prevalence of iron-deficiency anemia in adult men and women is as low as 0.2% to 3% (Cook et al. , 1986 ) Better iron balance has been achieved by the practice of fortifying flour, the use of iron-fortified formulas for infants, and the prescription of medicinal iron supplements during pregnancy

Iron-deficiency anemia results from a dietary intake of iron that is inadequate to meet normal

requirements (nutritional iron deficiency), blood loss, or some interference with iron absorption Most nutritional iron deficiency in the United States is mild Moderate-to-severe iron deficiency is usually the result of blood loss, either from the gastrointestinal tract or, in the woman, from the uterus Impaired absorption of iron from food results most often from partial gastrectomy or

malabsorption in the small intestine

Iron deficiency in infants and young children can lead to behavioral disturbances and

developmental delays Chronic developmental defects may not be fully reversible Iron deficiency

in children also can lead to an increased risk of lead toxicity secondary to pica and an increased absorption of heavy metals Premature and low-birth-weight infants are at greatest risk for

developing iron deficiency, especially if they are not breast-fed and/or do not receive iron-fortified formula After age 2 to 3, the requirement for iron declines until adolescence, when rapid growth combined with irregular dietary habits again increases the risk of iron deficiency Adolescent girls are at greatest risk; the dietary iron intake of most girls ages 11 to 18 is insufficient to meet their

The recognition of iron deficiency rests on an appreciation of the sequence of events that lead to depletion of iron stores (Hillman and Finch, 1997) A negative balance first results in a reduction of iron stores and, eventually, a parallel decrease in red-cell iron and iron-related enzymes (Figure 54–5) In adults, depletion of iron stores may be recognized by a plasma ferritin of less than 12 g per liter and the absence of reticuloendothelial hemosiderin in the marrow aspirate Iron-deficient erythropoiesis, defined as a suboptimal supply of iron to the erythron, is identified by a decreased saturation of transferrin to less than 16% and/or by an increase above normal in red-cell

protoporphyrin Iron-deficiency anemia is associated with a recognizable decrease in the

concentration of hemoglobin in blood However, the physiological variation in hemoglobin levels is

so great that only about half the individuals with iron-deficient erythropoiesis are identified from their anemia (Cook et al. , 1976 ) Moreover, "normal" hemoglobin and iron values in infancy and childhood are different because of the more restricted supply of iron in young children (Dallman et

al.

, 1980)

Figure 54–5 Sequential Changes (from Left to Right) in the Development of Iron Deficiency in the Adult Rectangles enclose abnormal test results RE marrow Fe, reticuloendothelial hemosiderin; RBC, red blood cells (After Hillman and Finch,

1997, as modified from Bothwell and Finch, 1962 Courtesy of F.A Davis Co With permission.)

The importance of mild iron deficiency lies more in identifying the underlying cause of the

deficiency than in any symptoms related to the deficient state Because of the frequency of iron deficiency in infancy and in the menstruating or pregnant woman, the need for exhaustive

evaluation of such individuals usually is determined by the severity of the anemia However, iron deficiency in the man or postmenopausal woman necessitates a search for a site of bleeding

Although the presence of microcytic anemia is the most commonly recognized indicator of iron

deficiency, laboratory tests—such as quantitation of transferrin saturation, red cell protoporphyrin, and plasma ferritin—are required to distinguish iron deficiency from other causes of microcytosis Such measurements are particularly useful when circulating red cells are not yet microcytic because

of the recent nature of blood loss, but iron supply is nonetheless limiting erythropoiesis More difficult is the differentiation of true iron deficiency from iron-deficient erythropoiesis due to

inflammation In the latter condition, the stores of iron are actually increased, but the release of iron from reticuloendothelial cells is blocked; the concentration of iron in plasma is decreased, and the supply of iron to the erythroid marrow becomes inadequate The increased stores of iron in this condition may be demonstrated directly by examination of an aspirate of marrow or may be inferred from determination of an elevated concentration of ferritin in plasma (Lipschitz et al. , 1974 )

Treatment of Iron Deficiency

General Therapeutic Principles

The response of iron-deficiency anemia to iron therapy is influenced by several factors, including the severity of anemia, the ability of the patient to tolerate and absorb medicinal iron, and the

presence of other complicating illnesses Therapeutic effectiveness can be best measured from the resulting increase in the rate of production of red cells The magnitude of the marrow response to iron therapy is proportional to the severity of the anemia (level of erythropoietin stimulation) and the amount of iron delivered to marrow precursors Studies by Hillman and Henderson (1969) demonstrated the importance of iron supply in governing erythropoiesis Using phlebotomy to induce a moderately severe anemia (hemoglobin 7 to 10 g/dl), erythropoiesis was reduced to less than one-third of normal when the serum iron fell below 70 g/dl In contrast, red cell production levels increased to more than three times the basal rate when the serum iron was maintained

between 75 and 150 g/dl Even higher levels of production were observed in patients with

hemolytic anemias or ineffective erythropoiesis

As regards oral iron therapy, the ability of the patient to tolerate and absorb medicinal iron is a very important factor in determining the rate of response There are clear limits to the gastrointestinal tolerance for iron The small intestine regulates absorption and, in the face of increasing doses of oral iron, limits the entry of iron into the bloodstream Therefore, there is a natural ceiling on how much iron can be supplied by oral therapy In the patient with a moderately severe iron-deficiency anemia, tolerable doses of oral iron will deliver, at most, 40 to 60 mg of iron per day to the

erythroid marrow This is an amount sufficient for production rates of two to three times normal

Complicating illness also can interfere with the response of an iron-deficiency anemia to iron

therapy Intrinsic disease of the marrow can, by decreasing the number of red cell precursors, blunt the response Inflammatory illnesses suppress the rate of red cell production, both by reducing iron absorption and reticuloendothelial release and by direct inhibition of erythropoietin and erythroid precursors Continued blood loss can mask the response as measured by recovery of the hemoglobin

or hematocrit

Clinically, the effectiveness of iron therapy is best evaluated by tracking the reticulocyte response and the rise in the hemoglobin or the hematocrit Since it takes time for the marrow to proliferate,

an increase in the reticulocyte count is not observed for 4 to 7 days or more after beginning therapy

A measurable increase in the hemoglobin level takes even longer A decision as to the effectiveness

of treatment should not be made for 3 to 4 weeks after the start of treatment An increase of 20 g per liter or more in the concentration of hemoglobin by that time should be considered a positive

response, assuming that no other change in the patient's clinical status can account for the

improvement It also assumes that the patient has not been transfused during this time.

