Vitamin B6 deficiency anemia resembles iron-deficiency anemia but does not activate intestinal HIF2α

Abstract

Microcytic hypochromic anemias, including iron-deficiency anemia (IDA) and vitamin B6-deficiency anemia (B6DA), share similar hematologic features but differ fundamentally in etiology and therapeutic responsiveness. IDA results from insufficient dietary iron, activating adaptive intestinal iron absorption via hypoxia-inducible factor 2α (HIF2α), whereas B6DA impairs heme biosynthesis without systemic iron depletion, rendering it refractory to iron therapy. Here, we investigated whether intestinal HIF2α activation distinguishes these anemias. Using mouse models, we established that severe dietary iron restriction (<5 ppm for 2 weeks) robustly induced intestinal Hif2α targets Dmt1, Dcytb, Fpn1, and Ncoa4, while suppressing hepatic hepcidin. In contrast, B6-deficient mice developed hypochromic microcytic anemia over 60 days without changes in intestinal Hif2α signaling, duodenal ferritin, or hepcidin. Parenteral B6 supplementation rapidly restored hemoglobin but did not alter intestinal iron gene expression, confirming that low iron, not anemia, drives Hif2α-mediated intestinal adaptation. These findings reveal a mechanistic distinction: IDA engages a co-ordinated systemic and intestinal iron response, whereas B6DA produces anemia through heme biosynthesis defects independent of iron sensing. Importantly, assessment of intestinal HIF2α activity or downstream targets provides a functional biomarker to differentiate iron-responsive from non–iron-responsive microcytic anemias. This approach could prevent misdiagnosis and guide more precise therapeutic strategies, highlighting the value of integrating molecular iron sensing into clinical evaluation of nutritional anemias.

Hemoglobin (Hb), an integral red blood cell (RBC) protein, is the main oxygen-carrying molecule in our body [1]. Normal adult Hb is a tetramer composed of two α- and two β-polypeptide chains, each bound to a heme group. The heme groups are of particular importance, as they contain ferrous iron capable of reversibly binding one oxygen molecule, allowing seamless oxygen delivery to tissues [2]. According to the World Health Organization, approximately 30% of the global population exhibits low Hb levels, resulting in reduced oxygen transport to organs and tissues [3–5]. This is a global health concern caused by a wide variety of disorders, including a large subset known as microcytic anemias. Microcytic anemias are defined by the production of smaller-than-normal RBCs due to impaired Hb synthesis [6]. Although multiple factors can contribute to reduced Hb levels, nutritional deficiencies are the most common cause [7]. In particular, inadequate iron intake limits Hb production, leading to iron-deficiency anemia (IDA), the most prevalent nutritional disorder worldwide [8]. Because mammals cannot synthesize iron de novo and rely entirely on dietary sources, consistent and sufficient iron intake is essential to prevent IDA. IDA is also referred to as microcytic hypochromic anemia, as its hallmark features include microcytosis (reduced mean corpuscular volume [MCV]) and hypochromia (pale RBCs due to low Hb content). Iron replacement remains the most effective and economic treatment; however, not all cases of IDA respond to iron therapy, suggesting the involvement of additional, complex mechanisms [9,10]. In such situations, careful evaluation of other causative factors (alone or in combination with iron deficiency) leading to microcytic hypochromic anemias is critical for effective patient management.

The intestine plays a central role by modulating dietary iron absorption in response to systemic and local iron status. Hypoxia-inducible factor 2α (HIF2α) functions as a transcriptional master regulator in enterocytes, stabilizing under conditions of low iron or hypoxia and upregulating genes essential for iron uptake, including divalent metal transporter 1 (DMT1), duodenal cytochrome b (Dcytb), ferroportin 1 (Fpn1), and nuclear receptor coactivator 4 (NCOA4), a regulator of ferritinophagy [11–13]. Through these mechanisms, HIF2α ensures efficient intestinal iron absorption to restore systemic iron homeostasis. Despite its central role, critical questions remain. What is the severity and duration of iron deficiency necessary to activate intestinal HIF2α? Can anemia itself, independent of iron deficiency, activate the intestinal HIF2α program?

