Plasma Iron Levels Research Articles

Preterm Piglets Born by Cesarean Section as a Suitable Animal Model for the Study of Iron Metabolism in Premature Infants.

Preterm infants are most at risk of iron deficiency. However, our knowledge of the regulation of iron homeostasis in preterm infants is poor. The main goal of our research was to develop and validate an animal model of human prematurity to assess iron status in preterm infants. We performed a cesarean section on sows on the 109th day of pregnancy, which corresponds to the last trimester of human pregnancy. Preterm piglets showed decreased body weight, red blood cell indices, plasma iron level and transferrin saturation. Interestingly, higher hepatic and splenic non-heme iron content and plasma and hepatic ferritin levels were found in premature piglets compared with term ones. In addition, premature piglets showed higher mRNA levels of iron-regulatory hormone hepcidin in the liver than term animals, which have not been reflected in higher plasma hepcidin-25 levels. We also showed changes in hepcidin regulators, including hepatic bone morphogenetic protein 6, plasma erythroferrone and growth differentiation factor 15 in preterm piglets. Consequently, no difference was observed in iron-exporter ferroportin levels in the spleen and liver. Overall, it seems that premature piglets show a pattern of iron metabolism characteristic of functional iron deficiency and iron accumulation in the tissue.

In-depth cerebrovascular lipidomics profiling for discovering novel biomarkers and mechanisms in moyamoya and intracranial atherosclerotic disease.

Despite considerable research efforts, the precise etiology and underlying pathways contributing to Moyamoya Disease (MMD) remain poorly understood. Moreover, the overlapping vascular pathologies shared between MMD and Intracranial Atherosclerotic Disease (ICAD) pose challenges in clinical differentiation, even with gold-standard cerebral angiography. An in-depth exploration of lipidomic alterations in cerebral intracranial MMD vessels could offer valuable insights into the pathogenesis of MMD-related mechanisms, encompassing MMD and ICAD, and unveil novel biomarkers and potential therapeutic targets. However, to date, comprehensive lipidomic profiling has been lacking. To discover novel biomarkers and unravel the pathophysiological mechanisms underlying MMD, we conducted a lipidomics analysis to characterize various lipid species in matched human extracranial and intracranial artery tissues from patients diagnosed with MMD (n=99) and ICAD (n=12). Our analysis identified 569 lipid species and delineated a robust panel of lipidomic biomarkers capable of effectively distinguishing MMD from ICAD (area under curve=0.98), as determined by receiver operating characteristic curve analysis. Notably, we observed a significantly more pronounced positive correlation of diacylglycerols and a negative association of triglycerides in intracranial artery tissues of MMD patients compared to those with ICAD, suggesting a potential role of dysregulated diacylglycerol-induced signaling in MMD pathogenesis. Furthermore, our investigation into the correlations of critical differential intracranial artery vessel lipid species between MMD and ICAD and clinical parameters revealed negative associations with plasma iron levels, implying a potential link between plasma iron metabolism and artery lipid homeostasis during MMD pathogenesis. These findings offer promising prospects for advancing clinical diagnosis for enhanced differentiation between the two disease conditions. Additionally, they shed light on the fundamental mechanisms implicated in MMD pathogenesis and suggest potential therapeutic avenues through targeting artery vessel lipids or plasma iron levels.

Ablation of mitophagy receptor FUNDC1 accentuates septic cardiomyopathy through ACSL4-dependent regulation of ferroptosis and mitochondrial integrity

Sepsis evokes compromised myocardial function prompting heart failure albeit target therapy remains dismal. Our study examined the possible role of mitophagy receptor FUNDC1 in septic cardiomyopathy. A sepsis model was established using cecal ligation and puncture (CLP) in FUNDC1 knockout (FUNDC1−/−) and WT mice prior to the evaluation of cardiac morphology, echocardiographic and cardiomyocyte contractile, oxidative stress, apoptosis, necroptosis, and ferroptosis. RNAseq analysis depicted discrepant patterns in mitophagy, oxidative stress and ferroptosis between CLP-challenged and control murine hearts. Septic patients displayed cardiac injury alongside low plasma FUNDC1 and iron levels. CLP evoked interstitial fibrosis, cardiac dysfunction (lowered ejection fraction, fractional shortening, shortening/relengthening velocity, peak shortening and electrically-stimulated intracellular Ca2+ rise, alongside increased LV end systolic diameter and relengthening duration), O2− buildup, apoptosis, necroptosis, and ferroptosis (downregulated GPX4 and SLC7A11), the responses of which were accentuated by FUNDC1 ablation. In particular, levels of lipid peroxidation enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) were upregulated following CLP procedure, with a more pronounced response in FUNDC1−/− mice. Co-immunoprecipitation and interaction interface revealed an evident interaction between FUNDC1 and ACSL4. In vitro studies revealed that the endotoxin lipopolysaccharide provoked cardiomyocyte contractile and lipid peroxidation anomalies, the responses were reversed by the mitophagy inducer oleanolic acid, inhibition of ACSL4 and ferroptosis. These findings favor a role for FUNDC1-ACSL4-ferroptosis cascade in septic cardiomyopathy.

