The magnitude of the plasma hepcidin response to oral iron supplements depends on the iron dosage.

BACKGROUND: Iron deficiency without anaemia is a common health problem, especially in young menstruating women. The efficacy of the usually recommended oral iron supplementation is limited due to increased plasma hepcidin concentration, which reduces iron absorption and leads to side effects such as intestinal irritation. This observation raises the question of how low-dose iron therapy may affect plasma hepcidin levels and whether oral iron intake dose-dependently affects plasma hepcidin production. METHODS: Fifteen non-anaemic women with iron deficiency (serum ferritin ≤30 ng/ml) received a single dose of 0, 6, 30, or 60 mg of elemental oral iron as ferrous sulfate on different days. Plasma hepcidin was measured before and seven hours after each dose. RESULTS: Subjects had an average age of 23 (standard deviation = 3.0) years and serum ferritin of 24 ng/ml (interquartile range = 16-27). The highest mean change in plasma hepcidin levels was measured after ingesting 60 mg of iron, increasing from 2.1 ng/ml (interquartile range = 1.6-2.9) to 4.1 ng/ml (interquartile range = 2.5-6.9; p < 0.001). Iron had a significant dose-dependent effect on the absolute change in plasma hepcidin (p = 0.008), where lower iron dose supplementation resulted in lower plasma hepcidin levels. Serum ferritin levels were significantly correlated with fasting plasma hepcidin levels (R2 = 0.504, p = 0.003) and the change in plasma hepcidin concentration after iron intake (R2 = 0.529, p = 0.002). CONCLUSION: We found a dose-dependent effect of iron supplementation on plasma hepcidin levels. Lower iron dosage results in a smaller increase in hepcidin and might thus lead to more efficient intestinal iron absorption and fewer side effects. The effectiveness and side effects of low-dose iron treatment in women with iron deficiency should be further investigated. This study was registered at the Swiss National Clinical Trials Portal (2021-00312) and ClinicalTrials.gov (NCT04735848).

Reference intervals for serum 11-oxygenated androgens in children.

OBJECTIVE: Classic androgens such as dehydroepiandrosterone, androstenedione, and testosterone are generally measured for diagnosis and treatment monitoring in children and adolescents with hyperandrogenism, as can occur in congenital adrenal hyperplasia, premature pubarche, or polycystic ovarian syndrome. However, adrenally-derived 11-oxygenated androgens also contribute to the androgen pool and should therefore be considered in clinical management. Nevertheless, paediatric reference intervals are lacking. Therefore, we developed a serum assay to establish reference intervals for four 11-oxygenated androgens in addition to four classic androgens. DESIGN: Reference interval study for serum 11-oxygenated androgens in children. METHODS: We developed and validated a sensitive LC-MS/MS assay and quantified eight serum androgens, including four 11-oxygenated androgens, in serum of 256 healthy children (aged 0-17 years old). An age-dependency for all androgens was observed, and therefore we divided the cohort based on age (prepubertal (n=133; 94 boys, 39 girls) and pubertal (n=123; 52 boys, 71 girls)) to compute reference intervals (2.5th - 97.5th percentiles). RESULTS: In the prepubertal group, there was no significant sex-difference for any of the measured androgens. In the pubertal group, androstenedione, testosterone, and dihydrotestosterone showed a significant difference between boys and girls. In contrast, adrenal androgens dehydroepiandrosterone, 11-hydroxyandrostenedione, 11-ketoandrostenedione, 11-hydroxytestosterone, and 11-ketotestosterone did not. CONCLUSIONS: We developed and validated an assay for 11-oxygenated androgens, in addition to four classic androgens and established reference intervals. This enables a comprehensive evaluation of serum androgen status in children with clinical symptoms of hyperandrogenism.

Effectiveness of low-dose iron treatment in non-anaemic iron-deficient women: a prospective open-label single-arm trial.