These questions have clinical relevance, as multiple nutritional anemias present with similar hematologic features (microcytic, hypochromic) but differ fundamentally in etiology and iron responsiveness. Among these, vitamin B6–deficiency anemia (B6DA) is of particular interest. Vitamin B6 (pyridoxine) is an essential cofactor for aminolevulinic acid synthase (ALAS), the first enzyme in heme biosynthesis [14]. Deficiency in B6 impairs heme synthesis, resulting in microcytic, hypochromic anemia similar to IDA [15]. Additionally, vitamin B6 deficiency is frequently associated with ring sideroblasts (iron-leaden erythroid precursors) in the bone marrow, highlighting impaired iron utilization during erythropoiesis [16]. Mutations in erythroid-specific ALA synthase gene, ALAS2, are associated with hereditary sideroblastic anemia, particularly X-linked sideroblastic anemia [17]. Severe vitamin B6 deficiency as a result of alcohol abuse, malnutrition, or malabsorption is likely to result in acquired sideroblastic anemia [17], which improves upon B6 supplementation [18,19]. Unlike IDA, however, B6DA does not involve systemic iron depletion and is refractory to iron therapy. compared with IDA, anemia due to isolated B6 deficiency is rare [20]. In many cases of hypochromic anemias, deficiencies of B6 and iron coexist [21,22]. For example, in conditions of high nutrient demand, such as women of reproductive age [23], pregnancy [24], and recovery phase following major gastrointestinal surgeries [25,26], combined deficiency of B6 is frequently encountered. Therefore, a high index of suspicion is required for deficiency anemias that display suboptimal response to iron supplementation [27]. Although population data related to the frequency of labeling B6DA as IDA are limited, clinical studies [24,28,29] caution against this based on their shared clinical features. The general recommendation in the field is to consider an empirical combination of iron and B6 supplementation where detailed diagnostic testing is infeasible or cost-ineffective [23,30]. Nevertheless, misdiagnosis of B6DA as IDA is a major concern that can lead to ineffective treatment, underscoring the need for physiological markers to differentiate anemia types.

In this work, we utilized a mouse model of B6DA to investigate whether non-IDA microcytic hypochromic anemias are associated with intestinal Hif2α activation. Our findings suggest that the absence of HIF signaling in B6-deficient states reflects a mechanistic distinction in how the body senses and responds to anemias that are hematologically similar. Particularly, one that does not trigger the typical hypoxia-induced iron absorption response. These observations highlight novel aspects of anemia diagnosis and management where assessment of HIF activity or downstream targets can be utilized to differentiate between IDA and anemias that are nonresponsive to iron, such as those due to impaired heme synthesis.

METHODS

Animals and Treatment

C57BL/6 mice were used for this study. Mice were fed ad libitum with iron-supplemented AIN-93G purified rodent diets (Dyets Inc.) and were maintained in a 12:12-hour dark/light cycle. For the gradient iron experiment, mice were treated with 4 iron diet groups: enriched (350 ppm), adequate (35 ppm), low marginal (10 ppm) or deficient (<5 ppm); each group was assigned to 4 time points i.e., 3, 7, 15, or 30 days, totaling 16 groups (5 mice/group). For the dietary vitamin B6 deficiency experiment, mice were fed with a pyridoxine-deficient 35 ppm iron diet (Dyets Inc. Cat# 511476), with or without vitamin B6, until the B6-deficient mice became anemic, as confirmed by in-life complete blood count (CBC) analysis. After the establishment of anemia, half of the B6-deficient mice were supplemented with pyridoxine by intraperitoneal (IP) administration of pyridoxal hydrochloride (PHC) (Cat # 35379, Cayman Chemical) for 7 days. All animal studies were carried out in accordance with Association for Assessment and Accreditation of Laboratory Animal Care International guidelines and approved by the University Committee on the Use and Care of Animals at the University of Michigan.