If the response to oral iron is inadequate, the diagnosis must be reconsidered A full laboratory evaluation should be carried out, and such factors as the presence of a concurrent inflammatory disease or poor compliance by the patient must be assessed A source of continued bleeding

obviously should be sought If no other explanation can be found, an evaluation of the patient's ability to absorb oral iron should be considered There is no justification for merely continuing oral iron therapy beyond 3 to 4 weeks if a favorable response has not occurred

Once a response to oral iron is demonstrated, therapy should be continued until the hemoglobin returns to normal Treatment may be extended if it is desirable to establish iron stores This may require a considerable period of time, since the rate of absorption of iron by the intestine will

decrease markedly as iron stores are reconstituted The prophylactic use of oral iron should be reserved for patients at high risk, including pregnant women, women with excessive menstrual blood loss, and infants Iron supplements also may be of value for rapidly growing infants who are consuming substandard diets and for adults with a recognized cause of chronic blood loss Except for infants, in whom the use of supplemented formulas is routine, the use of "over-the-counter" mixtures of vitamins and minerals to prevent iron deficiency should be discouraged

Therapy with Oral Iron

Orally administered ferrous sulfate, the least expensive of iron preparations, is the treatment of choice for iron deficiency (Callender, 1974; Bothwell et al. , 1979 ) Ferrous salts are absorbed about three times as well as ferric salts, and the discrepancy becomes even greater at high dosage (Brise and Hallberg, 1962) Variations in the particular ferrous salt have relatively little effect on

bioavailability, and the sulfate, fumarate, succinate, gluconate, and other ferrous salts are absorbed

to approximately the same extent

Ferrous sulfate (iron sulfate;FEOSOL, others) is the hydrated salt, FeSO 4·7H2O, which contains 20%

iron Dried ferrous sulfate (32% elemental iron) also is available Ferrous fumarate (FEOSTAT, others) contains 33% iron and is moderately soluble in water, stable, and almost tasteless Ferrous

gluconate (FERGON, others) also has been successfully used in the therapy of iron-deficiency

anemia The gluconate contains 12% iron Polysaccharide–iron complex (NIFEREX, others), a

compound of ferrihydrite and carbohydrate, is another preparation with comparable absorption The effective dose of all of these preparations is based on iron content

Other iron compounds have utility in fortification of foods Reduced iron (metallic iron, elemental iron) is as effective as ferrous sulfate, provided that the material employed has a small particle size

Large-particle ferrum reductum and iron phosphate salts have a much lower bioavailability (Cook

et al , 1973), and their use for the fortification of foods is undoubtedly responsible for some of the

confusion concerning effectiveness Ferric edetate has been shown to have good bioavailability and

to have advantages for maintenance of the normal appearance and taste of food (Viteri et al. , 1978 ).The amount of iron, rather than the mass of the total salt in iron tablets, is important It is also essential that the coating of the tablet dissolve rapidly in the stomach Surprisingly, since iron usually is absorbed in the upper small intestine, certain delayed-release preparations have been reported to be effective and have been said to be even more effective than ferrous sulfate when taken with meals However, reports of absorption from such preparations vary Because a number

of different forms of delayed-release preparations are on the market and information on their

bioavailability is limited, the effectiveness of most such preparations must be considered

A variety of substances designed to enhance the absorption of iron has been marketed, including surface-acting agents, carbohydrates, inorganic salts, amino acids, and vitamins One of the more popular of these is ascorbic acid When present in an amount of 200 mg or more, ascorbic acid increases the absorption of medicinal iron by at least 30% However, the increased uptake is

associated with a significant increase in the incidence of side effects (Hallberg et al. , 1966 );

therefore, the addition of ascorbic acid seems to have little advantage over increasing the amount of iron administered It is inadvisable to use preparations that contain other compounds with

therapeutic actions of their own, such as vitamin B12, folate, or cobalt, since the patient's response to the combination cannot be easily interpreted

The average dose for the treatment of iron-deficiency anemia is about 200 mg of iron per day (2 to

3 mg/kg), given in three equal doses of 65 mg Children weighing 15 to 30 kg can take half the average adult dose, while small children and infants can tolerate relatively large doses of iron—for example, 5 mg/kg The dose used is a practical compromise between the therapeutic action desired and the toxic effects Prophylaxis and mild nutritional iron deficiency may be managed with modest doses When the object is the prevention of iron deficiency in pregnant women, for example, doses

of 15 to 30 mg of iron per day are adequate to meet the 3- to 6-mg daily requirement of the last two trimesters When the purpose is to treat iron-deficiency anemia, but the circumstances do not

demand haste, a total dose of about 100 mg (35 mg three times daily) may be used

The responses expected for different dosage regimens of oral iron are given in Table 54–5

However, these effects are modified by the severity of the iron-deficiency anemia and by the time

of ingestion of iron relative to meals Bioavailability of iron ingested with food is probably one-half

or one-third of that seen in the fasting subject (Grebe et al. , 1975 ) Antacids also reduce the

absorption of iron if given concurrently It is always preferable to administer iron in the fasting state, even if the dose must be reduced because of gastrointestinal side effects For patients who require maximal therapy to encourage a rapid response or to counteract continued bleeding, as much as 120 mg of iron may be administered four times a day The timing of the dose is important Sustained high rates of red cell production require an uninterrupted supply of iron Oral doses should be spaced equally to maintain a continuous high concentration of iron in plasma

The duration of treatment is governed by the rate of recovery of hemoglobin and the desire to create iron stores The former depends on the severity of the anemia With a daily rate of repair of 2 g of hemoglobin per liter of whole blood, the red cell mass is usually reconstituted within 1 to 2 months Thus, an individual with a hemoglobin of 50 g per liter may achieve a normal complement of 150 g per liter in about 50 days, whereas an individual with a hemoglobin of 100 g per liter may take only half that time The creation of stores of iron is a different matter, requiring many months of oral iron administration The rate of absorption decreases rapidly after recovery from anemia and, after 3 to 4 months of treatment, stores may increase at a rate of not much more than 100 mg per month Much

of the strategy of continued therapy depends on the estimated future iron balance of the individual The person with an inadequate diet may require continued therapy with low doses of iron The individual whose bleeding has stopped will require no further therapy after the hemoglobin has returned to normal For the individual with continued bleeding, long-term, high-dose therapy is clearly indicated

Untoward Effects of Oral Preparations of Iron

Intolerance to oral preparations of iron is primarily a function of the amount of soluble iron in the

upper gastrointestinal tract and of psychological factors Side effects include heartburn, nausea, upper gastric discomfort, constipation, and diarrhea A good policy, particularly if there has been previous intolerance to iron, is to initiate therapy at a small dosage, to demonstrate freedom from symptoms at that level, and then gradually to increase the dosage to that desired With a dose of 200

mg of iron per day divided into three equal portions, symptoms occur in approximately 25% of individuals, compared with an incidence of 13% among those receiving placebos; this increases to approximately 40% when the dosage of iron is doubled Nausea and upper abdominal pain are increasingly common manifestations at high dosage Constipation and diarrhea, perhaps related to iron-induced changes in the intestinal bacterial flora, are not more prevalent at higher dosage, nor is heartburn If a liquid is given, one can place the iron solution on the back of the tongue with a dropper to prevent transient staining of teeth

Toxicity caused by the long-continued administration of iron, with the resultant production of iron overload (hemochromatosis), has been the subject of a number of case reports (for example,

seeBothwell et al. , 1979 ) Available evidence suggests that the normal individual is able to control absorption of iron despite high intake, and it is only individuals with underlying disorders that augment the absorption of iron who run the hazard of developing hemochromatosis However, recent data indicate that hemochromatosis may be a relatively common genetic disorder, present in 0.5% of the population