Associations between multiple metals exposure and cognitive function in the middle-aged and older adults from China: A cross-sectional study

The rapidly rising risk of cognitive decline is a serious challenge for the elderly. As the wide-distributed environmental chemicals, the effects of metals exposure on cognitive function have attracted much attention, but the results remain inclusive. This study aimed to investigate the roles of multiple metals co-exposure on cognition. We included a total of 6112 middle-aged and older participants, detected their plasma levels of 23 metals by using inductively coupled plasma mass spectrometry, and assessed their cognitive function by using the Mini-Mental State Examination (MMSE). The results showed that increased plasma levels of iron (Fe) and zinc (Zn) were positively associated with MMSE score, but the increased levels of nickel (Ni) and lead (Pb) were associated with decreased MMSE score (all FDR < 0.05). Subjects exposed to both high levels of Ni and Pb showed the lowest MMSE score [β (95% CI) = −0.310 (−0.519, −0.100)], suggesting that Ni and Pb had a synergistic toxic effect on cognitive function. In addition, the hazardous roles of Ni and Pb were mainly found among subjects with low plasma level of Zn, but were not significant among those with high-Zn level [Ni: β (95% CI) = −0.281 (−0.546, −0.015) vs. −0.146 (−0.351, 0.058); Pb: β (95% CI) = −0.410 (−0.651, −0.169) vs. −0.060 (−0.275, 0.155)], which suggested that Zn could attenuate the adverse effects of Pb and Ni on cognitive function. The cognitive function was gradually decreased among subjects with increased number of adverse exposures to the above four metals (Ptrend < 0.001). In conclusion, our findings revealed the individual, interactive, and combined effects of Fe, Ni, Pb, and Zn on cognitive function, which may provide new perspectives on cognitive protection, but further prospective cohort studies and biological researches are needed to validate these findings.

Comparison of the Effectiveness of Single Anastomosis Sleeve Ileal Bypass and Roux-en-Y Gastric Bypass in Obese Patients with Type 2 Diabetes.

The rapid increase in obesity and related problems has heightened the importance of obesity and metabolic surgery, resulting in a continuing search for the ideal surgical method. This study aimed to compare the effectiveness of Roux-en-Y gastric bypass (RYGB), which is the gold standard method, and the relatively newer method of single anastomosis sleeve ileal (SASI) bypass. The medical records of patients who underwent RYGB and SASI bypass were reviewed and compared regarding excess weight loss (EWL) and HgA1c, fasting glucose, serum iron, ferritin, hemoglobin (Hgb), and vitamin B12 levels. The body mass index was significantly lower (p = 0.003), and the %EWL was significantly higher (p = 0.023) at 6months postoperative in patients who underwent SASI bypass. Both groups had similar results at the other follow-up visits. The mean HgA1c levels at the 6- and 12-month follow-up visits were significantly lower in SASI patients (p = 0.037 and p = 0.012, respectively). At the 24-month follow-up, no difference was detected in HgA1c levels between the groups (p = 0.476). In patients who underwent RYGB, ferritin (p < 0.001), plasma iron (p = 0.001), and Hgb levels (p = 0.001) were significantly lower at 12, 24, and 12months postoperatively, respectively. SASI bypass is effective and comparable to RYGB in terms of EWL and metabolic control and has the advantage of not producing excluded segments and reducing nutritional deficiencies.

Effect of Dietary Energy Level during Late Gestation on Mineral Contents in Colostrum, Milk, and Plasma of Lactating Jennies.

