NUTRITIONAL ISSUES IN THE PATIENT WITH DIABETES AND FOOT ULCERS

MARY D. LITCHFORD , in Levin and O'Neal's The Diabetic Foot (Seventh Edition), 2008

Transferrin

Plasma transferrin is an iron-transport protein with a half-life of 8 to 10 days that reflects both protein and iron status. Transferrin increases with iron deficiency and decreases when iron status improves or with protein-energy malnutrition. If a patient has concurrent iron deficiency, it is difficult to determine whether a low transferrin level reflects iron status or protein status. In mild to moderate protein-energy malnutrition, transferrin values may vary, limiting the usefulness of this test. However, markedly low transferrin levels indicate severe protein-energy malnutrition. A value less than 100 mg/dL may be considered a reliable index of severe protein-energy malnutrition. 55

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Metabolism of Iron and Heme

N.V. Bhagavan , Chung-Eun Ha , in Essentials of Medical Biochemistry (Second Edition), 2015

Alterations of Plasma Transferrin Concentration

Plasma transferrin levels are commonly measured in the evaluation of disorders of iron metabolism (discussed later). It is customary to measure transferrin concentration indirectly from the maximum (or total) iron-binding capacity (TIBC) of plasma (reference interval for adults, 250–400   μg/dL). It can also be measured directly by immunological methods (reference interval for adults, 220–400   mg/dL). Hypertransferrinemia (or increased TIBC) can occur with diminished body iron stores, as in iron deficiency anemia or during pregnancy (because of enhanced mobilization of storage iron to supply maternal and fetal demands). Hypertransferrinemia of iron deficiency is corrected by oral iron supplementation, whereas that due to pregnancy is not. Exogenous administration of estrogens (e.g., oral contraceptives) also causes hypertransferrinemia.

Hypotransferrinemia can result from protein malnutrition and accompanies hypoalbuminemia. Since transferrin has a much shorter half-life (8 days) than albumin (19 days), measurement of the transferrin level may be a more sensitive indicator of protein malnutrition than albumin measurement (see also Chapter 15). Hypotransferrinemia also results from excessive renal loss of plasma proteins (e.g., in nephrotic syndrome).

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Iron deficiency anemia, anemia of chronic disorders and iron overload

MJ Pippard , in Blood and Bone Marrow Pathology (Second Edition), 2011

Assessment of iron status

There is no single measure of iron status that is applicable in every situation. Combinations of measures of iron stores (macrophage and hepatocyte), iron supply to the tissues, and functional hemoglobin iron are often needed to arrive at a clear assessment of iron status. 82 The measures used are summarized in Table 11.1. All are subject to potential confounding factors.

Serum transferrin receptors

Serum transferrin receptors are truncated soluble receptors that are shed into the circulation mainly from the erythroblasts in the marrow. 83 The measurement reflects both the iron status of individual eythroblasts and the total mass of the erythron. 84 It is likely to be of most value in distinguishing iron deficiency from the anemia of chronic disorders. 85

Serum ferritin

Serum ferritin is apoferritin made up from glycosylated ferritin light chains, the release of which from cells reflects current ferritin protein synthesis. 86 It is thus related to the intracellular labile iron that determines IRP affinity for the IRE, and only indirectly to iron stores through the release of ferritin iron either within the cytosol or through lysosomal degradation. 87 Ferritin protein synthesis also increases in response to inflammatory cytokines, behaving as an acute phase protein independently of iron stores. Damage to ferritin-rich tissues can release iron-containing ferritin into the circulation giving high ferritin values, e.g. in hepatitis, splenic infarction, or bone marrow infarction in sickle cell disease. Its use as a guide to the presence of increased iron stores is thus limited, 88 though a low serum ferritin is a clear indication that iron stores are absent. The dependence of ferritin protein synthesis on translational regulation by the IRP/IRE mechanism is illustrated by the rare hereditary hyperferritinemia/cataract syndrome, where autosomal dominant mutations in critical parts of the IRE stem loop are accompanied by uncontrolled synthesis of ferritin light chain: high serum ferritin values are seen with the development of cataracts, but there is no iron overload and transferrin saturations are not increased. 89 Other uncommon autosomal dominant causes of a high serum ferritin but normal transferrin saturation include loss of function 'ferroportin disease' and a benign hyperferritinemia associated with a point mutation in the coding sequence of L-ferritin. 90