Iron Poisoning

Large amounts of ferrous salts of iron are toxic but, in adults, fatalities are rare Most deaths occur

in childhood, particularly between the ages of 12 and 24 months (Bothwell et al. , 1979 ) As little as

1 to 2 g of iron may cause death, but 2 to 10 g is usually ingested in fatal cases The frequency of iron poisoning relates to its availability in the household, particularly the supply that remains after a pregnancy The colored sugar coating of many of the commercially available tablets gives them the appearance of candy All iron preparations should, therefore, be kept in childproof bottles

Signs and symptoms of severe poisoning may occur within 30 minutes or may be delayed for several hours after ingestion They consist largely of abdominal pain, diarrhea, or vomiting of brown or bloody stomach contents containing pills Of particular concern are pallor or cyanosis, lassitude, drowsiness, hyperventilation due to acidosis, and cardiovascular collapse If death does not occur within 6 hours, there may be a transient period of apparent recovery, followed by death in

12 to 24 hours The corrosive injury to the stomach may result in pyloric stenosis or gastric

scarring Hemorrhagic gastroenteritis and hepatic damage are prominent findings at autopsy In the evaluation of the child who is thought to have ingested iron, a color test for iron in the gastric contents and an emergency determination of the concentration of iron in plasma can be performed

If the latter is less than 63 M (3.5 mg per liter), the child is not in immediate danger However, vomiting should be induced when there is iron in the stomach, and an X-ray should be taken to evaluate the number of pills remaining in the small bowel (iron tablets are radioopaque) Iron in the upper gastrointestinal tract can be precipitated by lavage with sodium bicarbonate or phosphate solution, although the clinical benefit is questionable When the plasma concentration of iron is greater than the total iron-binding capacity (63 M; 3.5 mg per liter), deferoxamine should be administered; dosage and routes of administration are detailed in Chapter 67: Heavy Metals and Heavy-Metal Antagonists Shock, dehydration, and acid-base abnormalities should be treated in the conventional manner Most important is the speed of diagnosis and therapy With early effective treatment, the mortality from iron poisoning can be reduced from as high as 45% to about 1%

Therapy with Parenteral Iron

When oral iron therapy fails, parenteral iron administration may be an effective alternative

(Bothwell et al. , 1979 ) The rate of response to parenteral therapy is similar to that which follows usual oral doses (Pritchard, 1966) Predictable indications are iron malabsorption (sprue, short

bowel, etc.), severe oral iron intolerance, as a routine supplement to total parenteral nutrition, and in

patients with renal disease who are receiving erythropoietin (Eschbach et al. , 1987 ) Parenteral iron also has been given to iron-deficient patients and pregnant women to create iron stores, something

that would take months to achieve by the oral route Parenteral iron therapy should be used only

when clearly indicated, since acute hypersensitivity, including anaphylactic and anaphylactoid reactions, can occur in from 0.2% to 3% of patients The belief that the response to parenteral

iron, especially iron dextran, is faster than oral iron is open to debate (Pritchard, 1966) In otherwise healthy individuals, the rate of hemoglobin response is determined by the balance between the severity of the anemia (the level of erythropoietin stimulus) and the delivery of iron to the marrow from iron absorption and iron stores When a large intravenous dose of iron dextran is given to a severely anemic patient, the hematologic response can exceed that seen with oral iron for 1 to 3 weeks (Henderson and Hillman, 1969) Subsequently, however, the response is no better than that seen with oral iron This reflects the relative availability of the iron dextran stored in the

reticuloendothelial system Furthermore, inflammatory cytokines suppress both sources of iron supply equally, canceling any advantage

Iron dextran injection (INFED, DEXFERRUM) is the parenteral preparation currently in general use in the United States It is a colloidal solution of ferric oxyhydroxide complexed with polymerized dextran (molecular weight approximately 180,000), resulting in a dark brown, viscous liquid,

containing 50 mg/ml of elemental iron It can be administered by either intramuscular or

intravenous injection When given by deep intramuscular injection, it is gradually mobilized via the

lymphatics and transported to reticuloendothelial cells; the iron is then released from the dextran complex A variable portion (10% to 50%) may become locally fixed in the muscle for several weeks or months, especially if there is a local inflammatory reaction Intravenous administration gives a more reliable response and is preferred Given intravenously in a dose of less than 500 mg, the iron dextran complex is cleared exponentially with a plasma half-life of 6 hours When 1 g or more is administered intravenously as total dose therapy, reticuloendothelial cell clearance is

constant at 10 to 20 mg/hour This slow rate of clearance results in a brownish discoloration of the plasma for several days and an elevation of the serum iron level for 1 to 2 weeks

Once the iron is released from the dextran within the reticuloendothelial cell, it is either

incorporated into stores or transported via transferrin to the erythroid marrow The rate of release is

variable While a portion of the processed iron is rapidly made available to the marrow, a significant fraction is only gradually converted to usuable iron stores (Henderson and Hillman, 1969) All of the iron is eventually released (Kernoff et al. , 1975 ), although many months are required before the process is complete During this time, the appearance of visible iron dextran stores in

reticuloendothelial cells can confuse the clinician who attempts to evaluate the iron status of the patient

Intramuscular injection of iron dextran should only be initiated following a test dose of 0.5 ml (25

mg of iron) If no adverse reactions are observed, the injection can be given according to the

following schedule until the calculated total amount required has been reached Each day's dose should ordinarily not exceed 0.5 ml (25 mg of iron) for infants under 4.5 kg (10 lb), 1.0 ml (50 mg

of iron) for children under 9.0 kg (20 lb), and 2.0 ml (100 mg of iron) for other patients Iron

dextran should be injected only into the muscle mass of the upper outer quadrant of the buttock

using a z-track technique (displacement of the skin laterally prior to injection) However, local reactions, including long-continued discomfort at the site of injection and local discoloration of the skin, and the concern about malignant change at the site of injection (Weinbren et al. , 1978 ) make intramuscular administration inappropriate except when the intravenous route is inaccessible.