This study investigated the effects of dietary energy levels during late gestation on mineral content in the plasma, colostrum, and milk of jennies postpartum. Twenty-four pregnant multiparous DeZhou jennies, aged 6.0 ± 0.1 years, with a body weight of 292 ± 33 kg, an average parity number of 2.7 ± 0.1, and similar expected dates of confinement (74 ± 4 days), were randomly allocated to three groups and fed three diets: high energy (12.54 MJ/kg, HE), medium energy (12.03 MJ/kg, ME), and low energy (11.39 MJ/kg, LE). Blood samples were collected from the jugular vein of each jenny at time points of 0 h, 24 h, 48 h, 5 d, 7 d, and 14 d after parturition. Additionally, milk samples were collected through manual milking, and an analysis of the mineral content was conducted. The results showed that compared with HE, both ME and LE significantly increased the levels of calcium (Ca), phosphorus (P), zinc (Zn), selenium (Se), molybdenum (Mo), and cobalt (Co) in the plasma and Ca, P, magnesium (Mg), copper (Cu), manganese (Mn), Zn, selenium (Se), molybdenum (Mo), and Co in the milk of jennies postpartum (p < 0.05); ME also increased the levels of potassium (K), iron (Fe), and Mn in plasma and K and Fe in milk (p < 0.05). The levels of Ca, K, Mg, P, Fe, Cu, Mn, Co, Se, Zn, and Mo in plasma and milk gradually decreased with increasing postpartum time. Their contents were the highest at 0 h postpartum, rapidly decreased after 24 h postpartum, and declined to the lowest on day 14 postpartum. The interaction between dietary energy level and postpartum time showed that although the concentrations of the minerals Ca, P, K, Mg, Fe, Cu, Mn, Zn, Co, Se, and Mo decreased in jennies' plasma and milk in the treatment groups with different energy levels as postpartum time increased, the pattern of change was also influenced by dietary energy level. The influence of dietary energy level in late gestation on the mineral content of milk and plasma during the postpartum colostrum phase was higher than that during the milk phase. In conclusion, this study demonstrated that, under the current experimental conditions, the mineral content of the colostrum, milk, and plasma of jennies after parturition was dependent on the dietary energy level during late gestation.

Social defeat stress impairs systemic iron metabolism by activating the hepcidin-ferroportin axis.

Chronic psychological stress has been reported to decrease circulating iron concentrations and impair hematopoiesis. However, the underlying mechanisms remain unclear. This study aimed to investigate the effects of psychological stress on biological iron metabolism by using the social defeat stress (SDS) model, a widely used model of depression. Compared with control mice, mice subjected to SDS (SDS mice) had lower social interaction (SI) behavior. The SDS mice also showed impaired hematopoiesis, as evidenced by reduced circulating red blood cell counts, elevated reticulocyte counts, and decreased plasma iron levels. In the SDS mice, the iron contents in the bone marrow decreased, whereas those in the spleen increased, suggesting dysregulation in systemic iron metabolism. The concentrations of plasma hepcidin, an important regulator of systemic iron homeostasis, increased in the SDS mice. Meanwhile, the concentrations of ferroportin, an iron transport protein negatively regulated by hepcidin, were lower in the spleen and duodenum of the SDS mice than in those of the control mice. Treatment with dalteparin, a hepcidin inhibitor, prevented the decrease in plasma iron levels in the SDS mice. The gene expression and enzyme activity of furin, which converts the precursor hepcidin to active hepcidin, were high and positively correlated with plasma hepcidin concentration. Thus, furin activation might be responsible for the increased plasma hepcidin concentration. This study is the first to show that psychological stress disrupts systemic iron homeostasis by activating the hepcidin-ferroportin axis. Consideration of psychological stressors might be beneficial in the treatment of diseases with iron-refractory anemia.

