14.11: Emerging iron status indicators (17.11)

Emerging indicators, currently mainly used in a research setting, include hepcidin, non-trans­ferrin-bound iron, and some reticulocyte indices. More work is required to understand the diagnostic value of these emerging indicators before they become more widely available for public health or clinical use.

17.11.1 Hepcidin

Hepcidin is a protein encoded by the HAMP gene. It plays a major role in controlling physio­logical iron homeo­stasis, as noted earlier. Hepcidin binds and subsequently causes degradation of the iron-exporter ferroportin, thereby inhibiting both absorp­tion of dietary iron from the gastro­intestinal tract and the release of iron from body stores (Section 17.1.2). In con­ditions of iron depletion or increased iron demand, hepatic synthesis of hepcidin is reduced, thus facilitating iron absorp­tion and mobil­ization from body stores via active ferro­portin. In contrast when iron stores are replete (or in renal impairment when excretion of hepcidin is suppressed), hepcidin levels are increased which block the release of iron into the circu­lation. Instead, iron is lost with the cell when it is shed from the villus. Hepcidin produc­tion by the liver is also regulated by inflam­mation and erythro­poietic activity.

Hepcidin may be useful in the clinical diagnosis of iron-refractory anemia (i.e. the type of iron defi­ciency anemia that typically does not improve with oral iron treatment), in dif­fer­entiating between iron defi­ciency anemia and anemia of chronic disease and in patients with iron overload syndromes. As hepcidin is a determinant of dietary iron absorp­tion, hepcidin may also provide guidance on safe iron sup­ple­mentation in countries with a high infection burden (van der Vorm et al., 2016; Pfeiffer and Looker, 2017). Studies have also investigated the diagnostic potential of serum hepcidin as an index of iron defi­ciency in preg­nancy (Bah et al., 2017). Never­the­less, whether hepcidin assays provide an advantage over serum ferritin and the other more common methods for assessing nutritional iron status, particularly when iron status is replete, remains uncertain (Pfeiffer and Looker, 2017; Lynch et al., 2018).

Inflam­mation raises levels of pro-inflam­matory cytokines in the systemic circu­lation, which in turn increase serum hepcidin levels, an acute-phase protein, just like serum ferritin. Elevated hepcidin levels reduce both iron intestinal absorp­tion and the release of iron from body stores, thus decreasing the amount of circu­lating serum iron and thereby limiting erythro­poiesis. Hence, the risk of iron defi­ciency increases, even though body iron stores are normal.

Obesity is associated with low grade inflam­mation and thus elevated hepcidin levels. Results presented in Table 17.39

Table 17.39. Inflam­matory markers and iron biomarkers in normal weight healthy young women (n=22) and obese women (n=25).
WBC, White Blood Cell Count; IL-6, interleukin-6; CRP, C-reactive protein; MCV, mean corpuscular volume (mean cell volume); TIBC, total iron-binding capacity; TSAT, trans­ferrin satu­ration.
¹ Data available for 40 women (18 normal weight vs. 22 obese);
² Data available for 42 women (20 normal weight vs. 22 obese); * Geometric mean (95% CI).
Abstracted from Aguree and Reddy (2021).

	Normal Weight Women (BMI: 18.5‑24.9 kg/m^2)	Obese Women (BMI: >29.9 kg/m^2)	p Value
Variable	Mean ± SD	Mean ± SD
Inflam­matory markers
WBC (×10^3µL)	5.6 ± 1.6	7.7 ± 2.0	< 0.001
IL-6 (pg/mL) ^1*	1.46 [1.13, 1.89]	2.16 [1.86, 2.51]	0.003
CRP (mg/L) ^1*	8.2 [3.1, 21.8]	69.9 [41.1, 118.9]	< 0.001
Iron bio­markers
Hb (g/dL)	13.6 ± 1.1	13.3 ± 1.1	0.166
MCV (fL)	86.3 ± 4.5	82.4 ± 8.9	0.030
Serum iron (µg/dL) ^2	112.0 ± 41.4	92.4 ± 33.9	0.044
TIBC (µg/dL) ^2	394.4 ± 61.5	371.3 ± 50.4	0.086
TSAT (%) ^2	29.7 ± 13.3	25.2 ± 9.1	0.094
Ferritin (ng/mL) ^1*	34.0 [21.0, 55.1]	37.7 [26.9, 52.9]	0.355
Hepcidin (ng/mL) ^1	6.21 [4.39, 8.77]	11.21 [7.04, 17.83]	0.024