A test dose injection also should precede intravenous administration of a therapeutic dose of iron dextran After establishing secure intravenous access, 0.5 ml of undiluted iron dextran or an

equivalent amount (25 mg of iron) diluted in saline should be administered The patient should be observed during the injection for signs of immediate anaphylaxis, and for an hour following

injection for any signs of vascular instability or hypersensitivity, including respiratory distress, hypotension, tachycardia, or back or chest pain When widely spaced, total-dose infusion therapy is employed, a test dose injection should be given prior to each infusion, since hypersensitivity can appear at any time Furthermore, the patient should be closely monitored throughout the infusion for signs of cardiovascular instability Delayed hypersensitivity reactions also are observed, especially

in patients with rheumatoid arthritis or a history of allergies Fever, malaise, lymphadenopathy, arthralgias, and urticaria can develop days or weeks following injection and last for prolonged periods of time Therefore, iron dextran should be used with extreme caution in patients with

rheumatoid arthritis, other connective tissue diseases, and during the acute phase of an

inflammatory illness Once hypersensitivity is documented, iron dextran therapy must be

abandoned

Before initiating iron dextran therapy, the total dose of iron required to repair the patient's deficient state should be calculated Factors to be taken into account are the hemoglobin deficit, the need to reconstitute iron stores, and continued excess losses of iron, as seen with hemodialysis and chronic gastrointestinal bleeding A manufacturer-recommended formula for the calculation of the total treatment dose for an iron deficient anemia patient is as follows:

iron-[0.0476 x lean body weight in kg x hemoglobin deficit]+ 1 ml per 5 kg body weight (to maximum of

14 ml to reconstitute iron stores) = total dose in ml of iron dextran solution

An alternative formula, which calculates the total dose in mg of iron, is as follows:

[0.66 x lean body weight in kg]x[100 – (patient's hemoglobin in g/dl x 100/14.8)]= Total dose in mg

of iron

Iron dextran solution (50 mg/ml of elemental iron) can then be administered undiluted in daily doses of 2.0 ml until the total dose is reached or given as a single total-dose infusion In the latter case, the iron dextran should be diluted in 250 to 1000 ml of 0.9% saline and infused over an hour

or more

When hemodialysis patients are started on erythropoietin, oral iron therapy alone is generally

insufficient to guarantee an optimal hemoglobin response It is recommended, therefore, that

sufficient parenteral iron be given to maintain a plasma ferritin level between 100 and 800 g/l and

a percent saturation of transferrin between 20% and 50% One approach is to administer an initial intravenous dose of 200 to 500 mg, followed by weekly or every-other-week injections of 25 to 100

mg of iron dextran to replace ongoing blood loss (Besarab et al. , 1999 ) With repeated doses of iron dextran—especially multiple total-dose infusions, as sometimes used in the treatment of chronic gastrointestinal blood loss—accumulations of slowly metabolized iron dextran stores in

reticuloendothelial cells can be impressive The plasma ferritin level also can rise to levels

associated with iron overload Whether this is of any clinical importance is uncertain While

disease-related iron overload (hemochromatosis) has been associated with an increased risk of both infections and cardiovascular disease, this has not been shown to be true in hemodialysis patients treated with iron dextran (Owen, 1999) It seems prudent, however, to withhold the drug whenever the plasma ferritin rises above 800 g/l.

Sodium ferric gluconate complex in sucrose (FERRLECIT) was approved by the FDA for use in the treatment of iron deficiency in patients undergoing chronic hemodialysis who are receiving

supplemental erythropoietin therapy

Reactions to intravenous iron include headache, malaise, fever, generalized lymphadenopathy, arthralgias, urticaria, and, in some patients with rheumatoid arthritis, exacerbation of the disease Phlebitis may occur with prolonged infusions of a concentrated solution or when an intramuscular preparation containing 0.5% phenol is used in error Of greatest concern, however, is the rare anaphylactic reaction, which may be fatal in spite of treatment While only a few such deaths have been reported, it remains a deterrent to the use of iron dextran Thus, there must be specific

indications for the parenteral administration of iron

Copper

Copper deficiency is extremely rare in human beings (Evans, 1973) The amount present in food is more than adequate to provide the needed body complement of slightly more than 100 mg There is

no evidence that copper ever needs to be added to a normal diet, either prophylactically or

therapeutically Even in clinical states associated with hypocupremia (sprue, celiac disease,

nephrotic syndrome), effects of copper deficiency usually are not demonstrable However, anemia due to copper deficiency has been described in individuals who have undergone intestinal bypass surgery (Zidar et al. , 1977 ), in those who are receiving parenteral nutrition (Dunlap et al. , 1974 ), in malnourished infants (Holtzman et al. , 1970 ; Graham and Cordano, 1976), and in patients ingesting excessive amounts of zinc (Hoffman et al. , 1988 ) While an inherited disorder affecting the

transport of copper in human beings (Menkes' disease; steely hair syndrome) is associated with reduced activity of several copper-dependent enzymes, this disease is not associated with

granulocytopenia, and anemia Concentrations of iron in plasma are variable, and the anemia is not always microcytic When a low plasma copper concentration is determined in the presence of leukopenia and anemia and in a setting conducive to a deficiency of the element, a therapeutic trial with copper is appropriate Daily doses up to 0.1 mg/kg of cupric sulfate have been given by mouth,

or 1 to 2 mg per day may be added to the solution of nutrients for parenteral administration Copper deficiency usually occurs concurrently with multiple nutritional deficiencies, so that its specific role

in the production of anemia may be difficult to ascertain

Pyridoxine

The first case of pyridoxine-responsive anemia was described in 1956 by Harris and associates

Subsequent reports suggested that the vitamin might improve hematopoiesis in up to 50% of

patients with either hereditary or acquired sideroblastic anemias (Horrigan and Harris, 1968)

Characteristically, these patients show an impairment in hemoglobin synthesis and an accumulation

of iron in the perinuclear mitochondria of erythroid precursor cells, so-called ringed sideroblasts Hereditary sideroblastic anemia is an X-linked recessive trait with variable penetrance and

expression Affected men typically show a dual population of normal red cells and microcytic, hypochromic cells in the circulation In contrast, idiopathic acquired sideroblastic anemia and the sideroblastosis seen in association with a number of drugs, inflammatory states, neoplastic

disorders, and preleukemic syndromes show a variable morphological picture Moreover,

erythrokinetic studies demonstrate a spectrum of abnormalities, from a hypoproliferative defect with little tendency to accumulate iron to marked ineffective erythropoiesis with iron overload of the tissues (Solomon and Hillman, 1979a)

Oral therapy with pyridoxine is of proven benefit in correcting the sideroblastic anemias associated with the antituberculosis drugs isoniazid and pyrazinamide, which act as vitamin B6 antagonists A daily dose of 50 mg of pyridoxine completely corrects the defect without interfering with treatment,

and routine supplementation of pyridoxine is often recommended (seeChapter 48: Antimicrobial

Agents: Drugs Used in the Chemotherapy of Tuberculosis, Mycobacterium avium Complex