BACKGROUND: Iron deficiency without anaemia is highly prevalent and is particularly associated with fatigue, cognitive impairment, or poor physical endurance. Standard oral iron therapy often results in intestinal irritation with associated side effects and premature discontinuation of therapy, therefore, optimal oral iron therapy with sufficient iron absorption and minimal side effects is desirable. METHODS: Thirty-six iron-deficient non-anaemic premenopausal women (serum ferritin ≤30 ng/ml, haemoglobin ≥117 g/l) with normal body mass index (BMI) and no hypermenorrhea received 6 mg of elemental oral iron (corresponding to 18.6 mg ferrous sulphate) twice daily for 8 weeks. RESULTS: Participants treated with low-dose iron had an average age of 28 years and a BMI of 21 kg/m2. Their serum ferritin and haemoglobin increased significantly from 18 ng/ml to 33 ng/ml (p <0.001) and from 135 g/l to 138 g/l (p = 0.014), respectively. Systolic blood pressure increased from 114 mmHg to 120 mmHg (p = 0.003). Self-reported health status improved after 8 weeks (p <0.001) and only one woman reported gastrointestinal side effects (3%). CONCLUSION: This prospective open-label single-arm trial shows that oral iron treatment of 6 mg of elemental iron twice daily over 8 weeks is effective in iron-deficient non-anaemic women. Due to the negligible side effects, low-dose iron treatment is a valuable therapeutic option for iron-deficient non-anaemic women with normal BMI and menstruation. Further placebo-controlled studies with a larger number of participants are needed to confirm these results. CLINICALTRIALS: gov NCT04636060.

Hepcidin Status in Cord Blood: Observational Data from a Tertiary Institution in Belgium.

The hormone hepcidin plays an important role in intestinal iron absorption and cellular release. Cord blood hepcidin values reflect fetal hepcidin status, at least at the time of delivery, but are not available for the Belgian population. Therefore, we aimed (1) to provide the first data on cord blood hepcidin levels in a Belgian cohort and (2) to determine variables associated with cord blood hepcidin concentrations. A cross-sectional, observational study was performed at the University Hospital Leuven, Belgium. Cord blood samples were analyzed using a combination of weak cation exchange chromatography and time-of-flight mass spectrometry. Descriptive statistics, Spearman correlation tests, and Mann-Whitney U tests were performed. In total, 61 nonhemolyzed cord blood samples were analyzed. The median hepcidin level was 17.6 µg/L (IQR: 18.1; min-max: 3.9-54.7). A moderate correlation was observed between cord blood hepcidin and cord blood ferritin (r = 0.493) and hemoglobin (r = -0.342). Cord blood hepcidin was also associated with mode of delivery (p = 0.01), with higher hepcidin levels for vaginal deliveries. Nonetheless, larger studies are needed to provide more evidence on the actual clinical value and benefit of cord blood hepcidin measurements.

NTBI levels in C282Y homozygotes after therapeutic phlebotomy.

C282Y homozygotes exposed to sustained elevated transferrin saturation (TS) may develop worsening clinical symptoms. This might be related to the appearance of non-transferrin bound iron (NTBI) when TS≥50% and labile plasma iron (LPI) when TS levels reach 75-80%. In this study, NTBI levels were examined in 219 randomly selected untreated and treated C282Y homozygotes. Overall, 161 of 219 had TS ≥ 50%, 124 of whom had detectable NTBI (≥0.47 µM, 1.81 µM [0.92-2.46 µM]) with a median serum ferritin 320 µg/L (226-442 µg/L). Ninety of 219 homozygotes had TS ≥ 75%, and all had detectable NTBI (2.21 µM [1.53-2.59 µM] with a median ferritin 338 µg/L [230-447 µg/L]). Of 125 homozygotes who last had phlebotomy ≥12 months ago (42 months [25-74 months], 92 had TS levels ≥ 50%, and 70 of these had NTBI ≥ 0.47 µM (2.06 µM [1.23-2.61µM]). Twenty-six of these 70 had a normal ferritin. Fifty-five of 125 had TS ≥ 75%, and NTBI was detected in all of these (2.32 µM [1.57-2.77 µM]) with a median ferritin 344 µg/L (255-418 µg/L). Eighteen of these 55 had a normal ferritin. In summary, NTBI is frequently found in C282Y homozygotes with TS ≥ 50%. Furthermore, C282Y homozygotes in the maintenance phase often have TS ≥ 50% together with a normal ferritin. Therefore, monitoring the TS level during the maintenance phase is recommended as an accessible clinical marker of the presence of NTBI.