compare inflam­matory and iron status bio­markers in a cross-sectional study of normal weight and obese healthy U.S. women. The data highlight the higher mean serum concen­trations of CRP, inter­leukin‑6, and hepcidin but lower levels of MCV and serum iron (but not ferritin) in the obese women compared to their normal weight counterparts (Aguree and Reddy, 2021).

Enhanced erythro­poiesis suppresses hepcidin because of the increased iron demand. Conse­quently, iron absorp­tion is increased and iron is mobilized from body stores. Numerous con­ditions are associated with an increase in erythroid proliferation; some examples are given in Table 17.28. Of concern are individuals with severe transfusion-dependent HbE β‑thalassemia and β‑thalassemia carriers (but not HbE carriers) because the increased uptake of iron resulting from severe and mildly suppressed hepcidin, respectively, may increase the risk of iron overload. As an example, Table 17.40 highlights the suppression

Table 17.40. Demographic, iron, erythropoietic, and inflam­matory indices for patients with HbE β‑thalassemia stratified by severe and moderate phenotype, NTBI, Non-trans­ferrin-bound iron. Values are *mean (range) or **geometric mean (range). ^†signif­icant difference between moderate and severe patients (P < 0 .05). ^‡signif­icant difference between controls and severe patients, and controls and moderate patients. Data from Jones et al. (2015).

Parameter	Severe (n=28; 17 female)	Moderate (n=41; 25 female)	P	Local controls (n=25)
Hb g/dL*	5.8 (4.4‑7.6)	6.4 (4.9‑8.3)	.1397	15.0 (14.5, 15.5)^‡
Ferritin mg/L**	1356 (328, 9790)	732 (143, 3260)	.0021^†	48.2 (38, 61.2)^‡
Liver iron mg/g dwt**	11.3 (1, 54.2)	5.30 (0.7, 29.0)	.0006^†
NTBI µM/L*	5.16 (−1.67, 10.27)	4.36 (−2.67, 23.75)	.4456
Hepcidin ng/mL**	2.24 (0.1, 51.8)	1.95 (0.1, 36.3)	.7441	28.23 (18.83, 42.3)^‡
sTfR nM/L**	6.51 (1, 35.3)	8.87 (1.4, 35.3)	.1472
CRP mg/L**	1.66 (0.23, 34.23)	1.58 (0.20, 13.01)	.8433
Labile plasma iron µM/L*	3.60 (0.1, 7.7)	4.29 (−1.07, 13.61)	.9320
Trans­ferrin SAT, %*	97.3 (48.3, 100.0)	86.6 (31.9, 100.0)	.0319^†

of hepcidin in Sri Lankan patients with HbE β‑thalassemia classified as severe or moderate clinical phenotypes. Patients in both groups had lower concen­trations of serum hepcidin and Hb and higher serum ferritin concen­trations when compared to the values for 25 iron-replete, non‑thalassemic Sri Lankan male controls.

Even among Sri Lankan schoolchildren, hepcidin was mildly suppressed among those with increased erythro­poiesis associated with β‑thalassemia trait (but not with HbE) compared to controls, suggesting an enhanced propensity to accumulate iron (Jones et al., 2015). Hence, in Asian countries where β‑thalassemia trait syndromes and thalassemia carriers are prevalent, risk of causing an inadvertent burden of iron overload should be considered when planning public health iron inter­ventions to improve the iron status of high risk popu­lation groups (Jones et al., 2015), as noted earlier.