Disease, and Leprosy) In contrast, if pyridoxine is given to counteract the sideroblastic abnormality associated with administration of levodopa, the effectiveness of levodopa in controlling Parkinson's disease is decreased Pyridoxine therapy does not correct the sideroblastic abnormalities produced

by chloramphenicol and lead

Patients with idiopathic acquired sideroblastic anemia generally fail to respond to oral pyridoxine, and those individuals who appear to have a pyridoxine-responsive anemia require prolonged

therapy with large doses of the vitamin, 50 to 500 mg per day Unfortunately, the early enthusiasm for treatment with pyridoxine was not reinforced by results of later studies (Chillar et al. , 1976 ; Solomon and Hillman, 1979a) Moreover, even when a patient with sideroblastic anemia responds, the improvement is only partial, since both the ringed sideroblasts and the red cell defect persist, and the hematocrit rarely returns to normal However, in view of the low toxicity of oral pyridoxine,

a therapeutic trial with pyridoxine is appropriate

As shown in studies of normal subjects, oral pyridoxine in a dosage of 100 mg three times daily produces a maximal increase in red cell pyridoxine kinase and the major pyridoxal phosphate–dependent enzyme glutamic-aspartic aminotransferase (Solomon and Hillman, 1978) For an

adequate therapeutic trial, the drug must be administered for at least 3 months while the response is monitored by measuring the reticulocyte index and the concentration of hemoglobin It has been suggested that the occasional patient who is refractory to oral pyridoxine may respond to parenteral administration of pyridoxal phosphate (Hines and Love, 1975) However, oral pyridoxine in doses

of 200 to 300 mg per day produces intracellular concentrations of pyridoxal phosphate equal to or greater than those generated by therapy with the phosphorylated vitamin (Solomon and Hillman, 1979b) Pyridoxine is discussed further in Chapter 63: Water-Soluble Vitamins: The Vitamin B Complex and Ascorbic Acid

riboflavin deficiency is undoubtedly rare, if it occurs at all It has been described in combination with infection and protein deficiency, both of which are capable of producing a hypoproliferative anemia However, it seems reasonable to include riboflavin in the nutritional management of

patients with gross, generalized malnutrition Riboflavin is discussed further in Chapter 63: Soluble Vitamins: The Vitamin B Complex and Ascorbic Acid

Water-Vitamin B12, Folic Acid, and the Treatment of Megaloblastic Anemias

Vitamin B12 and folic acid are dietary essentials A deficiency of either vitamin results in defective synthesis of DNA in any cell in which chromosomal replication and division are taking place Since tissues with the greatest rate of cell turnover show the most dramatic changes, the hematopoietic system is especially sensitive to deficiencies of these vitamins An early sign of deficiency is a megaloblastic anemia Abnormal macrocytic red blood cells are produced, and the patient becomes severely anemic Recognition of this pattern of abnormal hematopoiesis—more than 100 years ago

—permitted the initial diagnostic classification of such patients as having "pernicious anemia" and spurred investigations that subsequently led to the discovery of vitamin B12 and folic acid Even today, the characteristic abnormality in red blood cell morphology is important for diagnosis and as

a therapeutic guide following administration of the vitamins

History

The discovery of vitamin B12 and folic acid is a dramatic story that starts more than 170 years ago and includes two Nobel prize–winning discoveries The first descriptions of what must have been megaloblastic anemias came from the work of Combe and Addison, who published a series of case reports beginning in 1824 It is still common practice to describe megaloblastic anemia as

Addisonian pernicious anemia Although Combe suggested that the disorder might have some relationship to digestion, it was Austin Flint who, in 1860, first described the severe gastric atrophy

and called attention to its possible relationship to the anemia The name progressive pernicious

anemia was coined in 1872 by Biermer, and this colorful term has persisted.

Following the observation by Whipple in 1925 that liver is a source of a potent hematopoietic substance for iron-deficient dogs, Minot and Murphy carried out their Nobel Prize–winning

experiments that demonstrated the effectiveness of the feeding of liver to reverse pernicious anemia Within a few years, Castle defined the need for both intrinsic factor, a substance secreted by the parietal cells of the gastric mucosa, and extrinsic factor, the vitamin-like material provided by crude liver extracts However, nearly 20 years passed before Rickes and coworkers and Smith and Parker isolated and crystallized vitamin B12; Dorothy Hodgkin then determined its crystal structure by X-ray diffraction and subsequently received the Nobel prize for this work

As attempts were being made to purify extrinsic factor, Wills and her associates described a

macrocytic anemia in women in India that responded to a factor present in crude liver extracts but not in the purified fractions known to be effective in pernicious anemia (Wills et al. , 1937 ) This factor, first called Wills' factor and later vitamin M, is now known to be folic acid The actual term

folic acid was coined by Mitchell and coworkers in 1941, following its isolation from leafy

vegetables

More recent work has shown that neither vitamin B12 nor folic acid as purified from foodstuffs is the active coenzyme for human beings During extraction procedures, active, labile forms are converted

to stable congeners of vitamin B12 and folic acid, cyanocobalamin and pteroylglutamic acid,

respectively These congeners must then be modified in vivo to be effective While a great deal has

been learned about the intracellular metabolic pathways in which these vitamins function as

required cofactors, many questions remain The most important of these is the relationship of vitamin B12 deficiency to the neurological abnormalities that occur with this disorder (Chanarin et

al.

, 1985)

Relationships between Vitamin B12 and Folic Acid

The major roles of vitamin B12 and folic acid in intracellular metabolism are summarized in Figure 54–6 Intracellular vitamin B12 is maintained as two active coenzymes: methylcobalamin and deoxyadenosylcobalamin (Linnell et al. , 1971 ) Deoxyadenosylcobalamin (deoxyadenosyl B12) is a cofactor for the mitochondrial mutase enzyme that catalyzes the isomerization of L-methylmalonyl CoA to succinyl CoA, an important reaction in both carbohydrate and lipid metabolism (Weissbach and Taylor, 1968) This reaction has no direct relationship to the metabolic pathways that involve folate In contrast, methylcobalamin (CH3B12) supports the methionine synthetase reaction, which is essential for normal metabolism of folate (Weir and Scott, 1983) Methyl groups contributed by methyltetrahydrofolate (CH3H4PteGlu1) are used to form methylcobalamin, which then acts as a methyl group donor for the conversion of homocysteine to methionine This folate–cobalamin interaction is pivotal for normal synthesis of purines and pyrimidines and, therefore, of DNA The methionine synthetase reaction is largely responsible for the control of the recycling of folate cofactors; the maintenance of intracellular concentrations of folylpolyglutamates; and, through the

synthesis of methionine and its product, S-adenosylmethionine, the maintenance of a number of

methylation reactions

Figure 54–6 Interrelationships and Metabolic Roles of Vitamin B12 and Folic Acid See text for explanation and Figure 54–9 for structures of the various folate coenzymes FIGLU is formiminoglutamic acid, which arises from the catabolism

of histidine TcII is transcobalamin II

Since methyltetrahydrofolate is the principal folate congener supplied to cells, the transfer of the methyl group to cobalamin is essential for the adequate supply of tetrahydrofolate (H4PteGlu1), the

substrate for a number of metabolic steps Tetrahydrofolate is a precursor for the formation of intracellular folylpolyglutamates; it also acts as the acceptor of a one-carbon unit in the conversion

of serine to glycine, with the resultant formation of 5,10-methylenetetrahydrofolate

(5,10-CH2H4PteGlu) The latter derivative donates the methylene group to deoxyuridylate (dUMP) for the synthesis of thymidylate (dTMP)—an extremely important reaction in DNA synthesis In the

process, the 5,10-CH2H4PteGlu is converted to dihydrofolate (H2PteGlu) The cycle is then

completed by the reduction of the H2PteGlu to H4PteGlu by dihydrofolate reductase, the step that is blocked by folate antagonists such as methotrexate (seeChapter 52: Antineoplastic Agents) As shown in Figure 54–6, several other pathways also lead to the synthesis of 5,10-

methylenetetrahydrofolate These pathways are important in the metabolism of formiminoglutamic

acid (FIGLU) and both purines and pyrimidines (See reviews by Weir and Scott, 1983; Chanarin et

al.

