Iron requirements based upon iron absorption tests are poorly predicted by haematological indices in patients with inactive inflammatory bowel disease

Abstract

Iron deficiency (ID) and iron deficiency anaemia (IDA) are common in patients with inflammatory bowel disease (IBD). Traditional clinical markers of iron status can be skewed in the presence of inflammation meaning that a patient’s iron status can be misinterpreted. Additionally, iron absorption is known to be down-regulated in patients with active IBD. However, whether this is the case for quiescent or mildly active disease has not been formally assessed. This study aimed to investigate the relationship between iron absorption, iron requirements and standard haematological indices in IBD patients without active disease. Twenty nine patients with quiescent or mildly active IBD and 28 control subjects undertook an iron absorption test which measured sequential rises in serum iron over four hours following ingestion of 200 mg ferrous sulphate. At baseline, serum iron, transferrin saturation, non-transferrin bound iron (NTBI), ferritin and soluble transferrin receptor were all measured. Thereafter (30-240 minutes) only serum iron and NTBI were measured. Iron absorption did not differ between the two groups (P=0.9; RM-ANOVA). In control subjects baseline haematological parameters predicted iron absorption (i.e. iron requirements) but this was not the case for patients with IBD.

Iron absorption is normal in quiescent or mildly active IBD patients but standard haematological parameters do not accurately predict iron requirements.

Introduction

Iron deficiency (ID) and iron deficiency anaemia (IDA) are common in patients with inflammatory bowel disease (IBD) (circa 20-30%) ^{(1, 2)}. Reasons are multifactorial but protein/blood losses in the gut and low dietary iron intakes are major drivers ⁽³⁾. Iron absorption is clearly down-regulated in patients with active inflammation due to anaemia of chronic disease ⁽⁴⁾, but it is not clear whether iron absorption is altered in patients who are in remission. The absorption of iron in patients with quiescent or mildly active IBD compared to healthy controls was first assessed in a pilot study, the results of which were inconclusive ⁽⁵⁾. The outcome of a more recent study implies that the absorption of iron from ferrous calcium citrate, but not iron bisglycinate, is similar in patients with quiescent Crohn’s disease compared to healthy subjects ⁽⁶⁾. However, this study was not designed to compare iron absorption in patients with IBD and healthy controls.

If iron absorption is normal in a cohort of patients with quiescent or mildly active IBD versus control subjects, then further analysis can consider the relationship between iron absorption and standard haematological parameters that are used to predict ID or iron repletion (IR). Apart from assessing bone marrow stores, which is ethically difficult, iron absorption probably provides the most sensitive test of iron requirements (i.e. iron status) ⁽⁷⁾. Thus standard haematological parameters that are used to predict iron status, and may be perturbed in low grade chronic inflammation and/or relapsing-remitting inflammation ⁽⁸⁾, can be assessed for their predictive value or effectiveness. Hence, in this work, both iron absorption and its relationship to haematological parameters have been assessed in patients with IBD and control subjects. The method of sequential blood sampling following ingestion of ferrous sulphate was used as this provides a direct and relevant measure of iron absorption ⁽⁹⁾ as opposed to utilization (e.g. erythrocyte incorporation), which may be independently perturbed in inflammatory conditions. In addition, the method of sequential blood sampling allows non-transferrin bound iron (NTBI) to be measured. NTBI has been proposed to occur transiently in serum, following the ingestion of therapeutic supplements by iron deficient subjects ^{(10, 11)} and even in some subjects with normal iron stores ⁽¹¹⁾. The rationale is that the rate of absorption is too great for transferrin to completely bind the incoming iron, and thus a small proportion binds to albumin or citrate or even undergoes partial hydrolysis forming polyhydroxy iron species ⁽¹²⁾. In such forms (i.e. not bound to transferrin) iron may be prone to redox cycling and therefore promote oxidative stress within the circulation ^{(13, 14)}. It has been proposed that the antioxidant capacity of the mucosa and the circulation is depleted in IBD ^(15-17) such that the formation of NTBI could induce oxidative damage more readily than in control subjects.

This study aimed to investigate the relationship between iron absorption, iron requirements and standard haematological indices in IBD patients without active disease. Additionally, it assessed the formation of circulating NTBI in patients with IBD and controls following ingestion of ferrous sulphate.

