Month: November 2016

[Hemostasis] General – Diagnostic Approach to the Bleeding Disorders

November 21, 2016 Clinical Skills, Hematology No comments , , , , , , , , , , , , , ,

screen-shot-2016-10-10-at-10-38-16-amClinical Presentations and Clinical Distinction Between Platelet- or Vessel-Induced Bleeding and Coagulation-Induced Bleeding

Certain signs and symptoms are virtually diagnostic of disordered hemostasis. They can be divided arbitrarily into two groups: those seen more often in disorders of blood coagulation and those most commonly noted in disorders of the vessels and platelets. The latter group is often called purpuric disorders because cutaneous and mucosal bleeding usually are prominent. The clinical findings that are most valuable in distinguishing between these two broad categories are summarized in Table 45.1. Although these criteria are relative, they provide valuable clues to the probable diagnosis if they are applied to the predominant clinical features in a given patient.

Bleeding into Skin and Soft Tissues

Petechiae are characteristic of an abnormality of the vessels or the platelets and are exceedingly rare in the coagulation disorders. These lesions are small capillary hemorrhages ranging from the size of a pinhead to much larger. They characteristically develop and regress in crops and are most conspicuous in areas of increased venous pressure, such as the dependent parts of the body and areas subjected to pressure or constriction from girdles or stockings. Petechiae must be distinguished from small telangiectasias and angiomas. Vascular structures such as telangiectasias or angiomas blanch with pressure, whereas petechiae do not.

Hemarthrosis

Hemorrhage into synovial joints is virtually diagnostic of a severe inherited coagulation disorder, most commonly hemophilia A or hemophilia B, and is rare in disorders of the vessels and platelets or in acquired coagulation disorders. This disabling problem often develops with pain and swelling as chief symptoms but without discoloration or other external evidence of bleeding. Subperiosteal hemorrhages in children with scurvy and swollen painful joints that may developin some patients with allergic purpura occasionally may be confused with hemarthrosis.

Traumatic Bleeding

The unavoidable traumas of daily life and even minor surgical procedures are a greater challenge to hemostasis than any test yet developed in the laboratory. In contrast to "spontaneous" bleeding manifestations, bleeding after trauma in a person with a hemorrhagic diathesis differs in a quantitative way from that which would normally be expected in terms of the amount, duration, and magnitude of the inciting trauma. Such variables are extremely difficult to assess accurately by taking the patient's history. The need for transfusions and the number administered may serve as a rough guide. The patient's statement concerning the duration of bleeding is more reliable.

In individuals with a coagulation disorder, the onset of bleeding after trauma often is delayed. For example, bleeding after a tooth extraction may stop completely, only to recur in a matter of hours and to persist despite the use of styptics, vasoconstrictors, and packing. The temporary hemostatic adequacy of the platelet plug despite defective blood coagulation may explain this phenomenon of delayed bleeding, as well as the fact that patients with coagulation disorders seldom bleed abnormally from small superficial cuts scuh as razor nicks. In contrast, posttraumatic or postoperative surgical bleeding in thrombocytopenic patients usually is immediate in onset, as a rule responds to local measures, and rarely is as rapid or voluminous as that encountered in patients with coagulation disorders. However, it may persist for hours or days after surprisingly small injuries.

Anyway, the response to trauma is an excellent screening test for the presence of an inherited hemorrhagic disorder, and a history of surgical procedures or significant injury without abnormal bleeding is equally good evidence against the presence of such a disorder.


Clinical Features of Inherited and Acquired Bleeding Disorders

An inherited bleeding disorder is suggested by the 1) onset of bleeding symptoms in infancy and childhood, 2) a positive family history (particularly if it reveals a consistent genetic pattern), and 3) laboratory evidence of a single or isolated abnormality, most commonly the deficiency of a single coagulation factor.

Birth and the neonatal period provide unique challenges to the hemostatic mechanism, and bleeding during the first month of life often is the first evidence of an inherited disorder of hemostasis. Small cephalohematomas and petechiae are common in tne newborn as a result of the trauma of delivery. Large cephalohematomas that progressively increase in size may result from hemophilia but are more common in association with acquired bleeding disorders such as hemorrhagic disease of the newborn. Bleeding from the umbilical stump and after circumcision is common in the acquired coagulation disorders, and it also occurs in the inherited coagulation disorders, with the exception of hypofibrinogenemia, dysfibrinogenemia, and factor XIII deficiency. The onset of bleeding from the umbilical cord may be delayed in these latter disorders. In the evaluation of bleeding in the neonate, the clinician should remember that hematochezia and hematemesis may originate from swallowed blood of maternal origin. Simple tests to distinguish such maternal blood from fetal blood have been described. It must be paid attention that manuy infants with inherited coagulation disorders do not bleed significantluy in the neonatal period. In such patients, the disorder may become clinically silent for a time. Hematomas may first be seen only when the child becomes active. Hemarthrosis commonly does not develop until a child is 3 or 4 years of age.

The family history is of great importance in the evaluation of bleeding disorders. In disorders inherited as autosomal dominant traits with characteristic symptoms and high penetrance, such as hereditary hemorrhagic telangiectasia, an accurate pedigree spanning several generations can often be obtained. The presence of typical bleeding manifestations in male siblings and maternal uncles is virtually diagnostic of X-linked recessive inheritance, which characterizes hemophilia A and hemophilia B. But, the limitations of the family history, however, are greater than is commonly realized. Hearsay history is difficult to evaluate, and it is often impossible to assess the significance of easy bruising or to differentiate between manifestations of a generalized bleeding disorder and more common localized lesions, such as peptic ulcer and uterine leiomyomas. A negative family history is of no value in excluding an inherited coagulation disorder in an individual patient. As many as 30% to 40% of patients with hemophilia A have a negative family history. The family history usually is negative in the autosomal recessive traits, and consanguinity, which is commonly prsent in these kindreds, is notoriously difficult to document or exclude.


Approach to the Patient W/ Excessive Bleeding

Excessive bleeding may occur in both male and female patients of all ages and ethnicities. Symptoms can begin as early as the immediate newborn period (uncommonly even in utero) or anytime or anytime thereafter. The bleeding symptoms experienced are related in large part to the specific factor and level of deficiency.

  • [Spontaneous or induced] Bleeding can be spontaneous; that is, without an identified trigger, or may occur after a hemostatic challenge, such as delivery, injury, trauma, surgery, or the onset of menstruation.
  • [Anatomic location(s)] Furthermore, bleeding symptoms may be confined to specific anatomic sites or may occur in multiple sites.
  • [Family history] Finally, bleeding symptoms may be present in multiple family members or may occur in the absence of a family history. All of this information is important to arrive at a correct diagnosis rapidly and with minimal yet correctly sequenced laboratory testing.

Thus, a detail patient and family history is a vital component of the approach to each patient with a potential bleeding disorder.

History

Obtaining a detail patient and family history is crucial regardless of prior laboratory testing. The history includes a detailed discussion of specific bleeding and clinical symptoms. Information regarding bleeding symptoms should include location, frequency, and pattern as well as duration both in terms of age of onset and time required for cessation.