, 1985.)

In the presence of a deficiency of either vitamin B12 or folate, the decreased synthesis of methionine

and S-adenosylmethionine interferes with protein biosynthesis, a number of methylation reactions,

and the synthesis of polyamines In addition, the cell responds to the deficiency by redirecting folate metabolic pathways to supply increasing amounts of methyltetrahydrofolate; this tends to preserve essential methylation reactions at the expense of nucleic acid synthesis With vitamin B12

deficiency, methylenetetrahydrofolate reductase activity increases, directing available intracellular folates into the methyltetrahydrofolate pool (not shown in Figure 54–6) The methyltetrahydrofolate

is then trapped by the lack of sufficient vitamin B12 to accept and transfer methyl groups, and

subsequent steps in folate metabolism that require tetrahydrofolate are deprived of substrate This process provides a common basis for the development of a megaloblastic anemia with deficiency of either vitamin B12 or folic acid

The mechanisms responsible for the neurological lesions of vitamin B12 deficiency are less well understood (Reynolds, 1976; Weir and Scott, 1983) Damage to the myelin sheath is the most obvious lesion in this neuropathy This observation led to the early suggestion that the

deoxyadenosyl B12-dependent methylmalonyl CoA mutase reaction, a step in propionate

metabolism, is related to the abnormality However, other evidence suggests that the deficiency of methionine synthetase and the block of the conversion of methionine to S-adenosylmethionine is more likely to be responsible (Scott et al. , 1981 )

Nitrous oxide (dinitrogen monoxide; N2O), used for anesthesia (seeChapter 14: General

Anesthetics), can cause megaloblastic changes in the marrow and a neuropathy that resemble those

of vitamin B12 deficiency (Chanarin et al. , 1985 ) Studies with N2O have demonstrated a reduction

in methionine synthetase and reduced concentrations of methionine and S-adenosylmethionine The latter is necessary for methylation reactions, including those required for the synthesis of

phospholipids and myelin Significantly, the neuropathy induced with N2O can be prevented

partially by feeding methionine A neuropathy similar to that occurring with vitamin B12 deficiency has been reported in dentists who are exposed to N2O used as an anesthetic (Layzer, 1978)

Vitamin B12

Chemistry

The structural formula of vitamin B12 is shown in Figure 54–7 (Pratt, 1972) The three major

portions of the molecule are:

Figure 54–7 The Structures and Nomenclature of Vitamin B12 Congeners

1

A planar group or corrin nucleus—a porphyrin-like ring structure with four reduced pyrrole rings (A to D in Figure 54–7) linked to a central cobalt atom and extensively substituted with methyl, acetamide, and propionamide residues

2

A 5,6-dimethylbenzimidazolyl nucleotide, which links almost at right angles to the corrin nucleus with bonds to the cobalt atom and to the propionate side chain of the C pyrrole ring

3

A variable R group—the most important of which are found in the stable compounds

cyanocobalamin and hydroxocobalamin and the active coenzymes methylcobalamin and deoxyadenosylcobalamin

5-The terms vitamin B 12 and cyanocobalamin are used interchangeably as generic terms for all of the

cobamides active in human beings Preparations of vitamin B12 for therapeutic use contain either cyanocobalamin or hydroxocobalamin, since only these derivatives remain active following storage.Metabolic Functions

The active coenzymes methylcobalamin and 5-deoxyadenosylcobalamin are essential for cell growth and replication Methylcobalamin is required for the formation of methionine and its

derivative S-adenosylmethionine from homocysteine In addition, when concentrations of vitamin

B12 are inadequate, folate becomes "trapped" as methyltetrahydrofolate to cause a functional

deficiency of other required intracellular forms of folic acid (seeFigures 54–6 and 54–7 and

discussion above) The hematological abnormalities that are observed in vitamin B12–deficient patients are the result of this process (Herbert and Zalusky, 1962) 5-Deoxyadenosylcobalamin is required for the isomerization of L-methylmalonyl CoA to succinyl CoA (Figure 54–6)

Sources in Nature

Human beings depend on exogenous sources of vitamin B12 In nature, the primary sources are certain microorganisms that grow in soil, sewage, water, or the intestinal lumen of animals and that synthesize the vitamin Vegetable products are free of vitamin B12 unless they are contaminated with such microorganisms, so that animals are dependent on synthesis in their own alimentary tract

or the ingestion of animal products containing vitamin B12 The daily nutritional requirement of 3 to

5 g must be obtained from animal by-products in the diet At the same time, strict vegetarians rarely develop vitamin B12 deficiency Some vitamin B12 is available from legumes, which are contaminated with bacteria capable of synthesizing vitamin B12, and vegetarians generally fortify their diets with a wide range of vitamins and minerals

Absorption, Distribution, Elimination, and Daily Requirements

Dietary vitamin B12, in the presence of gastric acid and pancreatic proteases, is released from food and salivary binding protein and bound to gastric intrinsic factor When the vitamin B12–intrinsic factor complex reaches the ileum, it interacts with a receptor on the mucosal cell surface and is actively transported into circulation Adequate intrinsic factor, bile, and sodium bicarbonate

(suitable pH) all are required for ileal transport of vitamin B12 (Allen and Mehlman, 1973; Herzlich and Herbert, 1984) Vitamin B12 deficiency in adults is rarely the result of a deficient diet per se;

rather, it usually reflects a defect in one or another aspect of this complex sequence of absorption

(seeFigure 54–8) Achlorhydria and decreased secretion of intrinsic factor by parietal cells

secondary to gastric atrophy or gastric surgery is a common cause of vitamin B12 deficiency in adults Antibodies to parietal cells or intrinsic factor complex also can play a prominent role in producing a deficiency A number of intestinal diseases can interfere with absorption Vitamin B12

malabsorption is seen with pancreatic disorders (loss of pancreatic protease secretion), bacterial overgrowth, intestinal parasites, sprue, and localized damage to ileal mucosal cells by disease or as

a result of surgery

Figure 54–8 The Absorption and Distribution of Vitamin B12 Deficiency of vitamin B12 can result from a congenital or acquired defect in any one of the following: (1) inadequate dietary supply; (2) inadequate secretion of intrinsic factor (classical pernicious anemia); (3) ileal disease; (4) congenital absence of transcobalamin II (Tc II); or (5) rapid depletion of hepatic stores by interference with reabsorption of vitamin B12 excreted in bile The utility of measurements of the concentration of vitamin B12 in plasma to estimate supply available to tissues

can be compromised by liver disease and (6) the appearance of abnormal amounts

of transcobalamins I and III (Tc I and III) in plasma Finally, the formation of methylcobalamin requires (7) normal transport into cells and an adequate supply

of folic acid as CH3H4PteGlu1

Once absorbed, vitamin B12 binds to transcobalamin II, a plasma -globulin, for transport to tissues Two other transcobalamins (I and III) are also present in plasma; their concentrations are related to the rate of turnover of granulocytes They may represent intracellular storage proteins that are released with cell death (Scott et al. , 1974 ) Vitamin B12 bound to transcobalamin II is rapidly cleared from plasma and is preferentially distributed to hepatic parenchymal cells The liver is a storage depot for other tissues In the normal adult, as much as 90% of the body's stores of vitamin

