Management of anemia of chronic disease: beyond iron-only supplementation

1 “Scientific Clinical Institutes Maugeri”, IRCCS Lumezzane, Cardiac Rehabilitation Division, Lumezzane, Brescia, Italy; 2 Division of Human Anatomy and Physiopathology, Department of Clinical and Experimental Sciences, University of Brescia, Brescia, Italy; 3 Department of Biology and Biotechnology, University of Pavia, Pavia, Italy. 4 Center for Heart and Vessel Preclinical Studies, St. John Hospital and Medical Center, Wayne State University, Detroit, Michigan, USA. 5 Division of Cardiology, Richmond Veterans Affairs Medical Center (VAMC), Richmond, VA 23249, USA. 6 Department of Internal Medicine, University of Cagliari, Cagliari, Italy.


Iron and Heme
Iron (Fe), a requisite metal in almost all biological systems, is necessary for numerous critical processes such as DNA synthesis, heme and iron-sulfur cluster synthesis etc. Therefore, cellular regulation of iron concentration is essential for maintenance of normal physiology [13].
About 70% of body's iron is found in the red blood cells as haemoglobin, and in muscle cells as myoglobin. Iron, an essential element for blood production, is a crucial component of a very large class of metalloproteins containing heme, hence the name, hemoproteins.
Heme is an organic, ring-shaped molecules consisting of an iron ion coordinated to four pyrroles which are small pentagon-shaped molecules with 4 carbons and 1 nitrogen, which together form an iron-binding tetrapyrrole called porphyrin (Figure 1). Thus, heme is an iron-binding porphyrin [11]. Interestingly, the iron plays a balanced attractive force interacting with the nitrogen molecules of heme, thus, electrons stay balanced and the global molecule remains stable.
There are 4 different forms of heme in nature: heme-A, -B, -C and -O; they influence the function of the molecules in which heme is present. Although heme-B is the most common form, heme-A and -C are present in many molecules. Biochemical behaviors of the most common heme groups are regulated by differences of the functional groups in the side chains bound to carbon 3,8 and 18 [11].
protoporphyrin-IX. The enzyme ferrochelatase then inserts the iron atom, forming heme which is shuttled in the cytoplasm where it is uitilized in the synthesis of heme-based molecules [14]. The process of heme synthesis is illustrated in the Figure 2.

Functions of heme
Heme and hemoproteins have many biological functions. The presence of an iron atom serves as a source of electrons during electron transfer or redox chemistry, thereby giving heme the ability to transport diatomic gases and to exert chemical catalysis with electron transfer.
The hemoproteins participate in many diverse biological actions (such as oxygen transport) fundamental for life. Indeed, although haemoglobin and myoglobin are the two best known hemoproteins, other important, but often overlooked, enzymes which belong to hemoproteins include: cytochrome p450s, cytochrome-c oxidase, cyclooxygenase 2, catalase, peroxidases and endothelial nitric oxide synthase. In addition, as part of the electron transport chain, hemoproteins also enable electron transfer. A change in iron content affects important cell survival systems, illustrating that heme is not only important for oxygen transport, but also plays a fundamental role in other important metabolic pathways such as: energy production; transformation of many molecules and detoxification of aggressive molecules such as as oxygen free radicals; regulation of inflammation and/or vascular tone; and blood coagulation [11].

Other molecules involved in heme synthesis
CD, especially if associated with qualitative malnutrition, induce a hyper-catabolic state and consequent protein disarrangement, which can precipitate the development of anaemia secondary to a reduction in hemoglobin. A schematic representation of this link is proposed in Figure 3.
Independently from iron, other molecules which are strictly related to heme synthesis include: VITAMIN B1. Its' pyrophosphate ester, thiamine diphosphate (TPP) is a co-factor for enzymes that catalyse alpha-keto acids of molecules involved in the Kreb's cycle and its intermediary metabolism [15].
VITAMIN B6. It co-catalyses reactions related to the anabolism and catabolism of AAs, facilitating the reactions of transamination. Interestingly, it is involved in protein folding, interacting with the folate cycle. In addition, vitamin B6 is a scavenger of free oxygen radicals [16]. In light of these considerations, provision of the molecules involved in the synthesis of heme and hemoglobin is essential, even more so if patients are nutritionally deficient.