Location

The location may suggest the part of the hemostatic system affected; patients with disorders of primary hemostasis (platelets and vWF) often experience mucocutaneous bleeding, including easy bruising, epistaxis, heavy menstrual bleeding, and postpartum hemorrhage in women of child-bearing age; whereas patients with disorders of secondary hemostasis (coagulation factor deficiencies) may experience deep-tissue bleeding, including the joints, muscles, and central nervous system.

Pattern and Duration

The bleeding pattern and duration of each episode, particularly for mucus membrane bleeding, assist in the determination of the likelihood of the presence of an underlying bleeding disorder.

Onset

The onset of symptoms can suggest the presence of a congential versus acquired disorder. Although congenital conditions can present at any age, it is more likely that patients with a long history of symptoms or symptoms that begin in childhood have a congenital condition, whereas patients whose onset occurs at an older age are more likely to have an acquired condition. Congenital clotting factor deficiencies that do not present until later in life do occur and include mild factor deficiencies and coagulation factor deficiencies associated with variable bleeding patterns, most notably FXI deficiency.

Other

Additional important information to be collected includes the current use of medications and herbal supplements, as these may affect the hemostatic system; the presence or absence of a family history of bleeding; a history of hemostatic challenges, including surgery, dental procedures, and trauma; and a menstrual history in females.

The goal at the end of the history is to establish the likelihood of a bleeding disorder, as this will guide the direction of the laboratory investigation. Quantification of clinical bleeding is a challenge, particularly in the outpatient setting. In recent years, several bleeding assessment tools (BAT) have been developed to more accurately differentiate bleeding phenotypes in healthy individuals and in patients with bleeding disorders. These tools, which were originally designed for assessing bleeding in von Willebrand disease (vWD) do not appear to be diagnostic and are in the process of being validated for the ability to screen other bleeding disorders. However, it is increasingly clear that a normal bleeding score rules out the presence of a bleeding disorder. Therefore, if the bleeding score is indicative of excessive bleeding, it should be followed by an evaluation of a hematologist to evaluate the need for further laboratory tests.

Screening Tests

  • Platelet count, PT, aPTT

The laboratory evaluation for bleeding includes performance of initial screening tests. The most common screening tests utilized include the platelet count, prothrombin time (PT), and activated partial thromboplastin time (aPTT). When the PT or aPTT is prolonged, mixing studies are required via a one-to-one mix of patient plasma with known normal standard plasma. Test correction in the mixing study indicates a deficiency state, whereas lack of correction indicates an inhibitor, either one directed against a specific factor or a a global inhibitor as best exemplified by a lupus anticoagulant. Specific factor analyses are performed after mixing studies reveal a correction of prolonged coagulation screening test(s) indicative of a deficiency state or in the face of normal screening tests with a positive history. Screening tests are not sensitive and do not evaluate all abnormalities associated with bleeding including vWF, FXIII, PAI-1, and 𝛼2AP deficiencies and may be insensitive to mild FVIII and FIX deficiencies; therefore, a patient history strongly suggestive of a bleeding disorder may warrant testing for such deficiencies, including rare abnormalities regardless of screening test results. The most common screening tests utilized include the platelet count, prothrombin time (PT), and activated partial thromboplastin time (aPTT). When the PT or aPTT is prolonged >10 seconds, mixing studies are required via a one-to-one mix of patient plasma with known normal standard plasma. Test correction in the mixing study indicates a deficiency state, whereas lack of correction indicates an inhibitor.

  • Platelet function

Screen tests also are utilized to identify individuals with a high likelihood of vWD or platelet disorders. The bleeding time, once widely used, has become obsolete because of the lack of sensitivity and specificity. The PFA-100 (platelet function analyzer) has been proposed to have a role in screening individuals with suspected platelet dysfunction or vWD. Initial studies demonstrated the efficacy of the PFA-100 in the evaluation of patients with known severe platelet disorders or vWD. The PFA-100 induces high shear stress and simulates primary hemostasis by flowing whole blood through an aperture with a membrane coated with collagen and either ADP or epinephrine. Platelets adhere to the collagen-coated surface and aggregate forming a platelet plug that enlarges until it occludes the aperture, causing cessation of blood flow. The time to cessation of flow is recorded as closure time (CT). The sensitivity and spcificity of the CT of the PFA-100 were reported as 90% for severe platelet dysfunction or vWD, with vWD plasma levels below 25%. The utility of the PFA-100 as a screening tool, however, has been challenged based on the reported low sensitivity (24%-41%) of the device in individuals with mild platelet secretion defect, mild vWD or storage pool disorders. Additionally, a significant limitation of the PFA-100 is the fact that the platelet count and hemoglobin levels affect the CT. The CT will be abnormal if the platelet count is less than 100,000/𝜇L and the hemoglobin is <10 g/dL.

[Physiology][Hematology] General Concepts in Hemolytic Anemias

November 18, 2016 Hematology, Physiology and Pathophysiology No comments , , , , , , , , , , , , , , , , , , , , ,

Hemolysis is the accelerated destruction of red blood cells (RBCs), leading to decreased RBC survival. The bone marrow's response to hemolysis is increased erythropoiesis, reflected by reticulocytosis. If the rate of hemolysis is modest and the bone marrow is able to completely compensate for the decreased RBC life span, the hemoglobin concentration may be normal; this is called fully compensated hemolysis. If the bone marrow is unable to completely compensate for hemolysis, the anemia occurs. This is called incompletely compensated hemolysis.

PS (from wikipedia): The Reticulocyte production index (RPI) is a calculated value used in the diagnosis of anemia. This calculation is necessary because the raw reticulocyte count is misleading in anemic patients. The problem arises because the reticulocyte count is not really a count but rather a percentage: it reports the number of reticulocytes as a percentage of the number of red blood cells. In anemia, the patient's red blood cells are depleted, creating an erroneously elevated reticulocyte count.

Calculation of RPI

Step 1 – Reticulocyte Index is calculated using the formla on the left

Step 2 – The next step is to correct for the longer life span of prematurely released reticulocytes in the blood—a phenomenon of increased red blood cell production. This relies on a table:

Hematocrit (%) Retic survival (days) = maturation correction
36-45 1.0
26-35 1.5
16-25 2.0
15 and below 2.5

Step 3 – The Reticulocyte Production Index is calcualted using the formla below:

Hemolysis can be classified as extravascular or intravascular. Extravascular hemolysis, in which erythrocyte desstruction occurs by macrophages in the liver and spleen, is more common. Intravascular hemolysis refers to RBC destruction occurring primarily within blood vessels. The distinction between intravascular and extravascular hemolysis is not absolute because both occur simultaneously, at least to some degree, in the same patient, and the manifestations of both can overlap. The site of RBC destruction in different conditions can be conceptualized to occur in a spectrum between pure intravascular and pure extravascular hemolysis. Some hemolytic anemias are predominantly intravascular, and some are predominantly extravascular. Others have substantial components of both.

To understand better, the hemolytic anemias can be classified according to whether the cause of hemolysis is intrinsic or extrinsic to the RBC.Intrinsic causes of hemolysis include abnormalities in hemoglobin structure or function, the RBC membrane, or RBC metabolism (cytosolic enzymes). Extrinsic causes may be due to a RBC-directed antibody, a disordered vasculature, or the presence of infecting organisms or toxins. In general, intrinsic causes of hemolysis are inherited and extrinsic causes are acquired, but there are notable exceptions.