The reciprocal relationship between the synthesis of transferrin receptors and ferritin within cells that is mediated by the IRE/IBP mechanism (Fig. 11.3) has its counterpart in values for serum transferrin receptors (increased) and serum ferritin (reduced) during the development of iron deficiency. The sensitivity of these measures for assessing iron status is increased by expressing them as a ratio. 91,92

Tissue biopsy

Bone marrow biopsy is used to examine macrophage iron stores. It is primarily used for supporting a diagnosis of the anemia of chronic disorders rather than iron deficiency. Bone marrow biopsy touch preparations may give results comparable to aspirates. 93 Positive identification of the absence of iron stores may be inaccurate 94 unless careful examination of macrophages is made.

Liver biopsy provides the opportunity for histological examination of increased hepatocyte iron and any fibrotic or cirrhotic changes. The periportal distribution of iron accumulation in hemochromatosis may reflect selective expression of ferroportin in periportal hepatocytes and its degradation when hepcidin production is low. 64 In addition, chemical iron determination allows a quantitative assessment of the degree of iron loading. Quantitative phlebotomy in patients with thalassemia major who had undergone curative allogeneic bone marrow transplantation has confirmed that the liver iron concentration is a reliable indicator of total body iron stores in secondary iron overload resulting from red cell transfusions. 95 Magnetic resonance quantitative measurements (particularly T2*) of liver (and cardiac) iron 96 may reduce the future need for liver biopsy in iron overload disorders.

Potential clinical use of hepcidin assay

Difficulties in measuring urinary and serum hepcidin with lack of standardization 97 have so far restricted investigation of potential diagnostic uses. Plasma hepcidin concentrations are highly correlated with serum ferritin values in normal men, 98 and identification of inappropriately low hepcidin levels for existing iron stores might be diagnostically helpful in predicting the severity of the clinical course in iron loading disorders. However, like many other measures of iron status, confounding by the effects of inflammation is likely to make interpretation difficult. The possibility that hepcidin measurement might help to identify patients with anemia of chronic disease who also have an element of iron deficiency requires investigation. 99

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Metal Transporters

Ashley N. Luck , Anne B. Mason , in Current Topics in Membranes, 2012

5.1 Recombinant Expression and Purification of hTF

Isolation of hTF is economically feasible and relatively simple given the relative abundance in human serum (∼25 to 50   μM) (Sun et al., 1999). However, the potential for exposure to blood-borne pathogens and the inability to introduce mutations in serum-derived hTF has prompted the recombinant production of hTF in a number of expression systems. Naturally, one of the first expression platforms utilized to produce recombinant hTF is also one of the most common, Escherichia coli. The numerous advantages of using a bacterial expression system are well established. However, production of hTF in E. coli met with extremely limited success (de Smit et al., 1995; Hershberger et al., 1991; Hoefkens et al., 1996; Ikeda, Bowman, Yang, & Lokey, 1992; Steinlein & Ikeda, 1993). Due to an inability to correctly form 19 disulfide bonds (Fig. 2), attempts to produce hTF in E. coli yielded low amounts of improperly folded protein.

Another common expression system for recombinant proteins is the yeast, Pichia pastoris. Attempts to produce the isolated N-lobe of hTF in this eukaryotic system produced significant amounts (50–250   mg/l) of functional protein (Mason et al., 1996; Steinlein, Graf, & Ikeda, 1995). Although no effect on function was observed, analysis of the purified protein by electrospray mass spectrometry identified an O-linked glycosylation on residue Ser32, not present in the naturally derived protein (Mason et al., 1996). While the P. pastoris expression system offers several advantages (high yields and low cost), until recently no significant amount of full-length hTF has been produced in this system (Mizutani et al., 2010). Recent efforts using common baker's yeast, Saccharomyces cerevisiae, have produced homogenous non-glycosylated hTF at significantly higher yields (∼1.5   g/l) than previously observed in any yeast system (Finnis et al., 2010).