Menstrual cycle affects iron homeostasis and hepcidin following interval running exercise in endurance-trained women.

PURPOSE: Menstrual cycle phase affects resting hepcidin levels, but such effects on the hepcidin response to exercise are still unclear. Thus, we investigated the hepcidin response to running during three different menstrual cycle phases. METHODS: Twenty-one endurance-trained eumenorrheic women performed three identical interval running protocols during the early-follicular phase (EFP), late-follicular phase (LFP), and mid-luteal phase (MLP). The protocol consisted of 8 × 3 min bouts at 85% of the maximal aerobic speed, with 90-s recovery. Blood samples were collected pre-exercise and at 0 h, 3 h and 24 h post-exercise. RESULTS: Data presented as mean ± SD. Ferritin were lower in the EFP than the LFP (34.82 ± 16.44 vs 40.90 ± 23.91 ng/ml, p = 0.003), while iron and transferrin saturation were lower during the EFP (58.04 ± 19.70 µg/dl, 14.71 ± 5.47%) compared to the LFP (88.67 ± 36.38 µg/dl, 22.22 ± 9.54%; p < 0.001) and the MLP (80.20 ± 42.05 µg/dl, 19.87 ± 10.37%; p = 0.024 and p = 0.045, respectively). Hepcidin was not affected by menstrual cycle (p = 0.052) or menstrual cycle*time interaction (p = 0.075). However, when comparing hepcidin at 3 h post-exercise, a moderate and meaningful effect size showed that hepcidin was higher in the LFP compared to the EFP (3.01 ± 4.16 vs 1.26 ± 1.25 nMol/l; d = 0.57, CI = 0.07-1.08). No effect of time on hepcidin during the EFP was found either (p = 0.426). CONCLUSION: The decrease in iron, ferritin and TSAT levels during the EFP may mislead the determination of iron status in eumenorrheic athletes. However, although the hepcidin response to exercise appears to be reduced in the EFP, it shows no clear differences between the phases of the menstrual cycle (clinicaltrials.gov: NCT04458662).

IRIDA Phenotype in TMPRSS6 Monoallelic-Affected Patients: Toward a Better Understanding of the Pathophysiology.

Iron-refractory iron deficiency anemia (IRIDA) is an autosomal recessive inherited form of iron deficiency anemia characterized by discrepantly high hepcidin levels relative to body iron status. However, patients with monoallelic exonic TMPRSS6 variants have also been reported to express the IRIDA phenotype. The pathogenesis of an IRIDA phenotype in these patients is unknown and causes diagnostic uncertainty. Therefore, we retrospectively summarized the data of 16 patients (4 men, 12 women) who expressed the IRIDA phenotype in the presence of only a monoallelic TMPRSS6 variant. Eight unaffected relatives with identical exonic TMPRSS6 variants were used as controls. Haplotype analysis was performed to assess the (intra)genetic differences between patients and relatives. The expression and severity of the IRIDA phenotype were highly variable. Compared with their relatives, patients showed lower Hb, MCV, and TSAT/hepcidin ratios and inherited a different wild-type allele. We conclude that IRIDA in monoallelic TMPRSS6-affected patients is a phenotypically and genotypically heterogeneous disease that is more common in female patients. We hypothesize that allelic imbalance, polygenetic inheritance, or modulating environmental factors and their complex interplay are possible causes. This explorative study is the first step toward improved insights into the pathophysiology and improved diagnostic accuracy for patients presenting with IRIDA and a monoallelic exonic TMPRSS6 variant.

Ferritin-guided iron supplementation in whole blood donors: optimal dosage, donor response, return and efficacy (FORTE)-a randomised controlled trial protocol.