Hemolysis Due to Intrinsic Abnormalities of the RBC

screen-shot-2016-11-14-at-9-19-43-pmIntrinsic causes of hemolysis include abnormalities of hemoglobin structure or function, the RBC membrane, or RBC metabolism (cytosolic enzymes). Hemoglobin is the oxygen-carrying protein within RBCs. It is composed of four globular protein subunits, called globins, each with an oxygen-binding heme group. The two main types of globins are the alpha-globins and the beta-globins, which are made in essentially equivalent amount in precursors of RBCs. Normal adult hemoglobin (Hb A) has two alpha-globins and two beta-globins (alpha2beta2).

Abnormalities of Hemoglobin

Disorders of hemoglobin can be classified as qualitative or quantitative disorders. Qualitative abnormalities of hemoglobin arise from mutations that change the amino acid sequence of the globin, thereby producing structrual and functional changes in hemoglobin. There are four ways in which hemoglobin can be qualitatively abnormal: (i) decreased solubility, (ii) instability, (iii) altered oxygen affinity, (iv) altered maintenance of the oxidation state of the heme-coordinated iron. Hemolytic anemia (qualitative abnormalities) result from decreased solubility and instability of hemoglobin. Qualitative hemoglobin disorders often are referred to as hemoglobinopathies, even though the term technicially can apply to both qualitative and quantitative disorders.

Quantitative hemoglobin disorders result from the decreased and imbalanced production of generally structurally normal globins. For example, if beta-globin production is diminished by a mutation, there will be a relative excess of alpha-globins. Such imbalanced production of alpha- and beta-globins damages RBCs and their precursors in the bone marrow. These quantitative hemoglobin disorders are called thalassemias.

Abnormalities of the RBC Membrane

Some hemolytic diseases is characterized by abnormal shape and flexibility of RBCs because of a deficiency or dysfunction of one or more of the membrane proteins, which leads to shortened RBC survival (hemolysis).

The RBC membrane consists of a phospholipid-cholesterol lipid bilayer intercalated by integral membrane proteins such as band 3 (the anion transport channel) and the glycophorins. This relatively fluid layer is stabilized by attachment to a membrane skeleton. Spectrin is the major protein of the skeleton, accounting for approximately 75% of its mass. The skeleton is organized into a hexagonal lattice. The hexagon arms are formed by fiber-like spectrin tetramers, whereas the hexagon corners are composed of small oligomers of actin that, with the aid of other proteins (4.1 and adducin), connect the spectrin tetramers into a two-dimensional lattice. The membrane cytoskeleton and its fixation to the lipid-protein bilayer are the major determinants of the shape, strength, flexibility, and survival of RBCs. When any of these constituents are altered, RBC survival may be shortened.

Abnormalities of RBC Metabolism (cytosolic enzymes)

Normal metabolism of the mature RBC involves two principal pathways of glucose catabolism: the glycolytic pathway and the hexose-monophosphate shunt. The three major functions of the products of glucose catabolism in the erythrocyte are (i) maintenance of protein integrity, cellular deformability, and RBC shape; (ii) preservation of hemoglobin iron in the ferrous form; and (iii) modulation of the oxygen affinity of hemoglobin. These functions are served by the regulation of appropriate production of five specific molecules: ATP, reduced glutathione, reduced NADH, reduced NADPH, and 2,3-BPG. Maintenance of the biochemical and structural integrity of the RBC depends on the normal function of >20 enzymes involved in these pathways as well as the availability of five essential RBC substrates: glucose, glutathione, NAD, NAD phosphate (NADP), and adenosine diphosphate (ADP).

  • ATP

The primary function of the glycolytic pathway is the generation of ATP, which is necessary for the ATPase-linked sodium-potassium and calcium membrane pumps essential for cation homeostasis and the maintenance of erythrocyte deformability.

  • 2,3-BPG

The production of 2,3-BPG is regulated by the Rapoport-Luebering shunt, which is controlled by bisphosphoglyceromutase, the enzyme that converts 1,3-BPG to 2,3-BPG. Concentration of 2,3-BPG in the RBC in the RBC in turn regulates hemoglobin oxygen affinity, thus facilitating the transfer of oxygen from hemoglobin to tissue-binding sites.

  • Reduced gluthione

The major function of the hexose-monophosphate shunt is preservation and regeneration of reduced gluthione, which protects hemoglobin and other intracellular and membrane proteins from oxidant injury.

Abnormalities of the glycolytic pathway

Defects in the glycolytic pathway lead to a decrease in the production of ATP or a change in the concentration of 2,3-BPG.

Deficiencies of erythrocyte hexokinase, glucose phosphate isomerase, phosphofructokinase, and pyruvate kinase (PK) all lead to a decrease in ATP concentration. Although genetic disorders involving nearly all of the enzymes of the glycolytic pathway have been described, PK accounts for >80% of the clinically significant hemolytic anemias from defects in this pathway. With the exception of phosphoglycerate kinase deficiency, which is X-linked, all other glycolytic enzyme defects are autosomal recessive.

PK deficiency is the most common congenital nonspherocytic hemolytic anemia caused by a defect in glycolytic RBC metabolism. The syndrome is both genetically and clinically heterogeneous. Both glucose phosphate isomerase and hexokinase deficiencies produce nonspherocytic hemolytic anemia associated with decreased ATP and 2,3-BPG content.

Abnormalities of the hexose-monophosphate shunt

G6PD deficiency is the most frequently encountered abnormality of RBC metabolism, affecting >200 million people worldwide. The gene for G6PD is carried on the X chromosome and exhibits extensive polymorphism. Enzyme deficiency is observed in males carrying a variant gene. Hemolysis in G6PD-deficient RBCs is due to a failure to generate adequate NADPH, leading to insufficient levels of reduced glutathione. This renders erythrocytes susceptible to oxidation of hemoglobin by oxidant radicals, such as hydrogen peroxide. The resulting denatured hemoglobin aggregates and forms intraerythrocytic Heinz bodies, which bind to membrane cytoskeletal proteins. Membrane proteins are also subject to oxidation, leading to decreased cellular deformability. Cells containing Heinz bodies are entrapped or partially destroyed in the spleen, resulting in loss of cell membranes through pitting of Heinz bodies and leading to hemolysis.

Abnormalities of nucleotide metabolism

Pyrimidine-5'-nucleotidase deficiency is an enzymatic abnormality of pyrimidine metabolism associated with hemolytic anemia. The peripheral blood smear in patients with this defect often shows RBCs containing coarse basophilic stippling. Lead intoxication also inactivates the enzyme, leading to an acquired variant of pyrimidine-5'-nucleotidase deficiency.

Adenosine deaminase (ADA) excess is an unusual abnormality. It is caused by a genetically determined increase in the activity of a normal erythrocyte enzyme. The excessive deaminase activity prevents normal salvage of adenosine and causes subsequent depletion of ATP and hemolysis.