Baculovirus-infected insect cells are another frequently used expression system. Although the production of hTF in suspended insect cells provides the convenience of fast growth rates (as in yeast and bacterial expression systems) and the ability to posttranslationally modify the expressed protein, maximum production of hTF in this system only reached ∼20   mg/l (Ali, Joao, Csonga, Hammerschmid, & Steinkasserer, 1996; Retzer, Kabani, Button, Yu, & Schryvers, 1996).

By far, the most well-defined and characterized expression system used to produce recombinant hTF is the mammalian system developed in our laboratory (Funk, MacGillivray, Mason, Brown, & Woodworth, 1990; Mason, Funk, MacGillivray, & Woodworth, 1991; Mason et al., 1993, 2004). This system utilizes baby hamster kidney (BHK) cells transfected with the pNUT expression vector (Palmiter et al., 1987) containing the complementary DNA (cDNA) sequence for hTF. Importantly, the pNUT vector contains an ampicillin resistance gene that allows for propagation in E. coli. The hTF gene expressed in the pNUT vector by our laboratory also possesses a number of key features, the first of which is the hTF 19-amino acid signal peptide. This sequence is critical to the natural secretion of hTF from the liver into the serum. Normally within the body, this signal peptide is cleaved. In order to assure cleavage of the signal peptide, the first four amino acids of hTF (Val-Pro-Asp-Lys) precede the N-terminal hexa-histidine tag (used for purification). Next, our hTF construct contains a factor Xa cleavage sequence (Ile-Glu-Gly-Arg) to allow for facile removal of the hexa-His tag. Finally, the plasmid contains the polynucleotide sequence coding for hTF (Yang et al., 1984). The resulting cDNA is then transfected into BHK cells using a standard calcium phosphate precipitation method (Mason et al., 1991). Of importance, the pNUT vector also includes a mutated dihydrofolate reductase enzyme that allows for rapid selection (1–2 weeks) with methotrexate of only those BHK cells containing the plasmid following transfection (Funk et al., 1990). The use of this recombinant system has provided the means to produce hTFs that are either incapable of binding (mutation of the two liganding Tyr residues to Phe precludes iron binding in one lobe to create authentic FeNhTF or FeChTF constructs) (Mason et al., 2004) or releasing iron from one of the two lobes (mutation of residues in the dilysine trigger or C-lobe triad prevent iron removal to create LockNhTF or LockChTF constructs) (Fig. 6) (Byrne, Chasteen, et al., 2010; Byrne & Mason, 2009; Halbrooks et al., 2003). Although production of the isolated N-lobe of hTF has been very successful and well documented (Funk et al., 1990; Mason et al., 1993), until recently, production of the isolated C-lobe has been far more problematic. Again, bacterial expression of the C-lobe is rendered nearly impossible by the need to correctly form 11 disulfide bonds (MacGillivray & Mason, 2002), although a low yield (∼5%) of C-lobe with questionable conformation was reported by one laboratory (Hoefkens et al., 1996). Other attempts to produce the C-lobe of hTF using bacterial, yeast and mammalian systems have met with limited success and poor yields (Hoefkens et al., 1996; Steinlein et al., 1995; Steinlein & Ikeda, 1993). Even previous attempts to express the C-lobe as a recombinant entity using the BHK expression system, although successful, produced limited amounts of protein (Mason et al., 1997). Moreover, the C-lobe produced contained a complex glycosylation pattern at each of the two N-linked glycosylation sites in the C-lobe, resulting in a heterogeneous sample, further exacerbating the purification process. The Aisen laboratory produced a small amount of isolated C-lobe using the BHK cell system and an hTF construct with a factor Xa cleavage site in the bridge between the N- and C-lobes (Zak & Aisen, 2002). Using the BHK system, we followed a similar strategy in which the seven amino acids in the bridge were replaced by the tobacco etch virus (TEV) protease cleavage sequence, allowing utilization of the highly specific TEV protease to produce high yields of the isolated C-lobe (Steere, Roberts, et al., 2010). This particular C-lobe was recently crystallized (Noinaj et al., 2012).