BACKGROUND: Frequent whole blood donors have an increased risk of developing iron deficiency. Iron deficiency can have detrimental health effects when left untreated. Donation intervals are commonly too short to replenish iron stores and extending these reduces donor availability. Oral iron supplementation is known to shorten iron store recovery time but may also induce gastrointestinal complaints. We aim to optimise the effectiveness of iron supplements while minimising the risks of side effects. Therefore, we will evaluate the impact of different iron supplementation protocols in terms of dosage and frequency on ferritin and haemoglobin levels, gastrointestinal side effects, iron deficiency-related symptoms and donor return compared with placebo supplementation. METHODS: Twelve hundred whole blood donors with ferritin levels ≤30 µg/L are included into a double-blind, randomised controlled trial. Participants are randomly allocated to one of six arms, administering capsules containing 0 mg, 30 mg or 60 mg of iron, either on alternate days or daily for 56 days. At baseline and 56, 122 and 182 days of follow-up, ferritin and haemoglobin levels are measured, and compliance, donor return, dietary iron intake, gastrointestinal, iron deficiency-related symptoms and general health are assessed by questionnaire. ETHICS AND DISSEMINATION: This study will provide a comprehensive overview of the effects of different frequencies and dosages of administration of iron supplements on iron status and health effects, thereby considering individual differences in treatment adherence and lifestyle. The outcome will provide scientific evidence to guide the debate if and how oral iron supplements may support the recovery of whole blood donors with low ferritin levels. TRIAL REGISTRATION NUMBER: NL8590; The Dutch trial registry.

Investigating the Molecular Mechanisms of Renal Hepcidin Induction and Protection upon Hemoglobin-Induced Acute Kidney Injury.

Hemolysis is known to cause acute kidney injury (AKI). The iron regulatory hormone hepcidin, produced by renal distal tubules, is suggested to exert a renoprotective role during this pathology. We aimed to elucidate the molecular mechanisms of renal hepcidin synthesis and its protection against hemoglobin-induced AKI. In contrast to known hepatic hepcidin induction, incubation of mouse cortical collecting duct (mCCDcl1) cells with IL-6 or LPS did not induce Hamp1 mRNA expression, whereas iron (FeS) and hemin significantly induced hepcidin synthesis (p < 0.05). Moreover, iron/heme-mediated hepcidin induction in mCCDcl1 cells was caused by the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, as indicated by increased nuclear Nrf2 translocation and induced expression of Nrf2 downstream targets GCLM (p < 0.001), NQO1 (p < 0.001), and TXNRD1 (p < 0.005), which could be prevented by the known Nrf2 inhibitor trigonelline. Newly created inducible kidney-specific hepcidin KO mice demonstrated a significant reduction in renal Hamp1 mRNA expression. Phenylhydrazine (PHZ)-induced hemolysis caused renal iron loading and oxidative stress in both wildtype (Wt) and KO mice. PHZ treatment in Wt induced inflammatory markers (IL-6, TNFα) but not Hamp1. However, since PHZ treatment also significantly reduced systemic hepcidin levels in both Wt and KO mice (both p < 0.001), a dissection between the roles of systemic and renal hepcidin could not be made. Combined, the results of our study indicate that there are kidney-specific mechanisms in hepcidin regulation, as indicated by the dominant role of iron and not inflammation as an inducer of renal hepcidin, but also emphasize the complex interplay of various iron regulatory mechanisms during AKI on a local and systemic level.

Transferrin Saturation/Hepcidin Ratio Discriminates TMPRSS6-Related Iron Refractory Iron Deficiency Anemia from Patients with Multi-Causal Iron Deficiency Anemia.