Hematological toxicity of disinfection by-product iodoacetic acid

To clarify the effect of iodoacetic acid(IAA) on the blood system and electrolyte balance, hence further study the intrinsic relation of blood routine parameters and electrolyte levels, major hematological toxicity effects and their pattern after IAA treatment. Forty-eight 21-day-old male SPF grade Sprague-Dawley(SD) rats were gavaged with 0, 6.25, 12.5 and 25 mg/kg IAA for 31 days. After detections of blood routine and plasma inorganic ion levels, Spearman correlation coefficients were performed to evaluate their relationship. Changes in ferritin, transferrin, hepcidin, C-reactive protein and glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were assessed by enzyme-linked immunosorbent assays. The EDock bioinformatics tool was applied to docking model of IAA and GAPDH. Compared to the control, high-dose IAA exposure had obvious inhibition effect on rat leukocytes with the total number declined by 51.12%, and neutrophils were particularly sensitive to IAA with the number reduced by 73.66%(P&lt;0.01), and rat erythrocytes exhibited a small cell low pigment effect with hemoglobin and hematocrit decreased by 8.60% and 8.70%, respectively(P&lt;0.05). But IAA had little effects on the platelet. Plasma iron, phosphorus, zinc and potassium levels were repressed significantly, while chlorine, sodium and magnesium levels were elevated obviously through IAA exposure. However, plasma calcium levels were hardly affected by IAA. In comparison with the control, iron levels declined by 67.09%, whereas magnesium levels increased by 131.82% in the high-dose group(P&lt;0.01). Overall, correlation analyses uncovered that plasma iron metabolism was most strongly and positively correlated with levels of leukocyte, erythrocyte and platelet system parameters after IAA exposure, and the correlation coefficients of leukocyte number, mean hemoglobin content and mean erythrocyte volume were 0.637, 0.410 and 0.365, respectively(P&lt;0.05). Compared to the control, in the high-dose IAA group, the plasma content of C-reactive protein was significantly upregulated by 13.30%(P&lt;0.05), and plasma levels of transferrin and ferromodulin were also respectively elevated by 12.73% and 11.02%(P&lt;0.05). But plasma levels of ferritin and GAPDH did not differ between groups. The docking model exhibited that IAA could bind to the 150 Cys active site of rat GAPDH did. IAA not only had toxic effects on rat leukocytes and the plasma electrolyte balance, but also generated inflammation and iron deficiency, leading to smaller erythrocytes and lower pigment.

Effect of dolutegravir on ferritin, iron, and C-reactive protein among people living with HIV and co-infections.

Dolutegravir-based antiretroviral therapy (ART) is currently recommended as the preferred first-line ART in many resource-limited settings. However, little is known about the clinical experience of dolutegravir within a context of prevalent co-infections. To assess virological outcomes, and iron, ferritin and C-reactive protein (CRP) levels among people living with HIV (PLWH) and co-infections after initiating or re-initiating dolutegravir-based ART. This prospective study was conducted between August 2022 and August 2023. Study participants were recruited from an HIV opportunistic infection clinic. Screening for co-infections (syphilis, hepatitis B virus, cytomegalovirus and herpes simplex virus) was performed at baseline, prior to ART initiation. Plasma HIV viral load (VL), CRP, ferritin and iron levels were measured at baseline and at the 6-month follow-up period. A total of 100 participants (51 women and 49 men) were enrolled in this study. The median age of the participants was 39 years. The prevalence of co-infections was 30%. Prior to ART initiation, participants with co-infections had higher VL, CRP and ferritin, and lower iron levels, compared to those without co-infections (P < 0.001). Following 6 months of ART, CRP and ferritin levels decreased while iron levels increased, regardless of co-infection status. However, CRP and ferritin remained significantly higher in those with co-infections despite similar and high rates of virologic suppression in both groups. The presence of co-infections in PLWH is associated with higher VL and with chronic inflammation. Ferritin and CRP decreased on dolutegravir-based ART but remained higher in people with co-infections despite similar rates of virologic suppression.

Overnutrition is a risk factor for iron, but not for zinc or vitamin A deficiency in children and young people: a systematic review and meta-analysis

IntroductionTraditionally associated with undernutrition, increasing evidence suggests micronutrient deficiencies can coexist with overnutrition. Therefore, this work aimed to systematically review the associations between iron, zinc and vitamin A (VA) status...

Phytochemical Properties of Crataegus azarolus Berries Decoction Extract and Evaluation of its Protective Activity Against Acetic Acid-Induced Ulcerative Colitis in Rats.

The present study aims to evaluate the protective effect of Crataegus azarolus berries decoction extract (CAB-DE) against acetic acid-induced ulcerative colitis as well as the mechanisms implicated in such protection. Adult male Wistar rats were separated into seven groups: Control (H2O), acetic acid (AA), AA + various doses of CAB-DE (100, 200, and 400mg/kg, b.w.,p.o.), and AA + sulfasalazine (100mg/kg, b.w.,p.o.) or gallic acid (50mg/kg, b.w.,p.o.) during 10days. All rats were kept fasting overnight and ulcerative colitis was induced by rectal infusion of AA (300mg kg-1, b.w.) (3%, v/v, 5mL kg-1 b.w), for 30s. The colon was rapidly excised and macroscopically examined to measure ulcerated surfaces and the ulcer index. In vitro, we found that CAB-DE exhibited a high antioxidant activity against DPPH radical (IC50 = 164.17 ± 4.78μg/mL). In vivo, pretreatment with CAB-DE significantly protected the colonic mucosa against AA-induced damage by stimulating mucus secretion, reducing ulcer index as well as histopathological changes. Also, CAB-DE limited the oxidative status induced by AA in the colonic mucosa, as assessed by MDA and H2O2 increased levels and the depletion of both enzymatic activities and non-enzymatic levels. In addition, AA intoxication increased iron and calcium levels in colonic mucosa and plasma, while CAB-DE pretreatment regulated all intracellular mediators deregulation and significantly reduced inflammatory markers such as CRP (1.175 ± .04 ─ .734 ± .06μg/dl) and ALP (161.53 ± 5.02 ─ 98.60 ± 4.21 UI/L) levels. We suggest that CAB-DE protected against AA-induced ulcerative colitis due in part to its antioxidant and anti-inflammatory properties.