Figure 6. Recombinant hTF constructs. Schematic representing recombinant hTF constructs. Mutations introduced into hTF to prevent iron binding in a lobe (as in the case of the FeNhTF, FeChTF and apohTF constructs) or prevent iron release from one lobe (as in the case of LockNhTF and LockChTF) are indicated. All constructs containing an N-terminal hexa-histidine tag are non-glycosylated and have all been previously described (Byrne, Chasteen, et al., 2010; Mason et al., 2004). For color version of this figure, the reader is referred to the online version of this book.

Recently, recombinant production of human proteins in plants has become an appealing alternative expression system and the topic of a great deal of research. Plant sources provide a relatively inexpensive animal-free method to the production of recombinant proteins for biopharmaceutical applications. Plant-derived recombinant proteins eliminate any potential contamination by animal pathogens. Furthermore, edible transgenic plants provide an attractive possibility for therapeutic purposes through direct oral delivery. Thus far, attempts to produce recombinant hTF in tobacco (Nicotiana tabacum) have met with rather limited success (estimated 0.25% total soluble protein) (Brandsma et al., 2010), while the production of hTF in rice (Oryza sativa) is estimated to be ∼40% of total soluble protein and is now available commercially under the trade name Optiferrin™ (Zhang et al., 2010). While Optiferrin™ appears to function almost identically to BHK-derived recombinant hTF in a number of aspects, the production of transferrins using this transgenic rice system requires ∼1 to 2 years, making it somewhat impractical for the production of mutant transferrins (Steere, A.N., Bobst, C.E., Zhang, D., Pettit, S., Kaltashov, I.A., Huang, N., & Mason, A.B., Journal of Inorganic Biochemistry, In Press.). It seems imperative that all recombinant hTF and TFR samples undergo a rigorous evaluation of their ability to function in a physiologically relevant manner.

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Transferrin Saturation

M.E. Elsayed , ... A.G. Stack , in Advances in Clinical Chemistry, 2016

2.2.1 The Transferrin Family

Human serum transferrin belongs to the transferrin family, which is a group of homologous glycoproteins that are related evolutionarily. Members of this family are widely distributed in the biological fluids of most vertebrates and invertebrates such as serum, egg white, mammalian milk, tears, and leukocytes [2,19–21]. Characteristically, they share not only a similar basic structure but also a unique ability to reversibly bind iron. They usually consist of 680–700 amino acids and show high degree of homology. This is evident by X-ray crystallography demonstrating that transferrins across various species share a 60–80% amino acid sequence homology [22,23]. For most of these proteins, the structure is mainly composed of two lobes that are further highly homologous. Each lobe is additionally arranged into two subdomains with a separating cleft that contains a metal-binding site. Homology between the two lobes indicated a duplication event of an ancestral ancient gene that resulted in the evolution of a monolobar protein to a bilobar form [23–25]. The transferrin family can be subdivided into several categories according to amino acid sequence, function, and occurrence in nature [26]. These include:

•

Serum transferrin (serotransferrin, siderophilin, or β-1-metal-binding globulin). This is the transferrin found in the serum of vertebrates and other biological fluids including cerebrospinal fluid, milk, and semen. Its main biological function lies in its ability to transfer iron between different biological tissues.

•

Lactotransferrin (lactoferrin): is mainly produced by mucosal epithelial cells of mammals. Thus it is found abundantly in mammalian milk and other secreted fluids, eg, tears, saliva, and pancreatic juice. It can also be present in specific granules of polymorphonuclear leukocytes. Lactoferrin exhibits variable properties that have antioxidant, antiinflammatory, and antimicrobial activities. Therefore, it plays an important role in body defense against infections [27–29].