Hematology

The CBC analysis was performed by the Unit for Laboratory Animal Medicine Pathology Core at the University of Michigan.

Real-Time Quantitative PCR

About 1 μg of total RNA extracted using Trizol reagent from mouse tissues (duodenal epithelial scrapes and liver) was reverse transcribed, and gene expression was analyzed by real-time reverse-transcription PCR PCR (RT-qPCR) with SYBR green master mix. All genes were normalized to β-actin, and the results are expressed as relative fold change. The primers are listed in Supplementary Table E1.

Western Blotting

Whole-cell and membrane lysate preparations were described previously [31] Lysates containing 30–40 μg of protein per well were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted overnight at 4°C with antibodies against ferritin H (Ftn) and tubulin (dilution of 1:1000). Horseradish peroxidase–conjugated secondary antibodies used were: anti-rabbit and anti-mouse at a dilution of 1: 2000, and immunoblots were developed using ChemiDoc touch imaging system (ChemiDoc, BioRad).

Quantification and Statistical Analysis

Statistical details of all experiments are mentioned in the figure legends. Results are expressed as the mean ± standard error of mean (SEM). Significance between the two groups was tested using a two-tailed, unpaired t test. Significance among multiple groups was tested using a one-way analysis of variance followed by Tukey post hoc test. GraphPad Prism 10.0 was used to conduct the statistical analyses. Statistical significance is described in the figure legends as: * p < 0.05, ** p < 0.01, *** p < 0.001.

RESULTS

A Time-Dependent Iron-Gradient Experiment Determines Optimal Responses to Dietary Iron Perturbations

Standard laboratory chow contains up to 350 ppm iron, whereas the physiological iron requirement for adult mice is 25–50 ppm [32,33]. In an IDA mouse model, adult mice (4–6 weeks old) typically develop anemia after approximately 2 weeks on a low-iron diet [34,35]. As an adaptive response, intestinal iron transport genes, including Dmt1 and Dcytb, are robustly upregulated in IDA mice [11]. To investigate the temporal and dose-dependent effects of dietary iron on murine iron homeostasis, we conducted a gradient iron diet experiment. Mice were assigned to 4 diet groups: enriched (350 ppm), adequate (35 ppm), low marginal (10 ppm), or deficient (<5 ppm), and each group was examined at 4 time points: 3, 7, 15, or 30 days (Figure 1A). Duodenal gene expression analysis showed that (1) Dmt1 and Dcytb levels were not significantly different between the enriched (350 ppm) and adequate (35 ppm) diet groups at any time point and (2) the deficient (<5 ppm) diet induced strong upregulation, with maximal expression observed at 15 and 30 days (Figure 1B). Pairwise comparison between the adequate and deficient diet groups confirmed robust induction of these genes after 15 days of iron restriction (Figure 1C). Consistent with the intestinal response, hepatic hepcidin was significantly downregulated in mice on the <5 ppm diet, reflecting the appropriate systemic adaptation to low-iron intake [36,37]. These results define the dietary iron threshold (<5 ppm) and duration (~2 weeks) required for robust intestinal HIF2α activation.

Figure 1.

Optimization of a dietary IDA mouse model. (A) Gradient iron diet experiment was set up by assigning mice (5 per group) to 4 diet groups (e.g., 350, 35, 10, <5 ppm iron) with each group subdivided into 4 time points (e.g., 3, 7, 15, 30 days); (B) Duodenal gene expression heat map of Dmt1 and Dcytb levels across diet groups and time points. Fifteen-day time point comparison between 35 ppm and <5 ppm groups by duodenal Dmt1 and Dcytb (C), and hepatic hepcidin gene expression (D) analysis. Bar graph represent the mean value ± SEM. **, P < 0.01; *, P < 0.05.