Experimental Methods

Participants

Patients with IBD (n=29: 5 with ulcerative colitis and 24 with Crohn’s disease) were recruited from gastrointestinal outpatient clinics at Guy’s and St Thomas’ NHS Hospital Trust (GSTT), London, UK. Control subjects (n=28) were recruited from a local newspaper advert.

Patients

Patients were aged 18 to 65 and in all cases IBD was diagnosed by histological and/or radiological criteria. Patients with other chronic diseases, hereditary disorders of iron metabolism (detected by assessment of common mutations in the HFE gene using ‘WAVE’ technology based on denaturating high performance liquid chromatography), pregnant and lactating females and those taking proton pump inhibitors or iron therapy/supplements within the previous 28 days were excluded. Additionally, only patients with inactive or mildly active disease using a Harvey Bradshaw Index (HBI) of less than eight ⁽¹⁸⁾ were recruited for the study. Patients fulfilling these criteria were invited to participate and then had a blood sample taken to assess iron status (full blood count, ferritin, serum iron, soluble transferrin receptor and total serum iron binding capacity [TIBC]) and inflammatory status (erythrocyte sedimentation rate and C-reactive protein).

Controls

An advert was placed in a freely available newspaper distributed predominantly within Greater London. Potential subjects responding to the advert were screened by telephone to exclude anyone with known chronic disease, gastrointestinal disorders, hereditary disorders of iron metabolism and those taking proton-pump inhibitor medication or iron therapy/supplements within the previous 28 days. Pregnant and lactating women were also excluded. Volunteers fulfilling these criteria were invited to participate and then had a blood sample taken to assess iron status and inflammatory status, as detailed above. Mutations of the HFE gene were also assessed.

This study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human subjects were approved by the St Thomas’ Hospital Local Research Ethics Committee (EC03/089). Written informed consent was obtained from all subjects.

Study design

Recruited subjects were invited to attend St Thomas’ Hospital, London, (UK) for a single four hour study appointment at a mutually agreeable time. Subjects were requested to not take any multivitamin or mineral supplements for the week preceding the study and were advised to avoid any iron-rich foods and tea on the day of their appointment. Subjects were not fasted as it has previously been shown that iron absorption from oral ferrous sulphate is similar with or without fasting ⁽¹⁰⁾.

Upon arrival, signed consent was obtained and each subject was cannulated in the forearm with a 21G Venflon Pro cannula (Becton Dickenson Infusion Therapy, AB SE-251 06, Helsingborg, Sweden) and a 20 ml baseline blood sample was taken using a plastic 20 ml syringe. The collected blood was aliquoted into labeled plain, EDTA, and SST Vacutainers (Becton Dickenson Vacutainer Systems. Plymouth, UK) for the baseline measures as outlined above. Following collection of the baseline blood sample, subjects were given a single ferrous sulphate capsule (200 mg; 65 mg elemental iron) with a glass of water. Further blood samples (10 ml) were collected at 30, 60, 120, 180, 210 and 240 minutes post iron ingestion to measure serum iron, TIBC and NTBI.

Laboratory analysis

Baseline samples for ferritin and C-reactive protein (SST Vacutainer) and full blood count (EDTA Vacutainer) were analysed by the Clinical Chemistry Laboratory using routine laboratory methods. Samples collected in plain Vacutainers for serum iron, NTBI and TIBC were immediately taken following collection (on ice) to a laboratory where the samples were allowed to clot prior to being centrifuged at 2500 rpm for 10 minutes at 4° C (Eppendorf 5804R, Germany). Following centrifugation, serum was transferred to labelled cryovials, frozen and stored at −80° C in labelled boxes. Samples for serum iron, TIBC and serum transferrin receptor analysis were analysed by routine methods at the Nutritional Biochemistry Laboratory, MRC Human Nutrition Research, Cambridge. Serum NTBI was analysed using a modified version of the method developed by Singh et al ⁽¹⁹⁾ in the Department of Pharmacy, King’s College London as previously described ⁽²⁰⁾. Transferrin saturation was calculated from TIBC using the following equation:

$$\text{Transferrin saturation}={100}^{\ast }(\text{Serum Iron}∕\text{TIBC}).$$

Data and statistical analysis

Subjects with non-iron deficiency anaemia, significant inflammation or homozygous mutations of the HFE gene were excluded from the study. Subjects where venous access could not be maintained for the duration of the study were excluded from analysis. Data were analysed using SPSS version 14 and are presented as mean ± SD or, where indicated, mean ± SEM. Unpaired t tests were used to make between-group comparisons of the peak serum iron and comparison of baseline markers, of iron status and inflammation, between control subjects and patients with IBD. Pearson’s product moment correlation coefficient was used to measure correlation between NTBI, serum iron and transferrin saturation. Significance was assumed where p<0.05.