Pathogenic TMPRSS6 variants impairing matriptase-2 function result in inappropriately high hepcidin levels relative to body iron status, leading to iron refractory iron deficiency anemia (IRIDA). As diagnosing IRIDA can be challenging due to its genotypical and phenotypical heterogeneity, we assessed the transferrin saturation (TSAT)/hepcidin ratio to distinguish IRIDA from multi-causal iron deficiency anemia (IDA). We included 20 IRIDA patients from a registry for rare inherited iron disorders and then enrolled 39 controls with IDA due to other causes. Plasma hepcidin-25 levels were measured by standardized isotope dilution mass spectrometry. IDA controls had not received iron therapy in the last 3 months and C-reactive protein levels were <10.0 mg/L. IRIDA patients had significantly lower TSAT/hepcidin ratios compared to IDA controls, median 0.6%/nM (interquartile range, IQR, 0.4-1.1%/nM) and 16.7%/nM (IQR, 12.0-24.0%/nM), respectively. The area under the curve for the TSAT/hepcidin ratio was 1.000 with 100% sensitivity and specificity (95% confidence intervals 84-100% and 91-100%, respectively) at an optimal cut-off point of 5.6%/nM. The TSAT/hepcidin ratio shows excellent performance in discriminating IRIDA from TMPRSS6-unrelated IDA early in the diagnostic work-up of IDA provided that recent iron therapy and moderate-to-severe inflammation are absent. These observations warrant further exploration in a broader IDA population.

Kidney tubule iron loading in experimental focal segmental glomerulosclerosis.

Kidney iron deposition may play a role in the progression of tubulointerstitial injury during chronic kidney disease. Here, we studied the molecular mechanisms of kidney iron loading in experimental focal segmental glomerulosclerosis (FSGS) and investigated the effect of iron-reducing interventions on disease progression. Thy-1.1 mice were injected with anti-Thy-1.1 monoclonal antibody (mAb) to induce proteinuria. Urine, blood and tissue were collected at day (D)1, D5, D8, D15 and D22 after mAb injection. Thy-1.1 mice were subjected to captopril (CA), iron-deficient (ID) diet or iron chelation (deferoxamine; DFO). MAb injection resulted in significant albuminuria at all time points (p < 0.01). Kidney iron loading, predominantly in distal tubules, increased in time, along with urinary kidney injury molecule-1 and 24p3 concentration, as well as kidney mRNA expression of Interleukin-6 (Il-6) and Heme oxygenase-1 (Ho-1). Treatment with CA, ID diet or DFO significantly reduced kidney iron deposition at D8 and D22 (p < 0.001) and fibrosis at D22 (p < 0.05), but not kidney Il-6. ID treatment increased kidney Ho-1 (p < 0.001). In conclusion, kidney iron accumulation coincides with progression of tubulointerstitial injury in this model of FSGS. Reduction of iron loading halts disease progression. However, targeted approaches to prevent excessive kidney iron loading are warranted to maintain the delicate systemic and cellular iron balance.

Streptococcus gallolyticus Increases Expression and Activity of Aryl Hydrocarbon Receptor-Dependent CYP1 Biotransformation Capacity in Colorectal Epithelial Cells.

Objective: The opportunistic pathogen Streptococcus gallolyticus is one of the few intestinal bacteria that has been consistently linked to colorectal cancer (CRC). This study aimed to identify novel S. gallolyticus-induced pathways in colon epithelial cells that could further explain how S. gallolyticus contributes to CRC development. Design and Results: Transcription profiling of in vitro cultured CRC cells that were exposed to S. gallolyticus revealed the specific induction of oxidoreductase pathways. Most prominently, CYP1A and ALDH1 genes that encode phase I biotransformation enzymes were responsible for the detoxification or bio-activation of toxic compounds. A common feature is that these enzymes are induced through the Aryl hydrocarbon receptor (AhR). Using the specific inhibitor CH223191, we showed that the induction of CYP1A was dependent on the AhR both in vitro using multiple CRC cell lines as in vivo using wild-type C57bl6 mice colonized with S. gallolyticus. Furthermore, we showed that CYP1 could also be induced by other intestinal bacteria and that a yet unidentified diffusible factor from the S. galloltyicus secretome (SGS) induces CYP1A enzyme activity in an AhR-dependent manner. Importantly, priming CRC cells with SGS increased the DNA damaging effect of the polycyclic aromatic hydrocarbon 3-methylcholanthrene. Conclusion: This study shows that gut bacteria have the potential to modulate the expression of biotransformation pathways in colonic epithelial cells in an AhR-dependent manner. This offers a novel theory on the contribution of intestinal bacteria to the etiology of CRC by modifying the capacity of intestinal epithelial or (pre-)cancerous cells to (de)toxify dietary components, which could alter intestinal susceptibility to DNA damaging events.