[Clinical Art][Pharmacokinetics] Interpretation of Plasma Drug Concentrations (Steady-State)

November 11, 2016 Clinical Skills, Critical Care, Pharmacokinetics, Practice No comments , , , , , , , , , , , ,

Plasma drug concentration are measured in the clinical setting to determine whether a potentially therapeutic or toxic concentration has been produced by a given dosage regimen. This process is based on the assumption that plasma drug concentrations reflect drug concentrations at the receptor and, therefore, can be correlated with pharmacologic response. This assumption is not always valid. When plasma samples are obtained at inappropriate times or when other factors (such as delayed absorption or altered plasma binding) confound the usual pharmacokinetic behavior of a drug, the interpretation of serum drug concentrations can lead to erroneous pharmacokinetic and pharmacodynamic conclusions and utimately inappropriate patient care decisions. These facors are discussed below.

Confounding Factors

To properly interpret a plasma concentration, it is essential to know when a plasma sample was obtained in relation to the last dose administered and when the drug regimen was initiated.

  • If a plasma sample is obtained before distribution of the drug into tissue is complete, the plasma concentration will be higher than predicted on the basis of dose and response. (avoidance of distribution)
  • Peak plasma levels are helpful in evaluating the dose of antibiotics used to treat severe, life-threatening infections. Although serum concentrations for many drugs peak 1 to 2 hours after an oral dose is administered, factors such as slow or delayed absorption can significantly delay the time at which peak serum concentrations are attained. Large errors in the estimation of Css max can occur if the plasma sample is obtained at the wrong time. Therefore, with few exceptions, plasma samples should be drawn as trough or just before the next dose (Css min) when determining routine drug concentration in plasma. These trough levels are less likely to be influenced by absorption and distribution problems. (slow or delayed absorption)
  • When the full therapeutic response of a given drug dosage regimen is to be assessed, plasma samples should not be obtained until steady-state concentrations of the drug have been achieved. If drug doses are increased or decreased on the basis of drug concentrations that have been measured while the drug is still accumulating, disastrous consequences can occur. Nevertheless, in some clinical situations it is appropriate to measure drug levels before steady state has been achieved. If possible, plasma samples should be drawn after a minimum of two half-lives beause clearance values calculated from drug levels obtained less than one half-life after a regimen has been initiated are very sensitive to small differences in the volume of distribution and minor assay errors. (Whether steady-state attained)
  • The impact of drug plasma protein binding on the interpretation of plasma drug coencentration has been discussed in thread "The Plasma Protein Concentration and The Interpretation of TDM Report" before.

Revising Pharmacokinetic Parameters

The process of using a patient's plasma drug concentration and dosing history to determine patient-specific pharmacokinetic parameters can be complex and difficult. A single plasma sample obtained at the appropriate time can yield information to revise only one parameter, either the volume of distribution or clearance, but not both. Drug concentrations measured from poorly timed samples may prove to be useless in estimating a patient's V or Cl values. Thus, the goal is to obtain plasma samples at times that are likely to yield data that can be used with confidence to estimate pharmacokinetic parameters. In addition, it is important to evaluate available plasma concentration data to determine whether they can be used to estiamte, with some degree of confidence, V and/or Cl. The goal in pharmacokinetic revisions is not only to recognize which pharmacokinetic parameter can be revised, but also the accuracy or confidence one has in the revised or patient-specific pharmacokinetic parameter. In the clinical setting, based on the way drugs are dosed and the recommended time to sample, bioavailability is almost never revised, volume of distribution is sometimes revised, and most often clearance is the pharmacokientic parameter that can be revised to determine a patient-specific value.

Volume of Distribution

A plasma concentration that has been obtained soon after administration of an initial bolus is primarily determined by the dose administered and the volume of distribution. This assumes that both the absorption and distribution phases have been avoided.

C1 = (S) (F) (Loading Dose) x e(-kt1) / V (IV Bolus Model)

When e(-kt1) approches 1 (i.e., when t1 is much less than t1/2), the plasma concentration (C1) is primarily a function of the administered dose and the apparent volume of distribution. At this point, very little drug has been eliminated from the body. As a clinical guideline, a patient's volume of distribution can usually be estimated if the absorption and distribution phase are avoided and t1, or the interval between the administration and sampling time, is less than or equal to one-third of the drug's half-life. As t1 exceeds one-third of a half-life, the measured concentration is increasingly infuenced by clearance. As more of the drug is eliminated (i.e., t1 increases), it is difficult to estimate the patient's V with any certainty. The specific application of this clinical guideline depends on the confidence with which one knows clearance. If clearance is extremely variable and uncertain, a time interval of less than one-third of a half-life would be necessary in order to revise volume of distribution. On the other hand, if a patient-specific value for clearance has already been determined, then t1 could exceed one-third of a half-life and a reasonably accurate estimate of volume of distribution could be obtained. It is important to recognize that the pharmacokinetic parameter that most influences the drug concentration is not determined by the model chosen to represent the drug level. For example, even if the dose is modeled as a short infusion, the volume of distribution can still be the important parameter controlling the plasma concentration. V is not clearly defined in the equation (see it below); nevertheless, it is incorporated into the elimination rate constant (K).

C2 =[(S) (F) (Dose/tin) / Cl]*(1-e-ktin)(e-kt2)

Although one would not usually select this equation to demonstrate that the drug concentration is primarily a function of volume of distribution, it is important to recognize that the relationship between the observed drug concentration and volume is not altered as long as the total elapsed time (tin + t2) does not exceed one-third of a half-life.

Our assumption in evaluating the volume of distribution is that although we have not sampled beyond one-third of a t1/2, we have waited until the drug absorption and distribution process is complete.

Clearance

A plasma drug concentration that has been obtained at steady state from a patient who is receiving a constant drug infusion is determined by clearance.

Css ave = (S) (F) (Dose / tau) / Cl

So, the average steady-state plasma concentration is not influenced by volume of distribution. Therefore, plasma concentrations that represent the average steady-state level can be used to estimate a patient's clearnace value, but they cannot be used to estimate a patient's volume of distribution. Generally, all steady-state plasma concentrations within a dosing interval that is short relative to a drug's half-life (tau =<1/3 t1/2) approximate the average concentration. Therefore, these concentrations are also primarily a function of clearance and only minimally influenced by V.

Also the below equation could be used,

Css 1 =[(S)(F)(Dose)/V]/(1-e-kτ)*(e-kt1)

the expected volume of distribution should be retained and the elimination rate constant adjusted such that Css1 at t1 equals the observed drug plasma concentration.

Sensitivity Analysis

Whether a measured drug concentration is a function of clearance or volume of distribution is not always apparent. When this is difficult to ascertain, one can examine the sensitivity or responsiveness of the predicted plasma concentration to a parameter by changing one parameter while holding the other constant. For example, for maintenance infusion, a plasma concentration (C1) at some time intervnal (t1) after a maintenance infusion has been started should be:

C1=[(S)(F)(Dose/τ)/Cl]*(1-e-kt1)

when the fraction of steady that has been reached (1-e-kt1) is small, large changes in clerance are frequently required to adjust a predicted plasma concentration to the appropriate value. If a large percentage change in the clearance value results in a disproportionately small change in the predicted drug level, then something other than clearance is controlling (responsible for) the drug concentration. In this case, the volume of distribution and the amount of drug administered are the primary determinants of the observed concentration. Also in cases where the drug concentration is very low, it might be assay error or sensitivity that is the predominant factor in determining the drug concentration making the ability to revise for any pharmacokinetic parameter limited if not impossible.