Results

Seventy-two subjects (IBD n=36, controls n=36) were screened for study recruitment. Five subjects in the control group were not suitable due to non-iron related anaemia and two patients with IBD were excluded, one due to moderately active disease (HBI=12) and the other because he was homozygous for the primary haemochromatosis-susceptibility mutation, C282Y. Sixty-five subjects consented to take part but data were incomplete for eight (IBD n=5, controls n=3) due to difficulty in maintaining venous access for the duration of the study and were excluded from analysis. Of the remaining 57 subjects, 28 subjects were controls (12 male) with a mean age of 35 ± 11 years and BMI of 23.4 ± 3 kg/m². Twenty-nine had IBD (13 male) with a mean age of 42 ± 13 years, BMI 25.7 ± 6 kg/m² and HBI 4.2 ± 1.8. Five patients with IBD had ulcerative colitis and the remaining patients had Crohn’s disease (site involved: ileal n=4; ileocaecal n=3; ileocolonic n=8; colonic n=5; site not specified n=4).

Iron absorption was measured by the rise in serum iron over four hours, following ingestion of ferrous sulphate and was similar for patients with IBD (n=29) and control subjects (n=28; Fig. 1). According to a priori classification of iron status (see Methods) control subjects with ID (n=10) or IDA (n=5) had peak iron absorption around 180 minutes post dose and approximately 30 and 45 μmol/l above baseline levels, respectively. In contrast, control subjects without ID (i.e. iron replete; n=13) had iron absorption peaks that increased by less than 5 μmol/l (control subjects: IDA or ID versus IR P<0.001; Fig. 2). In patients with IBD and IDA (n=4), a significant increase in serum iron levels, peaking at 55 μmol/l above baseline, was observed while in non-anaemic patients with IBD with ID (n=11) or without ID (i.e. IR; n=14), similar iron absorption curves were observed with mean peak increases of approximately 15μmol/l (patients with IBD: ID versus IR P=0.8; Fig. 2).

Fig. 1.

Increase in serum iron (mean ± SEM) from baseline following ferrous sulphate administration in patients with IBD (n=29; ○) versus control subjects (n=28; ●) P=0.9.

Fig. 2.

Increase in serum iron (mean ± SEM) from baseline following ferrous sulphate administration in (a) control subjects (n=13 IR ○, n=10 ID●, and n=5 IDA Δ) and (b) patients with IBD (n=14 IR ○, n=11 ID ●, and n=4 IDA Δ) according to a priori classification of iron status. For patients with IBD, P=0.8 for IR versus ID and P<0.05 for IR versus IDA. For control subjects, P<0 .001 for IR versus ID or IDA. IR = iron replete, ID = iron deficient and IDA = iron deficient anaemic, all according to standard haematological criteria (see Experimental Methods).

Due to the inability of iron status markers to predict iron absorption, in the absence of anaemia in subjects with IBD (Figure 2) further assessment was undertaken. The peak rise in serum iron is a strong correlate for total iron absorption ⁽⁹⁾. Subjects were thus categorized according to whether they did or did not absorb iron and this was related to baseline ferritin, serum iron and transferrin saturation in all subjects. Briefly, the iron status of all subjects was determined using reference ranges for haematological values, so subjects with ID were classified as having (i) serum ferritin <20 μg/l or (ii) serum ferritin 20-55 μg/l and either serum iron < 14 μmol/l or transferrin saturation <10 % ⁽²¹⁾. Mild anaemia was defined as haemoglobin concentration 11-12.9 g/dl (male) and 10.5-12.4 g/dl (female). Additionally, subjects were also categorized as iron absorbers or non-absorbers defined as having a peak serum iron increase of more than 5 μmol/l or less than 5 μmol/l respectively, following ingestion of 65 mg of iron as ferrous sulphate. In the control subjects, all four markers (baseline ferritin, serum iron, transferrin saturation and haemoglobin) provided some significant prediction of iron requirements (i.e. absorption) as expected, whereas, there was no significant predictive power of these markers in patients with IBD (Table 1).

Table 1.