ACVR1: A Novel Therapeutic Target to Treat Anemia in Myelofibrosis.

Activin receptor type I (ACVR1) is a transmembrane kinase receptor belonging to bone morphogenic protein receptors (BMPs). ACVR1 plays an important role in hematopoiesis and anemia via the BMP6/ACVR1/SMAD pathway, which regulates expression of hepcidin, the master regulator of iron homeostasis. Elevated hepcidin levels are inversely associated with plasma iron levels, and chronic hepcidin expression leads to iron-restricted anemia. Anemia is one of the hallmarks of myelofibrosis (MF), a bone marrow (BM) malignancy characterized by BM scarring resulting in impaired hematopoiesis, splenomegaly, and systemic symptoms. Anemia and red blood cell transfusions negatively impact MF prognosis. Among the approved JAK inhibitors (ruxolitinib, fedratinib, momelotinib, and pacritinib) for MF, momelotinib and pacritinib are preferably used in cytopenic patients; both agents are potent ACVR1 inhibitors that suppress hepcidin expression via the BMP6/ACVR1/SMAD pathway and restore iron homeostasis/erythropoiesis. In September 2023, momelotinib was approved as a treatment for patients with MF and anemia. Zilurgisertib (ACVR1 inhibitor) and DISC-0974 (anti-hemojuvelin monoclonal antibody) are evaluated in early phase clinical trials in patients with MF and anemia. Luspatercept (ACVR2B ligand trap) is assessed in transfusion-dependent MF patients in a registrational phase 3 trial. Approved ACVR1 inhibitors and novel agents in development are poised to improve the outcomes of anemic MF patients.

Differences in some biochemical parameters in two herds of Karakachan sheep depending on environment

The aim of the present study was to estimate the values of some biochemical parameters - ALT, AST, ALP, Fe and glucose of two Karakachan sheep herds – IAS-Kostinbrod (I.) and Milanovo (M.), reared at different altitudes (740 and 1150 m above sea level, resp.) with different body condition score (BCS) and hematocrit (HCT) levels. Analysis of biochemical parameters was performed in extracted plasma with a BTS-350 Semi Automated Biochemical Analyser. The body condition score was performed during sampling. The BCS of the sheep from the herd of I. was better than that of the animals from M., as the difference was significant for the herds overall (p&lt;0.05). The HCT levels of the sheep from the I. herd were also higher (but not significantly) than those of the animals from M. The activity of ALT was higher than reference values and significantly lower in the sheep from M. compared to those from I. in general for the herds (p&lt;0.001) and in the group with low HCT (p&lt;0.01). The activities of AST and ALP were lower than reference values, as well as the levels of plasma iron and glucose. The differences were significantly higher in the sheep from M. compared to those from I. in general for the herds (AST, ALP, Fe and glucose - p&lt;0.001) and in the group with low HCT (ALP - p&lt;0.05, AST and Fe - p&lt;0.01 and AST - p&lt;0.001). In the group with a high HCT, there were no significant differences in all biochemical parameters.

Trace element levels and autism spectrum disorder in a sample of Algerian children: A case-control study investigation

The prevalence of autism has been increasing in recent years due to better screening. diagnosis. and awareness campaigns. Toxic metals are considered to be one of the environmental factors that may contribute to autism spectrum disorders (ASD). This study aimed to investigate the levels of toxic and essential trace elements in autistic children and their connection to autism. The study included 40 autistic children and 41 controls. both aged 4–9 years. The levels of toxic metals and metalloids (Arsenic. Cadmium. Chromium. Mercury. Lead) and trace elements (Copper. Iron. Selenium. Zinc) were measured in whole blood. plasma. and urine using ICP-MS multi-elemental techniques. The results showed that the concentrations of toxic and essential metals were significantly higher in autistic children compared to controls. This was demonstrated by higher levels of Cadmium. Chromium. Copper. Zinc. and Lead in blood. plasma. and urine. while plasma Iron levels were lower in autistic children. The results suggest a possible association between autism and metal exposure. but causality cannot be established.