Novel concepts in red blood cell clearance.

PURPOSE OF REVIEW: Red blood cell (RBC) clearance has been studied for decades in many different pathologies, which has revealed different routes of RBC degradation, depending on the situation. This review summarizes the latest mechanistic insights on RBC clearance in different contexts; during homeostatic removal, immune-mediated destruction, and systemic inflammation. RECENT FINDINGS: Besides the recognition of a variety of potential 'eat me' signals on RBCs, recent evidence suggests that normal RBC degradation is driven by the increase of the adhesive properties of RBCs, mediating the retention in the spleen and leading to RBC hemolysis. Furthermore, immune-mediated degradation of RBCs seems to be fine-tuned by the balance between the density of the antigens expressed on RBCs and the presence of 'don't eat me' signals. Moreover, besides RBC clearance by macrophages, neutrophils seem to play a much more prominent role in immune-mediated RBC removal than anticipated. Lastly, RBC clearance during systemic inflammation appears to be driven by a combination of extreme macrophage activity in response to proinflammatory cytokines as well as direct damage of RBC by the inflammation or inflammatory agent. SUMMARY: Recent studies on RBC clearance have expanded our knowledge on their destruction in different contexts.

Comparative Evaluation of Sucrosomial Iron and Iron Oxide Nanoparticles as Oral Supplements in Iron Deficiency Anemia in Piglets.

Iron deficiency is the most common mammalian nutritional disorder. However, among mammalian species iron deficiency anemia (IDA), occurs regularly only in pigs. To cure IDA, piglets are routinely injected with high amounts of iron dextran (FeDex), which can lead to perturbations in iron homeostasis. Here, we evaluate the therapeutic efficacy of non-invasive supplementation with Sucrosomial iron (SI), a highly bioavailable iron supplement preventing IDA in humans and mice and various iron oxide nanoparticles (IONPs). Analysis of red blood cell indices and plasma iron parameters shows that not all iron preparations used in the study efficiently counteracted IDA comparable to FeDex-based supplementation. We found no signs of iron toxicity of any tested iron compounds, as evaluated based on the measurement of several toxicological markers that could indicate the occurrence of oxidative stress or inflammation. Neither SI nor IONPs increased hepcidin expression with alterations in ferroportin (FPN) protein level. Finally, the analysis of the piglet gut microbiota indicates the individual pattern of bacterial diversity across taxonomic levels, independent of the type of supplementation. In light of our results, SI but not IONPs used in the experiment emerges as a promising nutritional iron supplement, with a high potential to correct IDA in piglets.

Genetic Diagnosis in Hereditary Hemochromatosis: Discovering and Understanding the Biological Relevance of Variants.

BACKGROUND: Hereditary hemochromatosis (HH) is a genetic disease, leading to iron accumulation and possible organ damage. Patients are usually homozygous for p. Cys282Tyr in the homeostatic iron regulator gene but may have mutations in other genes involved in the regulation of iron. Next-generation sequencing is increasingly being utilized for the diagnosis of patients, leading to the discovery of novel genetic variants. The clinical significance of these variants is often unknown. CONTENT: Determining the pathogenicity of such variants of unknown significance is important for diagnostics and genetic counseling. Predictions can be made using in silico computational tools and population data, but additional evidence is required for a conclusive pathogenicity classification. Genetic disease models, such as in vitro models using cellular overexpression, induced pluripotent stem cells or organoids, and in vivo models using mice or zebrafish all have their own challenges and opportunities when used to model HH and other iron disorders. Recent developments in gene-editing technologies are transforming the field of genetic disease modeling. SUMMARY: In summary, this review addresses methods and developments regarding the discovery and classification of genetic variants, from in silico tools to in vitro and in vivo models, and presents them in the context of HH. It also explores recent gene-editing developments and how they can be applied to the discussed models of genetic disease.