This type of sensitivity analysis is useful to reinforce the concept that the most reliable revisions in pharmacokinetic parameters are made when the predicted drug concentration changes by approximately the same percentage as the pharmacokinetic parameter undergoing revision.

When a predicted drug concentration changes in direct proportion, or inverse proportion to an alteration in only one of the pharmacokinetic parameters, it is likely that a measured drug concentration can be used to estimate that patient-specific parameter. But when both clearance and volume of distribution have a significant influence on th prediction of a measured drug concentration, revision of a patient's pharmacokinetic parameters will be less certain because there is an infinite number of combinations for clearance and volume of distribution values that could be used to predict the observed drug concentration. When this occurs, the patient's specific pharmacokinetic characteristics can be estimated by adjusting one or both of the pharmacokinetic parameters. Nevertheless, in most cases additional plasma level sampling will be needed to accurately predict the patient's clearance or volume of distribution so that subsequent dosing regimens can be adjusted.

When the dosing interval is much shorter than the drug's half-life, the changes in concentration within a dosing interval are relatively small, and any drug concentration obtained within a dosing interval can be used as an approximation of the average steady-state concentration. Even though Css max and Css min exist,

Css max =[(S)(F)(Dose)/V]/(1-e-kτ)

and

Css min =[(S)(F)(Dose)/V]/(1-e-kτ)*(e-kτ)

and could be used to predict peak and trough concentrations, a reasonable approximation could also be achieved by using the Css ave, that is

Css ave =(S)(F)(Dose/τ)/Cl

This suggests that even though Css max and Css min do not contain the parameter clearance per se, the elimination rate constant functions in such a way that the clearance derived from Css max or Css min and Css ave would all essentially be the same.

In the situation in which the dosing interval is greater than one-third of a half-life, the use of Css max and Css min are appropriate as not all drug concentrations within the dosing interval can be considered as the Css ave. However, as long as the dosing interval has not been extended beyond one half-life, clearance is still the primary pharmacokinetic parameter that is responsible for the drug concentrations within the dosing interval. Although the elimination rate constant and volume of distribution might be manipulated in Css max and Css min, it is only the product of those two numbers (i.e., clearance) that can be known with any certainty: Cl = (K) (V).

If a drug is administered at a dosing interval that is much longer than the apparent half-life, peak concentrations may be primarily a function of volume of distribution. Since most of the dose is eliminated within a dosing interval, each dose can be thought as something approaching a new loading dose. Of course for steady-state conditions, at some point within the dosing interval, the plasma concentration (Css ave) will be determined by clearance. Trough plasma concentrations in this situation are a function of both clearance and volume of distribution. Since clearance and volume of distribution are critical to the prediction of peak and trough concentrations when the dosing interval is much longer than the drug t1/2, a minimum of two plasma concentrations is needed to accurately establish patient-specific pharmacokinetic parameters and a dosing regimen that will achieve desired peak and trough concentrations.


Choosing A Model to Revise or Estimate A Patient's Clearance at Steady State

As previously discussed, a drug's half-life often determines the pharmacokinetic equation that should be used to make a revised or patient-specific estimate of a pharmacokinetic parameter. A common problem encountered clinically, however, is that the half-life observed in the patient often differs from the expected value. Since a change in either clearance or volume of distribution or both may account for this unexpected value, the pharmacokinetic model is often unclear. One way to approach this dilemma is to first calculate the expected change in plasma drug concentration assocaited with each dose:

delta C = (S) (F) (Dose) / V

where delta C is the change in concentration following the administration of each dose into the patient's volume of distribution. This change in concentration can then be compared to the steady-state trough concentration measured in the patient.

(S) (F) (Dose) / V versus Css min

or

delta C versus Css min

When the dosing interval (tau) is much less than the drug half-life, delta C will be small when compared to Css min. As the dosing interval increases relative to tau, delta C will increase relative to Css min. Therefore, a comparison of delta C or (S) (F) (Dose) / V to Css min can serve as a guide to estimating the drug t1/2 and the most appropriate pharmacokineitc model or technique to use for revision. With few exceptions, drugs that have plasma level monitoring are most often dosed at intervals less than or equal to their half-lives. Therefore, clearance is the pharmacokinetic parameter most often revised or calculated for the patient in question. The following guidelines can be used to select the pharmacokinetic model that is the least complex and therefore the most appropriate to estimate a patient-specific pharmacokientic parameter.

Condition 1

When, (S) (F) (Dose) / V =< 1/4 Css min

Then, tau =<1/3 t1/2

Under these conditions, Css min ≈ Css ave

And Cl can be estimated by Cl = (S) (F) (Dose / tau) / Css ave

Rules/Conditions: Must be at steady state.

Condition 2

When, (S) (F) (Dose) / V =< Css min

Then, tau =< t1/2

Under these conditions, Css min + (1/2) (S) (F) (Dose) / V ≈ Css ave

And Cl can be estimated by Cl = (S) (F) (Dose / tau) / Css ave

Rules/Conditions: Must be at steady state; C is Css min; Bolus model for absorption is acceptable (dosage form is not sustained release; short infusion model is not required, that is, tin =<1/6t1/2)

Conditon 3

When, (S) (F) (Dose) / V > Css min

Then, tau > t1/2

Under these conditions: Css min + (S) (F) (Dose) / V = Css max

where V is an assumed value from the literature.

K is revised (Krevised):

Krevised = ln {[(Css min + (S) (F) (Dose / V)] / Css min} / tau = ln (Css max / Css min) / tau

Rules/Conditions: Must be at steady state; C is Css min; Bolus model for absorption is acceptable (dosage form is not sustained release; short infusion model is not required, that is, tin =< 1/6 t1/2)

Note that the approaches used become more complex as the dosing interval increases relative to the drug half-life. If a drug is administered at a dosing interval less than or equal to one-third of its half-life and the technique in Condition 3 is used to revise clearance, the revised clearance would be correct. The calculation is not wrong, just unnecessarily complex. However, if a drug is administered at a dosing interval that exceeds one half-life and the technique in Condition 1 is used to revise clearance, the revised clearance value would be inaccurate because Css min cannot be assumed to be approximately equal to Css ave. While it could be argued that the technique used in Condition 3 would suffice for all the previous conditions, it is more cumbersome and tends to focus on the intermediate parameters, K and V rather than Cl. One should also be ware that as the dosing interval increases, relative to the drug's half-life, the confidence in a revised clearance diminishes because the volume of distribution, which is an assumed value from the literature, begins to influence the revised clearance to a greater degree. As a general rule, the confidence in Cl is usually good when the dosing interval is < t1/2, steady state has been achieved, and drug concentrations are obtained properly.