B12, from 1 to 10 mg, is in the liver Vitamin B12 is stored as the active coenzyme with a turnover rate of 0.5 to 8 g per day, depending on the size of the body stores (Heyssel et al. , 1966 ) The recommended daily intake of the vitamin in adults is 2.4 g Recommended daily intakes are

presented in Table XIII–2

Approximately 3 g of cobalamins is secreted into bile each day, 50% to 60% of which represents cobalamin analogs not destined for reabsorption This enterohepatic cycle is important, since

interference with reabsorption by intestinal disease can result in a continuous depletion of hepatic stores of the vitamin This process may help explain why patients will develop vitamin B12

deficiency within 3 to 4 years after major gastric surgery, even though a daily requirement of 1 to 2

g would not be expected to deplete hepatic stores of more than 2 to 3 mg during this time

The supply of vitamin B12 available for tissues is directly related to the size of the hepatic storage pool and the amount of vitamin B12 bound to transcobalamin II (Figure 54–8) Since the amount of vitamin B12 in liver cannot be measured easily, the concentration of vitamin B12 in plasma is the best routine measure of B12 deficiency Normal individuals have plasma concentrations of the vitamin ranging from 150 to 660 pM (about 200 to 900 pg/ml) Deficiency should be suspected whenever the concentration falls below 150 pM The correlation is excellent except when the concentrations

of transcobalamin I and III in the plasma increase—for example, as a result of hepatic disease or a myeloproliferative disorder Inasmuch as the vitamin B12 bound to these transport proteins has a very slow turnover rate and, therefore, is relatively unavailable to cells, tissues can become

deficient at a time when the concentration of vitamin B12 in plasma is normal or even high (Retief et

al.

, 1967) A congenital absence of transcobalamin II has been observed in at least two families (Hakami et al. , 1971 ; Hitzig et al. , 1974 ) Megaloblastic anemia was present despite relatively

normal concentrations of vitamin B12 in plasma Clinical responses to doses of parenteral vitamin

B12 that were sufficient to exceed renal clearance were observed

Defects in intracellular metabolism of vitamin B12 have been reported in children with

methylmalonic aciduria and homocystinuria Mechanisms involved may include an incapacity of cells to transport vitamin B12 or accumulate the vitamin because of a failure to synthesize an

intracellular acceptor, a defect in the formation of deoxyadenosylcobalamin, or a congenital lack of methylmalonyl CoA isomerase (Cooper, 1976)

Vitamin B12 Deficiency

Vitamin B12 deficiency is recognized clinically by its impact on both the hematopoietic and the nervous systems The sensitivity of the hematopoietic system relates to its high rate of turnover of

cells Other tissues with high rates of cell turnover (e.g., mucosa and cervical epithelium) have

similar high requirements for the vitamin

As a result of an inadequate supply of vitamin B12, DNA replication becomes highly abnormal Once a hematopoietic stem cell is committed to enter a programmed series of cell divisions, the defect in chromosomal replication results in an inability of maturing cells to complete nuclear divisions while cytoplasmic maturation continues at a relatively normal rate This results in the production of morphologically abnormal cells and death of cells during maturation, a phenomenon

referred to as ineffective hematopoiesis (Finch et al. , 1956 ) These abnormalities are readily

identified by examination of the bone marrow and peripheral blood Usually, the changes are most marked for the red cell series Maturation of red cell precursors is highly abnormal (megaloblastic erythropoiesis) Those cells that do leave the marrow also are abnormal, and many cell fragments, poikilocytes, and macrocytes appear in the peripheral blood The mean red cell volume increases to values greater than 110 fl ( m3) When deficiency is marked, all cell lines may be affected, and a pronounced pancytopenia results

The diagnosis of a vitamin B12 deficiency usually can be made using measurements of the serum vitamin B12 level and/or serum methylmalonic acid level The latter is somewhat more sensitive and has been used to identify metabolic deficiency in patients with normal serum vitamin B12 levels As part of the clinical management of a patient with a severe megaloblastic anemia, a therapeutic trial using very small doses of the vitamin can be used to confirm the diagnosis Serial measurements of the reticulocyte count, serum iron, and hematocrit are performed to define the characteristic

recovery of normal red cell production The Schilling test is used to quantitate the absorption of the vitamin and delineate the mechanism of the disease (Schilling, 1953) By performing the Schilling test with and without added intrinsic factor, it is possible to discriminate between intrinsic factor deficiency, by itself, and primary ileal cell disease

Vitamin B12 deficiency can result in irreversible damage to the nervous system Progressive

swelling of myelinated neurons, demyelination, and neuronal cell death are seen in the spinal column and cerebral cortex This causes a wide range of neurological signs and symptoms,

including paresthesias of the hands and feet, diminution of vibration and position senses with resultant unsteadiness, decreased deep tendon reflexes, and, in the later stages, confusion,

moodiness, loss of memory, and even a loss of central vision The patient may exhibit delusions, hallucinations, or even an overt psychosis Since the neurological damage can be dissociated from the changes in the hematopoietic system, vitamin B12 deficiency must be considered as a possibility

in elderly patients with dementia and psychiatric disorders, even if they are not anemic

B12 may be absorbed by simple diffusion, the oral route of administration cannot be relied upon for effective therapy in the patient with a marked deficiency of vitamin B12 and abnormal

hematopoiesis or neurological deficits Therefore, the preparation of choice for treatment of a vitamin B12–deficiency state is cyanocobalamin, and it should be administered by intramuscular or deep subcutaneous injection

Cyanocobalamin injection is a clear aqueous solution with a characteristic red color

Cyanocobalamin injection is safe when given by the intramuscular or deep subcutaneous route, but

it should never be given intravenously There have been rare reports of transitory exanthema and anaphylaxis following injection If a patient reports a previous sensitivity to injections of vitamin B12, an intradermal skin test should be carried out before the full dose is administered

Cyanocobalamin is administered in doses of 1 to 1000 g Tissue uptake, storage, and utilization

depend on the availability of transcobalamin II (see above) Doses in excess of 100 g are rapidly

cleared from plasma into the urine, and administration of larger amounts of vitamin B12 will not result in greater retention of the vitamin Administration of 1000 g is of value in the performance

of the Schilling test After isotopically labeled vitamin B12 is administered orally, the compound that

is absorbed can be quantitatively recovered in the urine if 1000 g of cyanocobalamin is

administered intramuscularly This unlabeled material saturates the transport system and tissue binding sites, so that more than 90% of the labeled and unlabeled vitamin is excreted during the next 24 hours