Malaria induces an increase in hepcidin concen­trations, likely as a result of suppressed erythropoietic activity, inflam­mation and perhaps additionally by a direct stimulatory effect of malaria parasites and their products (de Mast et al., 2009). Hepcidin concen­trations are elevated even when the malaria is asymptomatic (i.e., presence of parasitemia in the absence of fever or malaria-related symptoms) and in the absence of a marked acute phase response, as shown in Figure 17.18. Although an effect of mild inflam­mation cannot be excluded, never­the­less, other mech­anisms might also con­tribute to elevated hepcidin concen­trations such as the existence of an IL-6-independent pathway in malaria.

Figure 17.18. Serum concen­trations of hepcidin in children with asymptomatic P. falciparum parasitemia (n=73), asymptomatic P. vivaxparasitemia (n=18) and controls (n=17). Data depicted are before and 4 weeks after the start of antimalarial treatment; the line represents the mean. P values were determined using the Student's t-test and paired t-test. Redrawn from de Mast et al. (2009).

The results shown in Figure 17.18 are based on a group of Indonesian children (5‑15y) (n=1197) screened by microscopy for the presence of parasitemia, Hb, serum hepcidin, indices of iron status (ferritin, sTfR, mean cell volume) and inflam­mation (CRP) at baseline and 4 weeks after antimalarial treatment.

It is of interest that the presence of even mildly elevated hepcidin concen­trations appear to induce functional iron defi­ciency in these Indonesian children with asymptomatic parasitemia, as indicated by lower values of Hb, MCV, serum iron and trans­ferrin satu­ration. These findings suggest that in the presence of asymptomatic malaria, iron therapy for the treatment of malaria anemia may be less effective because absorp­tion of iron is compro­mised by the high hepcidin concen­trations. Moreover, the iron therapy may be even hazardous, increasing malaria-associated morbidity and mortality (de Mast et al., 2010).

Preg­nancy suppresses hepcidin by a mech­anism that is unclear (Koenig et al., 2014). During the first trimester, serum hepcidin concen­trations are within the reference range for nonpregnant women, but decrease in the second trimester to very low levels, where they remain in the third trimester, increasing again immediately after delivery and thereafter (van Santen et al., 2013; Bah et al., 2017; Fisher and Nemeth, 2017) (Figure 17.19).

Figure 17.19. Median (IQR) serum hepcidin concen­trations in 31 women during preg­nancy and postpartum. ***Compared with first-trimester values, P < 0.0001. Modified from Fisher and Nemeth (2017).

The fall in hepcidin appears to occur in advance of the onset of low iron stores in preg­nancy, as shown from the data for Gambian women presented in Table 17.29. Here, hepcidin, indices of iron stores, erythro­poiesis, and inflam­mation were all measured at 14, 20 and 30wks gestation(Bah et al., 2017). This suggests that maternal iron defi­ciency is not solely responsible for the sharp decline. Even mothers with replete iron stores have low hepcidin concen­trations at delivery, suggesting that maternal hepcidin may be actively suppressed during preg­nancy (Fisher and Nemeth, 2017).

How­ever, in pregnancies complicated by infection or inflammation (including obesity), maternal hepcidin concentrations are elevated compared to healthy controls. Hence, in these conditions maternal and fetal iron bioavailability could be compromised in pregnancy, limiting the amount of iron presented for uptake by the placenta and for transfer to the fetus (Koenig et al., 2014).

Interpretive criteria for hepcidin concen­trations are difficult to define because concentrations vary widely across assays, as for serum sTfR (Kroot et al., 2009). As a conse­quence, no usable inter­national reference ranges and reference limits for hepcidin for specified life-stage groups exist at the present time.

Measure­ment of serum hepcidin with good reproducibility in both plasma and urine is possible using mass spec­trom­etry or immuno­assays, although inter-assay variability is large, with no certified reference material currently widely available (Pfeiffer and Looker, 2017). Recently an inter­national calibrator for hepcidin with long-term stability has been developed (available at HepcidinAnalysis.com) which will greatly improve the equivalence between hepcidin measure­ment procedures in the future; see Van der Vorm et al. (2016); Diepevenn et al. (2019); and Aune et al., (2020) for further details.

Measure­ment of hepcidin in urine may provide a less-invasive screening for iron status and warrants more investigation. Concen­trations are elevated in anemic children with febrile malaria.