•

Ovotransferrin (conalbumin): is primarily present in bird and reptile oviduct secretions and their eggs. It constitutes about 12–13% of the egg white in birds. It has the same structural protein, but different glycan component, of serum transferrin as they are derived from the same gene. It has also antimicrobial property that is vital to bird's innate immunity [30–33].

•

Melanotransferrin (p97 cell surface protein): traces of this cell surface protein can be found in normal tissues; however, the majority of human melanomas express it. It is one of the first cell surface markers associated with melanomas. Although its exact biological function remains unknown, it is believed that it may have a role in cell proliferation, migration, angiogenesis, and tumorigenesis [34].

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Assessment of Protein and Energy Nutritional Status

Lara B. Pupim , ... T. Alp Ikizler , in Nutritional Management of Renal Disease, 2013

Serum Transferrin

The primary function of serum transferrin is to transport iron in the plasma. It has a half-life of 8–10 days and a small body pool, making it sensitive to nutritional changes [49,69,70]. It can be directly measured or estimated by measurements of the serum total iron-binding capacity (TIBC) as follows: serum transferring = (0.8 ∗ TIBC) – 43. The normal range for serum transferrin is between 250–300 mg/dL. Low levels of serum transferrin (<200 mg/dL) have been associated with poor clinical outcome in malnourished children and other hospitalized patient populations [71–72]. Increases in serum transferrin levels have also been observed with nutritional supplementation [73]. In patients with CKD, assessment of serum transferrin as a marker of protein stores is problematic [74]. Specifically, iron metabolism is altered in advanced CKD, which can significantly affect serum transferrin concentrations. In addition, routine iron loading in ESRD patients, losses due to the nephrotic syndrome and gastrointestinal diseases, and, as with all the visceral proteins, any condition associated with inflammatory response or liver failure will affect serum transferrin concentrations [75,76]. Therefore, serum transferrin is not a recommended tool for monitoring nutritional status in patients with ESRD [77].

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Iron Deficiency Anaemia and Iron Overload

Mark Worwood , ... Barbara J. Bain , in Dacie and Lewis Practical Haematology (Twelfth Edition), 2017

Iron deficiency

The major application of the serum transferrin receptor assay has been to detect patients with an absence of stored iron (ferritin and haemosiderin in cells). In infants (age 8–15 months) sTfR concentration increases with increased severity of iron deficiency. 71 When normal subjects undergo quantitative phlebotomy, serum ferritin concentrations decrease steadily as iron stores are depleted, but there is little change in sTfR concentration. As iron stores become exhausted (serum ferritin <   15   μg/l), sTfR levels increase and continue increasing as Hb decreases 72 ; in this study the increased rate of erythropoiesis during phlebotomy had little effect on sTfR levels as long as iron stores were adequate so that most of the increase in sTfR level was the result of iron deficiency rather than increased erythropoiesis. However, the rate of phlebotomy was only 250   ml per week (about 450   ml per week is usually removed during treatment of haemochromatosis) and higher rates might cause an immediate increase in sTfR levels during phlebotomy. The log[sTfR/serum ferritin] gives a linear relationship with storage iron that has considerable potential for assessing iron stores in epidemiological studies. 73

Circulating transferrin receptor levels increase, not only in patients with simple iron deficiency but also in patients with the anaemia of chronic disease who lack stainable iron in the bone marrow. 74 Identifying a lack of storage iron in patients with the anaemia of chronic disease is difficult without this measurement because serum iron concentrations are low regardless of iron stores and serum ferritin concentrations are higher than in patients not suffering from chronic disease who have similar levels of stainable iron in the bone marrow (see p. 315). Unfortunately, the sTfR has not proved to be superior to serum ferritin for detecting iron deficiency in all studies (Table 9-7). 75–86

In both iron deficiency anaemia and the anaemia of chronic disease, sTfR levels are also influenced by changes in the rate of erythropoiesis. Ineffective erythropoiesis – an increase in the proportion of immature red cells destroyed within the bone marrow – increases in iron deficiency anaemia. 87 In the anaemia of chronic disease, erythropoiesis is normal or depressed; 88 nevertheless, iron deficiency increases the number of receptors.