A number of multivitamin preparations are marketed either as nutritional supplements or for the treatment of anemia Many of these contain up to 80 g of cyanocobalamin without or with intrinsic factor concentrate prepared from the stomachs of hogs or other domestic animals One oral unit of intrinsic factor is defined as that amount of material that will bind and transport 15 g of

cyanocobalamin Most multivitamin preparations supplemented with intrinsic factor contain 0.5 oral unit per tablet While the combination of oral vitamin B12 and intrinsic factor would appear to be ideal for patients with an intrinsic factor deficiency, such preparations are not reliable Antibodies to human intrinsic factor may effectively counteract absorption of vitamin B12 With prolonged

therapy, some patients develop refractoriness to oral intrinsic factor, perhaps related to production

of an intraluminal antibody against the hog protein (Ramsey and Herbert, 1965) Patients taking such preparations must be reevaluated at periodic intervals for recurrence of pernicious anemia

Hydroxocobalamin given in doses of 100 g intramuscularly has been reported to have a more sustained effect than cyanocobalamin, with a single dose maintaining plasma vitamin B12

concentrations in the normal range for up to 3 months However, some patients show reductions of the concentration of vitamin B12 in plasma within 30 days, similar to that seen after

cyanocobalamin Furthermore, the administration of hydroxocobalamin has resulted in the

formation of antibodies to the transcobalamin II–vitamin B12 complex (Skouby et al. , 1971 )

Vitamin B12 has an undeserved reputation as a health tonic and has been used for a number of diverse disease states Effective use of the vitamin depends on accurate diagnosis and an

understanding of the following general principles of therapy:

1

Vitamin B12 should be given prophylactically only when there is a reasonable probability that a deficiency exists Dietary deficiency in the strict vegetarian, the predictable malabsorption of vitamin B12 in patients who have had a gastrectomy, and certain diseases of the small intestine constitute such indications When gastrointestinal function is normal, an oral prophylactic supplement of vitamins and minerals, including vitamin B12, may be indicated Otherwise, the patient should receive monthly injections of cyanocobalamin

2

The relative ease of treatment with vitamin B12 should not prevent a full investigation of the etiology of the deficiency The initial diagnosis is usually suggested by a macrocytic anemia or

an unexplained neuropsychiatric disorder Full understanding of the etiology of vitamin B12

deficiency involves studies of dietary supply, gastrointestinal absorption, and transport

3

Therapy always should be as specific as possible While a large number of multivitamin

preparations are available, the use of "shotgun" vitamin therapy in the treatment of vitamin B12 deficiency can be dangerous With such therapy, there is the danger that sufficient folic acid will

be given to result in a hematological recovery This can mask continued vitamin B12 deficiency and permit neurological damage to develop or progress

4

Although a classical therapeutic trial with small amounts of vitamin B12 can help confirm the diagnosis, the acutely ill, elderly patients may not be able to tolerate the delay in the correction

of a severe anemia Such patients require supplemental blood transfusions and immediate

therapy with both folic acid and vitamin B12 to guarantee rapid recovery

Treatment of the Acutely Ill Patient

The therapeutic approach depends on the severity of the patient's illness The individual with

uncomplicated pernicious anemia, in which the abnormality is restricted to a mild or moderate anemia without leukopenia, thrombocytopenia, or neurological signs or symptoms, will respond to the administration of vitamin B12 alone Moreover, therapy may be delayed until other causes of megaloblastic anemia have been ruled out and sufficient studies of gastrointestinal function have been performed to reveal the underlying cause of the disease In this situation, a therapeutic trial with small amounts of parenteral vitamin B12 (1 to 10 g per day) can confirm the presence of an uncomplicated vitamin B12 deficiency

In contrast, patients with neurological changes or severe leukopenia or thrombocytopenia associated with infection or bleeding require emergency treatment The older individual with a severe anemia (hematocrit less than 20%) is likely to have tissue hypoxia, cerebrovascular insufficiency, and congestive heart failure Effective therapy must not wait for detailed diagnostic tests Once the megaloblastic erythropoiesis has been confirmed and sufficient blood collected for later

measurements of concentrations of vitamin B12 and folic acid, the patient should receive

intramuscular injections of 100 g of cyanocobalamin and 1 to 5 mg of folic acid For the next 1 to

2 weeks the patient should receive daily intramuscular injections of 100 g of cyanocobalamin, together with a daily oral supplement of 1 to 2 mg of folic acid Since an effective increase in red cell mass will not occur for 10 to 20 days, the patient with a markedly depressed hematocrit and tissue hypoxia also should receive a transfusion of 2 to 3 units of packed red cells If congestive heart failure is present, phlebotomy to remove an equal volume of whole blood can be performed or diuretics can be administered to prevent volume overload

The therapeutic response to vitamin B12 is characterized by a number of subjective and objective changes Patients usually report an increased sense of well-being within the first 24 hours after the initiation of therapy Objectively, memory and orientation can show dramatic improvement,

although full recovery of mental function may take months or, in fact, may never occur In addition, even before an obvious hematological response is apparent, the patient may report an increase in strength, a better appetite, and reduced soreness of the mouth and tongue

The first objective hematological change is the disappearance of the megaloblastic morphology of the bone marrow As the ineffective erythropoiesis is corrected, the concentration of iron in plasma falls dramatically as the metal is used in the formation of hemoglobin This usually occurs within the first 48 hours Full correction of precursor maturation in marrow with production of an

increased number of reticulocytes begins about the second or third day and reaches a peak three to five days later When the anemia is moderate to severe, the maximal reticulocyte index will be

between three and five times the normal value—i.e., a reticulocyte count of 20% to 40% The

ability of the marrow to sustain a high rate of production determines the rate of recovery of the hematocrit Patients with complicating iron deficiency, an infection or other inflammatory state, or renal disease may be unable to correct their anemia It is important, therefore, to monitor the

reticulocyte index over the first several weeks If it does not continue at elevated levels while the hematocrit is less than 35%, plasma concentrations of iron and folic acid should again be

determined and the patient reevaluated for an illness that could inhibit the response of the marrow.The degree and rate of improvement of neurological signs and symptoms depend on the severity and the duration of the abnormalities Those that have been present for only a few months are likely

to disappear relatively rapidly When a defect has been present for many months or years, full return

to normal function may never occur

Long-Term Therapy with Vitamin B12

Once begun, vitamin B12 therapy must be maintained for life This fact must be impressed upon the patient and family and a system established to guarantee continued monthly injections of

cyanocobalamin An intramuscular injection of 100 g of cyanocobalamin every 4 weeks is

sufficient to maintain a normal concentration of vitamin B12 in plasma and an adequate supply for tissues Patients with severe neurological symptoms and signs may be treated with larger doses of vitamin B12 in the period immediately following the diagnosis Doses of 100 g per day or several times per week may be given for several months with the hope of encouraging faster and more complete recovery It is important to monitor vitamin B12 concentrations in plasma and to obtain peripheral blood counts at intervals of 3 to 6 months to confirm the adequacy of therapy Since refractoriness to therapy can develop at any time, evaluation must continue throughout the patient's life

Other Therapeutic Uses of Vitamin B12