Methods
Based on the aforementioned fundamental biochemical knowledge and in observation of "good medical practice" (www.gmc-uk.org), we conducted a controlled clinical trial which integrated personalised standard therapy with iron infusion, along with the administration of specialized mixtures rich in free EAAs [21] and vitamins (B1, B6, B9, D) to treat heme synthesis deficiency in a cohort of select elderly female patients (n=15; age 78.3 ±8.5 y.o.) with CHF. Written informed consent was obtained; ethical approval was not required under local legislation. The inclusion criteria were: 1) anemia (Hb >8.5/<11.5 mg/dl); 2) symptoms and signs of stable CHF for at least 3 months on a standard medical therapy with beta-blocker, diuretics, ACE-inhibitor or ARB; 3) protein disarrangement (albuminemia <3.5 g/dl), but normal BMI (>24); 4) iron deficiency (plasma iron <50 mg/dl, ferritin <100 mg/dl, or serum ferritin within range 100-299 mg/dl when transferrin saturation is <20%); 5) inflammation (by CRP >5 mg/l); and 6) vitamin D and/or folate lower than the normal ranges (15.2-90.1 pg/ml and >3.00 ng/ml respectively).
Since these patients, as well as the haemoglobin concentration, had levels of albumin and vitamins below the minimum, according to good medical practice, we treated them for 30 days with daily intravenous administration of 100 mg of ferric carboxymaltose, integrated with oral administration of 4g of specific free AAs mixture rich in essential ones (84%), 15mg of Vit.B9, 0. The baseline mean clinical biochemical data from two cohorts are summarized in Table 1.

Results
Baseline and post-intervention clinical biochemical data are summarized in Table 2. Increased levels of sideremia, ferritin, saturated transferrin were observed in both groups; there was no diffence in total transferrin values. However, only the experimental group receiving intravenous iron therapy PLUS integrated therapy demonstrated increased levels of haemoglobin (Figure 4A-B) compared to the standard iron therapy only group [see Table 3 and Figure 4C-D].
The Student t-Test was used to compare the data before and after treatment, with p-value <0.05 considered significant.

Discussion and Conclusion
The incidence of anemia (32%) is common in HF patients, with concurrent iron and folate deficiencies noted in 43% of anemic patients, compared to 15% of non-anemic patients [29].
Currently, the standard therapy of anemia is primarily based on the supplementation of iron, with or without erythropoietin for hematopoiesis stimulation. Previous randomized, controlled studies with intravenous iron in HF patients reported that haemoglobin increased after 4 or 6 months [7,10,30,31]. Indeed, if a deficiency of fundamental molecules (such as amino acids and vitamins) results in the lack of heme synthesis, iron supplementation alone will not lead to a proportional hemoprotein increase, or Hb in primis. In addition, an isolated increase in iron, without any accompanying augmentation in heme, could favor the persistence of oxidative stress (via Fenton/Haber-Weiss reactions), chronic inflammation and autophagy [32]. The mode of iron supplementation also appears to be important, as oral supplementation has not been shown to be effective in improving exercise capacity in patients with HF with reduced ejection fraction and iron deficiency [33].
Based on these preliminary data demonstrating a rapid escalation in hemoglobin level (within 30 days after interventions aimed at increasing iron and heme), we conclude that the effective approach to treating heme synthesis (including anaemia) in CD must consider not only the iron availability, but also integrate a therapeutic strategy which counteracts catabolism. Therefore, the standard intravenous or oral iron supplementation should incorporate the supply of specific mixtures of EAAs and vitamins involved in biochemical pathway of the heme synthesis as illustrated in the Figure 5. Consequently, the careful evaluation of nutritional status of patients, the presence of catabolism and of molecules involved in heme synthesis, as well as their integration, must therefore be the first step of the personalized therapeutic intervention aimed at correcting the state of anaemia in patient with CD such as CHF. Our therapeutical approach based on biochemical data should be confirmed in a large-scale clinical trial.

•
In chronic hypercatabolic diseases (such as CHF), the metabolism of iron and heme-protein is markedly impaired, inducing anaemia and likely impairment of many other hemoproteins involved in essential metabolic pathways.
• Heme is the metabolically active part of haemoglobin. It is characterised by the presence of iron atoms linked to tetrapyrrole groups.