17.11.2 Novel red blood cell indices

Novel red blood cell indices are generated by specific models of hematology automated cell counters and include among others, reticulocyte mean Hb content (RHcc) and mean reticulo­cyte volume (MRV). How­ever, their inter­pretation is complicated by the dif­fer­ent techniques used by the manufacturer and the lack of system­atic studies to determine their usefulness to evaluate nutritional anemias.

Reticulocyte Hb content (RHcc) reflects directly the synthesis of Hb in bone marrow precursors and is a measure of the adequacy of the availability of iron. It can be used to dif­fer­entiate iron defi­ciency from other causes of anemia. Reticulo­cyte Hb content is sometimes used in clinical settings to establish iron status in young children with anemia, when other common iron bio­marker assays may not be available; low values provide an early marker of iron deficient erythro­poiesis. The measure­ment of RHcc is not useful in individuals with α and β‑thalassemias (including individuals who are hetero­zygous) when the reticulocyte Hb count is reduced independently of iron stores.

Reticulocyte Hb content can also be used to measure the early response to intravenous iron therapy because it increases signif­icantly after only 48‑72h (Buttarello, 2016). The sensitivity and specificity of RHcc for diagnosing iron defi­ciency is only moderate, with no recom­mended cutoffs to define iron defi­ciency. How­ever, RHcc is less affected by inflam­mation than serum iron, trans­ferrin satu­ration, and ferritin, although it is influenced by any con­ditions that cause iron restricted erythro­poiesis (Gelaw et al., 2019).

Reticulocyte volume is also an index that can be measured by some newer hematology analyzers. Values for reticulocyte volume decrease rapidly with the devel­opment of iron-deficient erythro­poiesis and increase rapidly in individuals with depleted iron stores or nutritional macrocytosis after therapy with iron, or vitamin B₁₂ and/or folic acid, respectively. How­ever, numeric results depend on the analyzer manufacturer, making comparisons difficult (Buttarello, 2016).

17.11.3 Non-trans­ferrin bound iron

Iron in plasma is tightly bound to trans­ferrin, the iron trans­port protein that delivers iron throughout the body, as noted earlier (Section 17.6). Cells take up trans­ferrin-bound iron in proportion to the number of cell-surface trans­ferrin receptors (TfR1). When cellular iron levels are sufficient, the uptake of trans­ferrin decreases to limit further iron assimilation and prevent excessive iron accumulation; usually only 20‑30% of trans­ferrin is saturated with iron. How­ever, in iron overload con­ditions, such as hereditary hemo­chromatosis and thalassemia major, trans­ferrin is fully saturated and non-trans­ferrin-bound iron (NTBI) appears in serum. Note the high concen­trations of NTBI in the patients with HbE β‑thalassemia in Table 17.40 , which were inversely associated with hepcidin. This trend may reflect the increased release of iron into the plasma that occurs when suppressed hepcidin levels permit enhanced iron absorp­tion and mobilization. The NTBI also corre­lated with ferritin and liver iron concen­trations, suggesting that a high level of NTBI is associated with hepatic iron accumulation (Jones et al., 2015).

There is no known regulatory mech­anism for NTBI uptake so NTBI can enter cells readily, where it produces free radicles resulting in cellular and organ damage. Circulating NTBI may also cause an increase in bacterial-pathogenic infections owing to the free ion being utilized by the parasite. Concern has been raised over the rapid absorp­tion of iron sup­ple­ments given in non-physio­logical amounts, and their potential for exceeding the capacity for trans­ferrin to bind the circu­lating iron, resulting in the formation of NTBI concen­trations (Prentice et al., 2016).

The measure­ment of serum NTBI is fraught with problems, often with a 40-fold variation difference in methods, in part due to the hetero­geneous nature of the chemical forms of NTBI, as well as large analytical variation. A new automated NTBI assay has been developed which looks promising in terms of reproducibility and comparability with HPLC, one of the most reliable methods to quantify NTBI. Never­the­less, more research is required to define the most relevant forms of NTBI, and understand their clinical importance (Pfeiffer and Looker, 2017).