Although it has been claimed that sTfR measurements provide a sensitive indicator of iron deficiency in pregnancy, 89 questions remain about the decreased erythropoiesis in early pregnancy because this may mask iron deficiency at this time 90 and increases in sTfR in later pregnancy appear to relate to increased erythropoiesis rather than iron depletion. 69 Measurement of sTfR did not enhance the sensitivity and specificity for the detection of iron deficiency anaemia in pregnant women from Malawi, where anaemia and chronic disease are both common. 91

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Anemia in Heart Failure

A. Patel , N.L. Altman , in Encyclopedia of Cardiovascular Research and Medicine, 2018

Anemia of Chronic Disease

Defined by a reduced serum iron and transferrin concentration and a low total iron binding capacity along with a normal to high ferritin level, anemia of chronic disease is a prevalent entity in the heart failure population, with incidence varying in the literature. Erythropoiesis is dependent on a number of mechanisms, many of which can be altered in patients with heart failure. Erythropoietin is produced in the kidney. The primary stimulus for erythropoietin production is low serum oxygen content (Anand, 2008), which occurs due to a reduction in renal blood flow and glomerular filtration rate in patients with HF. However, the amount by which erythropoietin production rises is blunted in comparison with the severity of anemia (Opasich et al., 2005).

In addition, heart failure results in an overall active proinflammatory state, including elevated levels of tumor necrosis factor-α, interleukin (IL)-6, CRP, and other proinflammatory cytokines, which inhibit erythropoiesis in the kidney via a series of complex mechanisms (Jelkmann, 2009).

In patients with heart failure who develop anemia without ID, alternate methods of therapy are often considered. Trials using darbepoetin alfa (Swedberg et al., 2013; Pfeffer et al., 2009) to correct hemoglobin to a target level of 13.0   g/dL showed no reduction in death, cardiovascular events, or HF hospitalizations, but did result in a concomitant increase in fatal and nonfatal strokes. The medical community has therefore steered away from the use of EPO-analogs to treat mild anemia in heart failure.

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Disorders of Glycosylation

Hudson H. Freeze , ... Marc C. Patterson , in Swaiman's Pediatric Neurology (Sixth Edition), 2017

Diagnosis

Most CDG patients were first recognized by abnormal glycoforms of serum transferrin. Commercially available tests include isoelectric focusing, mass spectrometry, zone electrophoresis, and high performance liquid chromatography; however, electrospray ionization-mass spectrometry (ESI-MS) (Babovic-Vuksanovic and O'Brien, 2007) is the most informative because it differentiates absence of entire sugar chains from one or more monosaccharide units. Normal transferrin has two sugar chains, each containing two negatively charged Sia molecules, designated tetrasialotransferrin. Loss of one or two entire chains produces disialotransferrin or asialotransferrin respectively, but this is a misnomer because it is the loss of more than sialic acid. ESI-MS shows losses of 2200 or 4400 mass units, respectively. ESI-MS can also detect loss of single or multiple individual sugars. This distinction helps narrow gene candidates from whole exome or genome sequencing results. ESI-MS is the preferred method (Babovic-Vuksanovic and O'Brien, 2007) and is suitable for routine diagnostics.

Transferrin isoform analysis produces few false-positive results. Uncontrolled fructosemia, galactosemia, and recent heavy alcohol consumption produce a pattern typical of group I disorders. Sometimes, patients with genetically confirmed CDGs develop normal transferrin, and in some patients, previously abnormal patterns normalized in preadolescence. Thus a normal transferrin pattern should not exclude follow-up testing. Healthy neonates sometimes have a slightly abnormal transferrin pattern, which normalizes within a few weeks. Suspicious results in neonates should be repeated.

Some genetic centers and commercial laboratories now offer various CDG gene diagnostic panels (Greenwood Genetic Center, Baylor Medical Genetics, Emory Genetic Laboratory), but falling costs and improved bioinformatics make whole exome sequencing the first choice. However, the proven power of transferrin analysis should always accompany a putative genetic result. Prenatal testing is available for confirmed at-risk families.

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