[Clinical Art][Physiology] Iron Physiology

November 3, 2016 Cytogenetics, Hematology, Molecular Biology, Physiology and Pathophysiology No comments , , , , , , , , , , , , , , , , , , , , , ,

screen-shot-2016-11-02-at-10-03-03-pmGlobal Iron Homeostasis

Under normal conditions, dietary iron is usually 15-25 mg daily, of which 5%-10% (1-2 mg) is absorbed through the gastrointestinal (GI) tract and the same amount lost by desquanmation of GI epithelial cells, epidermal cells of the skin, and, in menstruating women, red bood cells. The average total body content of iron in men is 35-45 mg/kg; and lower in menstruating women. Most iron (about 1800 mg) is present in hemoglobin. Men and women, respectively, have approximately 2 or 1.5 g of erythrocyte iron. Iron is stored in cells, predominantly macrophages of the spleen, bone marrow, and liver, but also in hepatocytes, as ferritin or hemosiderin (partially denatured ferritin). At steady state, the serum ferritin level is a reasonably good reflection of total body iron stores. Total storage iron is approximately 1 g in men and 600 mg in women. Additional iron is found as myoglobin in muscle and in cytochromes and other enzymes.

PS: The loss of iron

Iron is eliminated only through the loss of epithelial cells from the gastrointestinal tract, epidermal cells of the skin, and, in menstruating women, red blood cells. On the basis of long-term studies of body iron turnover, the total average daily loss of iron has been estimated at ~1 to 2 mg in normal adult men and nonmenstruating women. Although iron is a physiologic component of sweat, only a tiny amount of iron (22.5 ug/L) is lost by this route. Urinary iron excretion amounts to <0.05 mg/day and is largely accounted for by sloughed cells. Menstruating women lose an additional, highly variable amount over each menstrual cycle, from 0.006 (average) to more than 0.025 mg/kg/day.

The release of iron into the circulation is regulated by ferroportin, expressed on the basolateral GI epithelial cell surface (and on cells of the reticuloendothelial system [RES] and hepatocytes). Ferroportin is downregulated by hepcidin, and when iron is low, hepcidin is low, allowing GI iron absorption to increase and stores to be mobilized from the RES. When iron is plentiful, hepcidin levels increase and result in decreased iron absorption and RES export. Hepcidin, in turn, is downregulated by the recently described hormone erythroferrone (ERFE), produced by erythroblasts during stress erythropoiesis. Absorbed iron is transported by transferrin and taken up into cells via the transferrin receptor. Each molecule of transferrin can bind two molecules of ferric (Fe3+) iron. Transferrin-bound iron turns over as iron is used, particularly by developing red blood cells in the bone marrow. The distribution of iron is influenced by multiple factors, and under normal conditions cells maintain a pool of labile iron by controlling uptake via expression of transferrin receptors and storage via ferritin. Most cells have no mechanism for iron efflux.

Iron balance is regulated such that the amount of iron absorbed equals the amount lost. There is, however, no physiologically regulated pathway for excretion of excess iron in iron overload.

Intestinal Iron Absorption

Iron is found in food as inorganic iron and heme (iron complexed to protoporphyrin IX). The typical diet consists of 90% inorganic and 10% heme iron, though diets in the industrial world can contain up to 50% heme iron from iron-rich meats. The bioavailability of inorganic but not heme iron is influenced by multiple factors such as other dietary constituents, for example, ascorbic acid (enhanced) and phytates and polyphenols in cereals and plants (inhibited). Iron absorption is strongly inhibited by tea, and less so by coffee.

PS: Intestinal Absorption of Iron

Iron is absorbed in the duodenum, and humans and other omnivorous mammals have at least two distinct pathways for iron absorption: one for uptake of heme iron and another for ferrous (Fe2+) iron. Heme iron is derived from hemoglobin, myoglobin, and other heme proteins in foods of animal origin, representing approximately 10% to 15% iron content in the typical Western diet, although heme-derived iron accounts for 2/3 of absorbed iron in meat-eating humans. Exposure to acid and proteases present in gastric juices frees the heme from its apoprotein. Heme is taken up by mucosal cells, but the specific receptor is still unkonwn. Once heme iron has entered the cell, the porphyrin ring is enzymatically cleaved by heme oxygenase. The liberated iron then probably follows the same pathways as those used by nonheme iron. A small proportion of the heme iron may pass into the plasma intact via heme exporter protein FLVCR (feline leukemia virus, subgroup C receptor), which transfers heme onto a heme-binding protein, hemopexin. Absorption of heme iron is relatively unafected by the overall composition of the diet.

Dietary nonheme iron is largely in the form of ferric hydroxide or loosely bound to organic molecules such as phytates, oxalate, sugars, citrate, lactate, and amino acids. Low gastric pH is thought be important for the solubility of inorganic iron. Dietary constituents may also have profound effects on the absorption of nonheme iron, making the bioavailability of food iron highly variable. Ascorbate, animal tissues, keto sugars, organic acids, and amino acids enhance inorganic iron absorption, whereas phytates, polyphenols, and calcium inhibit it. Depending on various combinations of enhancing and inhibitory factors, dietary iron assimilation can vary as much as tenfold.

The rate of iron absorption is influenced by several factors, including body iron stores, the degree of erythropoietic activity, blood hemoglobin and oxygen content, and the presence of inflammation. Iron absorption increases when stores are low or when increased erythropoietic activity is required, such as during anemia or hypoxemia. Conversely, the physiologically appropriate response to iron overload is downregulation of intestinal iron absorption; this downregulation fails in patients with hereditary hemochromatosis or chronic iron-loading anemias.

Iron is absorbed in the intestine via two pathways: one for inorganic iron and the other for heme-bound iron. Little is known about heme iron absorption. Nonheme iron in the diet is largely in the form of ferric-oxyhydroxides (Fe3+, ie, rust), but the intestinal epithelial cell apical iron importer, divalent metal transporter 1 (DMT1 or SLC11A2), transports only ferrous iron (Fe2+). DMT1 is a protein with 12 predicted transmembrane segments, which is expressed on the apical surface of absorptive enterocytes. Iron must therefore be reduced to be absorbed, and this is facilitated by duodenal cytochrome B (Dcytb), a heme-dependent ferrireductase. However, since knockout mice appear to have normal metabolism, Dcytb may not be the only ferrireductase enzyme involved in absorption of nonheme ironOnce transported across the apical border of the enterocyte, iron may be stored within the cell. For this purpose, iron is oxidized to Fe3+ by the H-subunit of ferritin and stored in this form. Eventually, the cell senesces and sloughs off into the feces, and stored iron is lost to the system. Alternatively, iron may be transported across the basolateral membrane into the portal circulation via ferroportin. Ferroportin 1 (FPN1) is the only known iron exporter in mammals and, like DMT1, transports only ferrous iron. FPN1 is a multi-transmembrane segment protein expressed on the basolateral surface of enterocytes. Aslo they are expressed in other tissues involved in handling large iron fluxes including macrophages, hepatocytes, and placental trophoblast. Similar to apical iron uptake, basolateral iron efflux is aided by an enzyme that changes the oxidation state of iron. Once reduced, ferrous iron is transported across the basolateral membrane by ferroportin, then oxidized to ferric iron by hephaestin. Intestinal iron absorption is regulated by hepcidin, which binds to ferroportin, inducing its internalization and degradation.

Cellular Iron Uptake, Storage, and Recycling

Each molecule of transferrin binds two ferric (Fe3+) iron atoms. Diferric transferrin (holotransferrin) binds to the transferrin receptor (TfR1) on target cells and enters by receptor-mediated endocytosis; it is then released from the TfR1 by acidification and transported into the cytoplasm by DMT1. The TfR is recycled to the cell surface.