Comparison of baseline markers of iron status (and inflammation) in control subjects and patients with IBD classified as iron absorbers and non-absorbers^*

	Control absorbers n=18				Control non-absorbers n=10				P-Values
	Mean	SD	Median	Range	Mean	SD	Median	Range
Haemoglobin (g/dl)	12.9	1.2	13.1	10.4-15.2	14.8	2.0	14.6	13.0-19.5	0.005
Ferritin (μg/l)	25	22	21	3.0-90.0	99	67	105	15-203	<0.001
Serum transferrin receptor (mg/l)	4.7	1.9	4.2	3.1-11.0	3.9	0.4	3.9	3.2-4.7	0.2
Serum Iron (μmol/l)	12.6	5.1	11.8	4.7-22.2	20.9	7.1	18.7	11.5-33.0	0.004
TIBC (μmol/l)	71	10	72	54.8-91.5	57	8	53	48.6-70.2	<0.001
NTBI (μmol/l)	0.3	0.4	0.2	−0.2-1.5	0.7	0.3	0.6	0.4-1.5	0.005
Transferrin Saturation (%)	23.1	8.2	22.0	10.0-38.0	34.0	11.8	36.0	21-50	<0.001
ESR (mm/h)	5	4	3	1.0-13.0	2	1	2	2-3.0	0.1
CRP (mg/l)	5.1	0.3	5.0	5.0-6.0	5.8	1.6	5.0	5.0-9.0	0.07
Platelets (10^9)	234	45	232	141-321	216	34	220	168-278	0.3

	IBD absorbers n=21				IBD non-absorbers n=8				P-Values
	Mean	SD	Median	Range	Mean	SD	Median	Range
Haemoglobin (g/dl)	13.3	1.5	13.3	8.8-16.0	14.0	0.8	14.0	13.0-14.9	0.3
Ferritin (μg/1)	39	23	39	5.0-94.0	39	14	38	22-62	0.7
Serum transferrin receptor (mg/1)	4.4	1.2	4.3	2.6-7.5	4.1	1.0	4.4	2.0-4.9	0.7
Serum Iron (μmol/l)	16.3	7.7	15.7	3.0-30.2	13.9	7.2	13.2	5.6-27.9	0.5
TIBC (μmol/1)	61	10	62	41.0-80.0	57	10	59	42.1-72.5	0.3
NTBI (μmol/1)	0.4	0.3	0.3	0.0-1.0	0.2	0.3	0.2	−0.1-2.7	0.3
Transferrin Saturation (%)	21.3	8.5	22.8	6.6-40.1	30.4	16.9	30.0	7.0-63.0	0.2
ESR (mm/h)	14	11	9	5.0-42.0	10	9	7	2.0-26.0	0.4
CRP (mg/1)	8.4	7.7	6.0	5.0-39.0	10.7	8.6	7.0	5.0-28.0	0.5
Platelets (10^9)	293	62	290	199-457	276	64	274	170-381	0.5

Abbreviations: TIBC, total iron binding capacity; NTBI, non-transferrin bound iron; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; IBD, inflammatory bowel disease.

^*See Experimental Methods section for categorization of iron absorbers and non-absorbers

Total iron absorption, transferrin saturation and NTBI were all strongly correlated for both subject groups (Fig. 3). Surprisingly, even baseline NTBI (i.e. pre-iron dose) was present in some individuals and, again, correlated with transferrin saturation (Fig. 3). NTBI in the absence of transferrin saturation with iron is controversial and, therefore, these data suggest its presence as either an artefact of NTBI assays or an equilibrium product with iron-transferrin, regardless of whether samples come from subjects who are healthy or with inflammatory bowel disease. This is further discussed below.

Fig. 3.

Correlation between NTBI and (a) non-baseline serum iron (r²=0.74; P<0.001) (b) non-baseline transferrin saturation (r²=0.77; P<0.001) (c) baseline serum iron (r²=0.24; P<0.001) (d) baseline transferrin saturation (r²=0.40; P<0.001) in patients with IBD (n=29; ○) and control subjects (n=28; ●). r² and P values are for patients with IBD and control subjects combined. Non-baseline refers to all values that were not at baseline (i.e. all time points post iron dose).

Discussion

Our findings provide important information on a number of aspects of iron status and iron absorption in subjects with IBD.

