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N.U. Stoffel et al.
high SHep even in anemic subjects.8,9 Nearly all experi- mental data are derived from cells or mice, where often very strong stimuli are applied (e.g. injection of LPS,2,3,5 phlebotomy,4 severe iron deficiency2,5). In humans, there is a lack of experimental data describing the effect of milder, physiological changes in these opposing stimuli on SHep and the net effects on iron recycling and iron absorption, but these interactions are common and relevant in many disorders.
Therefore, the objective of this prospective study was to assess changes in iron markers, SHep, iron absorption and erythrocyte iron incorporation during acute inflammation, in both non-anemic women and women with IDA. Previous human studies reported the effects of an acute inflammatory stimulus [e.g. infusions of LPS10 or inter- leukin-6 (IL-6)11] on SHep, but they did not assess effects on iron absorption, or how anemia or iron status may modulate this response. As the inflammatory stimulus in this study, we used vaccination, a practical, safe and stan- dardized model for the study of mild-to-moderate inflam- mation in humans.12,13 Our hypotheses were: 1) in non- anemic women, vaccination would induce acute inflam- mation and increase Shep. This would decrease iron absorption and produce hypoferremia; and 2) in contrast, in women with IDA, vaccination would induce acute inflammation, but would not increase SHep or affect serum iron or iron absorption. We used stable iron isotope techniques to quantify erythrocyte iron incorporation of dietary iron before and after vaccination.
Methods
Study subjects
We recruited women from the staff of the University Hospital Ibn Sina in Rabat, Morocco. Detailed inclusion criteria are described in the Online Supplementary Appendix. In this prospec- tive, 45-day study, in women (n=46, age 18-49 years) with IDA or without anemia, we compared iron and inflammation markers and SHep before and 8, 24 and 36 hours (h) after influenza/diph- theria-tetanus-pertussis (DTP) vaccination and erythrocyte iron incorporation from 57Fe-labeled test meals, before and 24 h after the vaccination as an acute inflammatory stimulus (Figure 1). The study was approved by the ethics committees of the ETH Zurich, Zurich, Switzerland and the University Mohammed V, Rabat, Morocco. All participants gave informed written consent.
On study day 1, an afternoon baseline blood sample was taken. On study day 2, after an overnight fast, a baseline morning blood sample was taken and we administered a test meal containing 6 mg labeled 57Fe as ethylenediaminetetraacetic acid ferric sodium salt (NaFeEDTA), added to a standardized test meal, given with bottled water, as described in the Online Supplementary Appendix. Blood samples were taken in the afternoon on day 2 as well as the next morning (day 3). After a 19-day isotope incorporation period, on study day 22, a blood sample was taken to measure erythro- cyte iron incorporation; this blood sample also served as the new baseline afternoon sample for the second absorption study. In the morning of day 23, a morning blood sample was taken. Then, all subjects received the trivalent Influenza Virus Vaccine Vaxigrip (Sanofi Pasteur, Lyon, France) and the DTP Virus Vaccine Dultavax (Sanofi Pasteur) given intramuscularly. Blood samples were taken at 8 h, 24 h and 36 h after vaccination. At 24 h after vaccination, on study day 24, an identical labeled test meal was administered, as described above. The final blood sample was taken on day 45. We assessed total and fractional iron absorption (FIA) by measur-
ing the amount of stable isotopic tracers incorporated in red blood cells 19 days after administration of the labeled test meals.14-16 Hemoglobin (Hb), iron- and inflammatory biomarkers were meas- ured as described in the Online Supplementary Appendix.
Assuming a standard deviation (SD) of 0.20 on differences in log transformed erythrocyte iron incorporation from previous ETH studies, a type I error rate of 5% and 80% power, we expected to detect a difference in FIA of 35% within groups with a sample size of 20 subjects per group. Assuming a drop-out rate of 20%, we enrolled 50 women (25 anemic and 25 non-anemic women).
Statistical analysis
We performed the statistical analyses using SPSS (IBM SPSS sta- tistics, v.22), as described in detail in the Online Supplementary Appendix. We used linear mixed effect model analysis to assess the effect of the group (anemic vs. non-anemic) and treatment (vacci- nation) on different variables. Group and treatment were defined as fixed effects, participants as random intercept effects using a variance component structure matrix. Regression analyses were performed with SHep, FIA and serum iron as dependent variables. Pearson and Spearman correlations were applied. For within- group effects, dependent sample t-tests or related samples non- parametric tests were used. P<0.05 was considered significant.
Results
We began recruiting on 1st September 2017, and from September 2017 to February 2018 we enrolled 50 women (28 non-anemic and 22 with IDA) into the study. We com- pleted the study on 29th March 2018. Six women in the non-anemic group left the study because they no longer wanted to participate in the study: three before the 19 day blood sample (when we measured erythrocyte iron incor- poration from the first test meal) and three after the 19 day blood sample. In the IDA group, one subject left the study before the 19 day blood sample because she no longer wanted to participate in the study (Figure 1). Data from the three non-anemic women who left the study after the 19 day blood sample were included in the analyt- ical models, resulting in a total of n=46 women (21 with IDA and 25 non-anemic).
Baseline characteristics of the subjects by group are shown in Table 1. There were no significant between- group differences in age, Body Mass Index or markers of inflammation, and no subject had increased markers of inflammation. There were significant between-group dif- ferences in hemoglobin (Hb), serum ferritin (SF), soluble transferrin receptor (sTfR), body iron stores (BIS), Shep, and erythropoietin (EPO) (all P<0.001). Eight of the women in the non-anemic group were iron-deficient, as defined by SF<15 μg/L.17
There was a significant vaccination effect (P<0.001) on IL-6, but no significant group effect or group-vaccination interaction (Table 2). There were no significant between- group differences in IL-6 measured at 4:00 pm the day before and at 8:00 am just before vaccination or at 8 h, 24 h, and 36 h after vaccination (Figure 2A). Median interquartile range (IQR) IL-6 (pg/mL) significantly increased in both groups comparing baseline to 24 h after vaccination (P<0.001): in the non-anemic group from 1.12 (0.92-1.66) to 3.37 (2.88-4.32), and in the IDA group from 1.53 (1.41-1.81) to 3.14 (2.48-4.33) (Table 2).
There was a significant group (P<0.001) and vaccination (P<0.001) effect on SHep, but no significant group-vacci-
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haematologica | 2019; 104(6)