Most iron in erythroid cells binds protoporphyrin to form heme, which complexes with globin proteins, forming hemoglobin. Erythrocytes survive in the circulation for approximately 120 days, after which aging red blood cells are phagocytized by macrophages of the RES. Hemoglobin is catabolized and iron released to transferrin via ferroportin, or stored within the RES as ferritin or hemosiderin.

PS: Reticuloendothelial System/RES

screen-shot-2016-11-02-at-1-20-03-pm

 

The main form of cellular iron storage is ferritin, a complex of subunits that binds iron and renders it insoluble and redox inactive. Circulating ferritin is present in a different subunit form than cellular storage ferritin. The function of circulating ferritin is incompletely understood.

Regulation of Iron Physiology

Because the total body iron content is largely determined by the efficiency of absorption of iron, the regulation of absorption has been of great interest for many years.

Hepcidin is a 25-amino-acid peptide produced in the liver and is the major regulator of iron absorption and storage. Hepcidin regulates cellular iron egress by binding to ferroportin, leading to its internalization and degradation. In this way, elevated levels of hepcidin inhibit iron absorption from the GI tract and promote storage (inhibit release) of iron within hepatocytes and macrophages. Hepcidin production is induced by interleukin (IL)-6 and IL-1 via the JAK/STAT pathway, and can be increased more than 100-fold in inflammatory states. The dysregulation of iron balance seen in the anemia of inflammation (anemia of chronic disease) can be attributed to an inappropriate increase in hepcidin levels, which leads to decreased circulating iron. Hepcidin levels are downregulated by anemia and iron deficiency. Hepcidin agonists and antagonists are under clinical development for the treatment of disorders of inappropriately low or high hepcidin levels, respectively.

Hepcidin, in turn, is negatively regulated by the recently described hormone ERFE. ERFE is produced by erythroblasts during stress erythropoiesis, feeding back on hepcidin and reducing its levels, thus allowing iron absorption and export via ferroportin to proceed.

The Molecular Mechanism for Hepcidin Regualtion

Systematic Mechanism

The two transferrin receptors TfR1 and TfR2, and HFE, an MHC class I-like membrane protein, may serve as holotransferrin sensors. HFE can interact with both transferrin receptors, but this interaction is modulated by holotransferrin concentrations. Because HFE and holotransferrin binding sites on TfR1 overlap, increasing concentrations of holotransferrin result in displacement of HFE from TfR1, and free HFE then interacts with TfR2. TfR2 protein is further stabilized by binding of holotransferrin. The holotransferrin/HFE/TfR2 complex then stimulates hepcidin expression through an incompletely understood pathway, possibly by potentiating BMP pathway signaling. HFE, however, may also regulate hepcidin expression without complexing with TfR2. The role of HFE or TfR2 in hepcidin regulation by iron is supported by genetic evidence: HFE and TfR2 mutations in humans or mice cause hepcidin deficiency and an adult form of hemochromatosis.

screen-shot-2016-11-03-at-3-19-33-pmThe BMP pathway with its canonical signaling via Smad proteins has a central role in the regulation of hepcidin transcription. BMP receptors are tetramers of serine/threonine kinase receptors, with two type I and two type II subunits. Recent data indicate that type I subunits Alk2 and Alk3 and type II subunit ActRIIA and BMPRII are specific BMP receptors involved in iron regulation. In the liver, BMP pathway signaling to hepcidin is modulated by a coreceptor hemojuvelin and, at least in mice, by the ligand BMP6. Loss of hemojuvelin or BMP6 in mice decrease hepcidin expression and impairs hepcidin response to acute or chronic iron loading. In humans, hemojuvelin mutations result in severe hepcidin deficiency and cause juvenile hemochromatosis. It remains to be clarified how BMP receptors and hemojuvelin interact with iron-sensing molecules that regulate hepcidin expression.

Local Mechanism (intestine)

In addtition to the regulaltion by systemic signals, iron absorption is subject to local regualtion by intracellular mechanisms in duodenal enterocytes. At least two mechanisms have been described: one related to the enterocyte iron levels and the other to the hypoxia pathway.

Regulation of the synthesis of multiple proteins involved in iron physiology, including TfR1, DMT1, FPN1, and ferritin, is controlled at a posttranscriptional level by influencing mRNA stability and translation. The mRNA of these proteins contain iron response elements (IREs), conserved nucleotide sequences with a stem-loop structure that binds iron regulatory proteins (IRPs)-1 and -2. The mRNAs for ferritin, DMT1, and FPN1 have IREs in the 5' untranslated region (UTR), and the mRNA for the TfR has multiple IREs in the 3' UTR. In low-iron states, IRP-1 is in a conformation that allows it to bind to IREs; for example, it binds the 3' IRE of the TfR mRNA, stabilizing it and allowing transcription of more TfR protein, and to the 5' UTR of ferritin mRNA, decreasing translation of ferritin for iron storage. Intracellular iron induces ubiquitination and degradation of IRP-2. Iron deficiency by this mechanism upregulates IRP activity via increased IRE binding, resulting in increased cellular iron uptake and decreased iron storage.

Dcytb mRNA does not contain an IRE but is strongly upregulated in iron-deficient duodenum, indicating additional regulation of transcription of iron-related genes. Hypoxia-inducible factor (HIF) transcriptin factors may serve as important local regulators of intestinal iron absorption. Iron deficiency induced HIF signaling in duodenum of mice, and caused increased Dcytb and DMT1 expression, and an increase in iron uptake. Accordingly, targeted deletion of Hif-2alpha in the intestine resulted in dramatic decrease in the expressions of DMT1-IRE and Dcytb mRNA. In vitro studies further demonstrated that HIF-2alpha directly binds to the promoters of DMT1 and Dcytb, activating their transcription.

Summary: Factors That Affecting Hepcidin Levels

Systemic mechanisms: 1) by transferrin levels, including holotransferrin/HFE/TfR2 complex + hemojuvelin (coreceptor) + BMP6 (ligand), which increases hepcidin gene expression; 2) by erythropoiesis, however, the mechanisms by which erythropoiesis regulates hepcidin production are not well understood, but it is thought that erythroid precursors in the bone marrow secrete a factor which exerts its effect on hepatocytes and causes hepcidin supression (so the ERFE previously mentioned?); and 3) by cytokines in inflammation state, via STAT-3 pathway (already described above).

Local mechanisms: 1) posttranscriptional level by influencing mRNA stability and tanslation, via IRPs; 2) HIF's impact on cis-acting regulatory elements of DMT1 and Dcytb genes.


Iron Cycle

screen-shot-2016-11-03-at-5-12-27-pmMost functional iron in the body is not derived from daily intestinal absorption (1 to 2 mg/day) but rather from recycling of iron (20 to 25 mg/day) from senescent erythrocytes and other cells. The most important source and destination of recycled iron is the erythron. At the end of a 4-month lifespan, effete erythrocytes are engulfed by reticuloendothelial macrophages, which lyse the cells and degrade hemoglobin to liberate their iron. This process is poorly understood, but it appears to involve the action of heme oxygenase for enzymatic degradation of heme. Some of this iron may remain stored in macrophages as ferritin or hemosiderin, but most is delivered to the plasma, and the rate of iron export is determined by the hepcidin-ferroportin interaction. In plasma, iron becomes bound to transferrin, completing the cycle. A small amount of iron, probably <2 mg, leaves the plasma each day to enter hepatic parenchymal cells and other tissue. Here, the iron is stored or used for synthesis of cellular heme proteins, such as myoglobin and the cytochromes.