Vitamin B6 Deficiency Causes Hypochromic Microcytic Anemia

To investigate a nutritional anemia model hematologically similar to IDA, we selected vitamin B6 due to its essential role in heme synthesis. Based on our gradient iron diet findings, 35 ppm iron was used as the control diet for the B6 study. Mice were fed either a standard 35 ppm iron diet or a B6-deficient 35 ppm iron diet until anemia developed. To confirm their responsiveness to B6, a subset of B6-deficient mice received in IP supplementation with PHC for 7 days after anemia onset. PHC is the transport form of B6, which converts to the physiologically active form, pyridoxal 5′-phosphate, following membrane transport [38,39]. For comparison, a low-iron diet experiment was conducted in parallel, with mice fed either 35 ppm or <5 ppm iron for 15 days (Figure 2A). Consistent with previous reports [34,35,40], mice on the <5 ppm iron diet developed hypochromic, microcytic anemia as indicated by reduced Hb levels and MCV, hallmark features of IDA (Figure 2B). B6-deficient mice maintained normal CBC values at day 30 (Supplementary Figure E1A); therefore, the experiment was extended for an additional 4 weeks. By day 60, B6-deficient mice exhibited hypochromic, microcytic anemia with Hb and MCV values nearly identical to those of IDA mice, whereas total RBC counts remained unchanged (Figure 2C). Importantly, IP pyridoxal (PHC) supplementation for 7 days rapidly corrected the anemia, confirming the critical role of vitamin B6 in hematopoiesis (Figure 2C).

Figure 2.

Both iron and B6 deficiencies cause hypochromic microcytic anemia. (A) Mouse models of dietary iron- or vitamin B6 deficiency anemias; (B) CBC analysis of mice on 35 ppm vs. <5 ppm iron; (C) End point CBC analysis of control, B6-deficient diet, and B6-supplemented (by pyridoxal hydrochloride (PHC) IP) control or B6-deficient mice. Bar graph represent the mean value ± SEM. ****, p < 0.0001; **, p < 0.01; *, p < 0.05. ns = not significant. Hb=Hemoglobin; MCH=major histocompatibility; MCV=mean corpuscular volume; RBC=red blood cell.

Intestinal Hif2α Signaling is only Activated by Iron Deficiency, Not by B6 Deficiency

HIF2α activation is the primary mechanism driving the adaptive increase in intestinal iron absorption under low-iron conditions [41,42]. Key genes involved in intestinal iron homeostasis, such as Dmt1, Dcytb, Fpn1, and Ncoa4, are direct transcriptional targets of Hif2α [11–13]. Gene expression analysis recapitulated these previous findings: intestinal expression of Dmt1, Dcytb, Fpn1, and Ncoa4 were robustly and significantly induced in mice fed a <5 ppm iron diet, confirming activation of intestinal HIF2α in this dietary IDA model (Figure 3A).

Figure 3.

Dietary B6 deficiency does not induce intestinal HIF2α targets. Gene expression analysis of principal intestinal iron homeostasis genes (DMT1, Dcytb, Fpn1, and NCOA4) in the IDA group (A), and in the B6 deficiency group (B). Hepatic hepcidin gene expression analysis of control, B6-deficient diet, and B6-supplemented control or B6-deficient mice. (C) Bar graph represent the mean value ±SEM. **, p < 0.01; *, p < 0.05. ns=not significant.

In contrast, during vitamin B6 deficiency, intestinal expression of these Hif2α target genes remained unchanged. Although parenteral B6 supplementation rapidly corrected B6DA, intestinal iron gene expression showed only a modest, nonsignificant upward trend (Figure 3B). Furthermore, hepatic hepcidin levels were unaltered under B6-deficient conditions (Figure 3C). Together, these findings indicate that despite exhibiting hematologic features consistent with IDA, B6DA does not engage intestinal HIF2α signaling.