First, in spite of speculation, iron absorption is normal in patients with IBD without significant inflammation when compared as a group to healthy controls, and as opposed to patients with active IBD ⁽⁵⁾. We recognize that the Harvey Bradshaw Index is used for assessing clinical activity (i.e. a proxy for inflammation) in Crohn’s disease and not patients with ulcerative colitis, but only 5/29 had ulcerative colitis in this study and the findings in the current study were the same with all IBD patients (n=29) or just the Crohn’s disease patients (n=24) (data not shown).

Secondly, IDA in patients with IBD, when assessed by traditional haematological criteria, is predictive of high iron needs (Figure 2) whereas ID alone is not (i.e. in the absence of concomitant anaemia) and iron repletion is not predictive of low iron needs (Figure 2). Thus, while patients in this cohort had quiescent or mild IBD and only slight increases in systemic inflammatory markers such as C reactive protein, erythrocyte sedimentation rate and platelets, this appears sufficient to affect measures based around iron, transferrin and ferritin as markers of iron requirements as is well known with more severe inflammation. Levels of serum soluble transferrin receptor have been proposed to be less influenced by inflammation than levels of circulating ferritin or iron/transferrin ⁽²²⁾, but this measure was also insensitive in assessing iron needs in IBD. Thus, in the absence of anaemia, current haematological parameters are unable to predict iron stores, and hence iron requirements, even in patients with quiescent or mildly active IBD. Given that oral ferrous iron supplementation has, on occasions, been associated with severe gastrointestinal adverse effects in IBD ⁽²³⁾, as also demonstrated in animal models of colitis ^(24-26), these data suggest that iron supplementation should only be undertaken in subjects with ID and anaemia where iron needs are clear. Even then, anaemia of chronic disease, which normally is associated with active IBD, should be ruled out because iron absorption is then markedly down-regulated ⁽⁵⁾. Diagnostic criteria for the assessment of ID and anaemia in IBD in the presence and absence of inflammation have been developed ⁽²⁷⁾.

Thirdly, in agreement with previous work ^{(10, 11)}, the present study has shown that following a single dose of oral ferrous sulphate, there is a rise in apparently detectable NTBI in the circulation of control subjects and in patients with IBD (Figs. 1&3). Transferrin is not just an iron transport protein but is also an anti-redox substrate, so any circulating iron that is not bound to transferrin (i.e. NTBI) has the capacity for systemic oxidative activity. Individuals with low mucosal and systemic anti-oxidant status, as has been proposed for patients with IBD ^(15-17) could, in theory, be especially prone to NTBI effects, including vascular damage and lipid peroxidation ^{(13, 14, 28)}. Unsurprisingly, the detection of NTBI in serum was strongly correlated with total iron uptake into serum and, therefore, the degree of transferrin saturation (Fig. 3). However, unexpectedly, baseline serum iron levels and transferrin saturation (i.e. fasting and pre-ferrous sulphate) also correlated with NTBI (Fig. 3). This raises the question of whether, outside of iron overload, NTBI genuinely exists as a function of partially saturated transferrin or is an artefact of NTBI assays (i.e. do NTBI assays compete for a small fraction of transferrin-bound iron in proportion to total serum iron concentration?). Although beyond the scope of this study, these issues should be resolved in further work as it is important to establish whether circulating NTBI really occurs following supplemental oral iron.

In summary, (i) there was no evidence for iron absorption to be dysfunctional in patients with IBD. (ii) In quiescent or mildly active IBD, IDA clearly indicates a need for iron repletion. (iii) In the absence of anaemia, standard haematological criteria cannot predict iron requirements in IBD. (iv) a signature for NTBI is detected in patients with IBD to the same extent as it is detected in control subjects, following ingestion of ferrous sulphate, but further work is required to determine the authenticity of this apparent NTBI.

Overall, the implications of this work are that if IBD patients with ID are to be supplemented with iron then they should have a concomitant anaemia consistent with IDA (typically a microcytic anaemia). Based on previous work, optimal iron absorption (i.e. supplemental effectiveness) will be inhibited in patients with active disease ⁽⁵⁾, while oral iron may still exacerbate symptoms ⁽²³⁾ so the choice between enteral and parenteral iron supplementation, and which patients to target, should be carefully considered.

Abbreviations

ID

iron deficiency

IDA

iron deficiency anaemia

IBD

inflammatory bowel disease

TIBC

total iron binding capacity

NTBI

non-transferrin bound iron

ESR

erythrocyte sedimentation rate

CRP

C-reactive protein