Macrophage Iron Recycling

Although there are many types of tissue macrophages, those that participate in the catabolism of red blood cells can be subdivided into two categories. One type, exemplified by pulmonary alveolar macrophages, is able to phagocytose erythrocytes or other cells and convert the iron they contain into storage forms, but lacks the ability to return the iron to the circulation. This type of macrophage appears to retain the iron throughout its life span. The second type of macrophage, comprising the reticuloendothelial system, acquires iron in a similar fashion but is able to return it to the plasma. The latter macrophages, found espeically in the sinuses of the spleen and the liver, play a primary role in the normal reutilization of iron from destroyed red cells, allowing completion of the iron cycle shown in Figure 23.4.


Plasma Transport

The plasma iron-binding protein, transferrin, is a glycoprotein with a molecular weight of approximately 80 kDa. Transferrin is synthesized chiefly in the liver and actively secreted by hepatocytes, but lesser amounts are made in other tissues, including the central nervous system, the ovary, the testis, and helper T lymphocytes (CD4+ subset). The rate of synthesis shows an inverse relationship to iron in stores; when iron stores are depleted, more transferrin is synthesized, and when iron stores are overfilled, the level of transferrin decreases. Transferrin keeps iron nonreactive in the circulation and extravascular fluid, delivering it to cells bearing transferrin receptors.

Transferrin can be measured directly using immunologic techniques; and normal concentration in the plasma is approximately 2 to 3 g/L. Alternatively, transferrin is quantified in terms of the amount of iron it will bind, a measure called the total iron-binding capacity (TIBC: normal values for plasma iron and TIBC are given in Appendix A). In the average subject, the plasma iron concentration is 100 ug/dL, and the TIBC is 300 ug/dL. Thus, only about one third of the available transferrin binding sites are occupied, leaving a large capacity to deal with excess iron. Plasma iron concentration varies over the course of the day, with the highest values in the morning and the lowest in the evening. Levels of serum transferrin are more constant, and there is no apparent diurnal variation in TIBC. General practice has been to evaluate transferrin saturation using a first morning, fasting sample to standardize the results, but this may not be helpful.

Transferrin has two homologous iron-binding domains, each of which binds an atom of trivalent (ferric) iron. The iron atoms are incorporated one at a time and appear to bind randomly at either or both of the two sites. When binding is complete, the iron lies in a pocket formed by two polypeptide loops. One mole of anion, usually carbonate or bicarbonate, is taken up, and 3 moles of hydrogen ion are released from each mole of iron bouond. There are functional differences between teh two iron-binding sites, but it is not clear that these have physiologic importance.

Under physiologic circumstances, ferric iron binds to transferrin with very high affinity, with an affinity constant of ~1-6 x 1022 M-1. The affinity of iron-transferrin interaction is pH-dependent, decreasing as pH is lowered. Other transition metals, such as copper, chromium, manganese, gallium, aluminum, indium, and cobalt, can be bound by transferrin but with less affinity than iron.

Iron Delivery to Erythroid Precursors

The biologic importance of transferrin in erythropoiesis is illustrated by abnormalities observed in patients and mice with congenital atransferrinemia. When transferrin is severely deficient, red cells display the morphologic stigmata of iron deficiency. This occurs despite the fact that intestinal iron absorption is markedly increased in response to a perceived need for iron for erythropoiesis. Nonhematopoietic tissues avidly assimilate the non-transferrin-bound metal. Similarly, mutant mice lacking tissue receptors for transferrin die during embryonic development from severe anemia, apparently resulting from ineffective iron delivery to erythroid precursor cells.

Transferrin delivers its iron to developing normoblasts and other cells by binding to specific cell-surface receptors. The transferrin receptor (TfR1) is a disulfide-linked homodimer of a glycoprotein with a single membrane-spanning segment and a short cytoplasmic segment. It is a type II membrane protein, with its N terminus located within the cell. The native molecular weight of TfR1 is ~180 kDa. Each TfR1 homodimer can bind two tranferrin molecules. Diferric transferrin is bound with higher affinity than monoferric transferrin. As a result, diferric transferrin has a competitive advantage in delivering iron to the erythroid precursors. Apotransferrin has little affinity for the receptor at physiologic pH but considerable affinity at lower pH, an important factor in intracellular iron release.

TfR1 numbers are modulated during erythroid cell maturation, reaching their peak in intermediate normoblasts. Very few TfR1 molecules are found on burst-forming-unit erythroid cells, and only slightly greater numbers are found on colony-forming-unit erythroid cells. However, by the early normoblast stage, approximately 300,000 receptors are found on each cell, increasing to 800,000 at the intermediate stages. The rate of iron uptake is directly related to the number of receptors. The number decreases as reticulocytes mature, and late in maturation, erythroid cells shed all remaining receptors by exocytosis and by proteolytic cleavage. The shed receptors, referred to as soluble transferrin receptors (sTfR) is a sensitive indicator of erythroid mass and tissue iron deficiency.

After ligand and receptor interact, iron-loaded transferrin undergoes receptor-mediated endocytosis. Specialized endocytic vesicles form, which are acidified to a pH of 5 to 6 by the influx of protons. The low pH facilitates release of iron from transferrin and strengthens the apotransferrin-receptor interaction. Released iron is reduced by an endosomal ferrireductase, STEAP3, and transferred to the cytosol by DMT1. Because DMT1 must cotransport protons with iron atoms, vesicle acidification is also important for the function of this transporter. After the iron enters the cytosol, the protein components of the endosome return to the membrane surface, where neutral pH promotes the release of apotransferrin to the plasma.

After Entering into Erythroid Precursor Cells

In the normal subject, ~80% to 90% of the iron that enters erythoid precursor cells is ultimately taken up by mitochondria and incorporated into heme. Most of the remainder is stored in ferritin. Granules of ferritin may sometimes be detected using the Prussian blue reaction. Normoblasts with Prussian blue-positive (siderotic) granules are called sideroblasts, and, if the granules persist after enucleation, the mature cells are called siderocytes. In normal individuals, approximately half of the normoblasts are sideroblasts, each containing less than five small granules.

It is intriguing that even though erythroid precursors require large amounts of iron and heme for hemoglobin synthesis, they have been found to express the heme exporter FLVCR and the iron exporter ferroportin. Studies suggest that FLVCR is necessary for survival of erythroid precursors. Because heme is toxic to cells at high concentrations, it is thought that FLVCR functions as a safety valve to prevent accumulation of excess heme early during erythroid differentiation.

Erythorid precursors also express ferroportin but its role in erythroid maturation is yet unclear. It is interesting that erythoid cells preferentially express non-IRE ferroportin transcript during the stages of rapid iron uptake, thus avoiding any iron-induced increase in ferroportin translation dependent on the IRE/IRP system.