Intestinal Iron Levels Remain Unchanged in B6 Deficiency

Intestinal iron levels are the primary driver of HIF2α-mediated systemic iron regulation. In addition to upregulating iron transport proteins, low dietary iron triggers NCOA4-dependent mobilization of iron from the storage protein ferritin (Ftn) [13,43]. As shown in Figure 4A, duodenal Ftn expression is strongly downregulated in mice fed a low-iron diet. In contrast, (1) duodenal Ftn levels in B6-deficient mice remained unchanged and (2) pyridoxine-supplemented B6-deficient mice also showed no alterations in duodenal Ftn levels (Figure 4B). Together, the data confirm that the vitamin B6 pathway does not involve intestinal iron transport or storage.

Figure 4.

Dietary B6 deficiency does not alter iron levels in the intestinal epithelium. Duodenal ferritin (Ftn) Western analysis of control vs. Fe-deficient mice (A), and B6-deficient vs. B6-supplemented mice with controls (B).

DISCUSSION

Nutritional anemia can result from either malabsorption disorders or a nutrient-deficient diet. Identifying malabsorption as the cause is relatively straightforward, as it is often accompanied by other intestinal complications, such as celiac disease [44]. In contrast, a nutrient-poor diet rarely corresponds to a single vitamin deficiency and is often confounded by socioeconomic factors [45–47]. Consequently, treating nutritional anemia without considering coexisting deficiencies may lead to ineffective therapies.

The most common form of nutritional anemia is IDA, which is typically identified using a CBC analysis. IDA presents as microcytic, hypochromic anemia, characterized by low Hb levels and MCV. B6DA represents an important differential diagnosis for IDA, as it also presents with microcytic, hypochromic anemia, with a degree of severity comparable with IDA. A key distinguishing feature of B6DA is the elevated iron content in RBCs, due to impaired incorporation into heme [17,48]. Another observable difference is the longer latency period required for B6 deficiency to result in anemia (Figure 2A).

However, B6 deficiency also points to a lack of intestinal HIF2α signaling. Under dietary iron deficiency, intestinal Hif2α activation upregulates iron transporters, including Dmt1, Dcytb, and Fpn1 (Figures 1C and 3A) [11]. Simultaneously, decreased hepatic hepcidin production derepresses intestinal Fpn1 activity, underscoring the critical role of the hepcidin-Fpn1 axis in systemic iron homeostasis (Figure 1D) [49]. In B6DA mice, supplementation with vitamin B6 restored Hb levels, yet did not alter the expression of HIF2α-dependent intestinal iron metabolism genes or hepatic hepcidin (Figure 3B, C).

These findings suggest that low Hb alone is insufficient to drive intestinal HIF2α activation; rather, low-iron levels in the duodenum or serum act as the upstream signal. Notably, HIF2 signaling in RBC and systemic HIF activation were not assessed in this study, representing a limitation. Given the comparable reductions in Hb and MCV observed in IDA and B6DA, a systemic hypoxic response might be expected. However, the lack of intestinal HIF2α activation in B6DA, as well as following B6 supplementation, indicates preservation of tissue-specific HIF responses. For example, during hypoxemia, known HIF targets such as erythropoietin, transferrin, and transferrin receptor are induced to promote erythropoiesis [50].

For iron-refractory forms of IDA, therapeutic targeting requires identification of the relevant tissue site for HIF modulation. For instance, intestinal HIF stabilization can effectively correct genetically iron-refractory IDAs, such as those caused by TMPRSS6 mutations or knockout [51]. Interestingly, regulation of ALAS2, the B6-dependent enzyme critical for heme biosynthesis, is complex: hypoxia upregulates ALAS2 expression through both HIF-dependent and independent pathways, whereas iron depletion downregulates it [50].

In conclusion, this study identifies B6DA as an important differential diagnosis for IDA, and highlights how assessment of intestinal HIF2α signaling can serve as both a diagnostic tool and a guide for therapeutic strategies in managing IDA-like anemias.

HIGHLIGHTS.

• Intestinal hypoxia-inducible factor 2α (HIF2α) is activated by specific thresholds of dietary iron depletion.

• Iron or B6 deficiencies result in similar and comparable degree of anemia.

• Intestinal HIF2α signaling differentiates iron-deficiency anemias (IDAs) from IDA-like anemias.