The Effect of Voxelotor on Erythrocyte Deformability, Neutrophil Phenotype and Endothelial Activation

Introduction Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis, caused by polymerization of abnormal sickle hemoglobin. The ongoing intravascular hemolysis leads to release of cell-free heme and iron, resulting in widespread oxidative damage, inflammation, neutrophil activation, endothelial activation and hypercoagulability, all contributing to vaso-occlusive events and end organ damage. Voxelotor, a small molecule reversibly binding to hemoglobin S (HbS) increases its oxygen affinity, thereby decreasing HbS polymerization and sickling, resulting in decreased hemolysis. The decrease in erythrocyte sickling might lead to improved erythrocyte deformability and decreased adhesion, while reductions in cell-free heme and labile plasma iron levels might result in decreased neutrophil activation, adhesion and aging and decreased endothelial activation. The aim of this study is to assess the effects of voxelotor on erythrocyte deformability, neutrophil activation, adhesion and aging and endothelial activation. Methods SCD patients (HbSS and HbSβ 0-thalassemia), with hemoglobin (Hb) ≤ 10.5 g/dL were treated with voxelotor 1500 mg in a single arm interventional study. Blood was collected at baseline and after 3 months of treatment. Erythrocyte deformability was assessed by ektacytometry (Lorrca, RR mechatronics), measuring elongation index (EI) and point of sickling (PoS). EI max and EI min indicate the highest and lowest erythrocyte deformability upon oxygenation and deoxygenation respectively. PoS is the oxygen tension at which 5% reduction of the EI max is observed. Antigen expression of neutrophil markers for activation, adhesion and aging were assessed with flow cytometry. Reactive oxygen species (ROS) production of neutrophils was measured by Amplex Red Assay (Molecular probes), measuring hydrogen peroxide (H 2O 2) production under unstimulated and stimulated (zymosan, streptozotocin (STZ), factor-n-formyl-MET-LEU-Phe (fMLP), Phorbol myristate acetate (PMA)) and platelet-activating factor-fMLP (PAF-fMLP)). Endothelial activation was assessed by measuring von Willebrand Factor antigen (vWF-Ag) using enzyme-linked immunosorbent assay (ELISA) (Glostrup). Prothrombin fragment 1+2 (F 1+2), thrombin-antithrombin complexes (TAT) and D-dimer were measured using ELISA. Data are presented as median with interquartile range (IQR), unless stated otherwise. A P value of &amp;lt;0.05 was considered significant. Results 15 SCD patients (11 HbSS, 4 HbSβ 0-thalassemia), 5 female, median age 35 years (IQR 27-46)) completed 3-month follow-up. Hb levels increased significantly after 3 months of treatment (from 7.4 (6.9 - 8.1) g/dL to 9.7 (7.9 - 11.0) g/dL, p=0.002), corresponding to significant decreases in LDH and bilirubin levels and reticulocyte counts as markers of hemolysis (p&amp;lt;0.05). PoS decreased significantly after 3 months (from 20.1 (17.5 - 26.1) mmHg to 16.1 (10.3 - 18.1) mmHg, p=0.006). Both EI max and EI min increased after 3 months of treatment (from 0.33 (0.24 - 0.42) to 0.38 (0.28 - 0.48), p=0.029 and from 0.13 (0.07 - 0.26) to 0.21 (0.16 - 0.41), p=0.007 respectively). Markers of neutrophil activation, adhesion and aging were generally lower after 3 months of treatment, though the differences were not statistically significant (Figure 1 A-C). Neutrophil ROS production in both unstimulated and stimulated condition remained unchanged (Figure 1D). After 3 months of treatment vWF-Ag significantly decreased (from 252 (187 - 284) % to 183 (153-205) %, p = 0.033). No changes were observed in markers of coagulation activation (F 1+2 and TAT, and D-dimer). Conclusion Voxelotor significantly improved erythrocyte deformability as suggested by decreased PoS and increased EI max and EI min, potentially leading to improved rheological properties. While markers of neutrophil activation seem to decrease, the differences are not statistically significant, possibly due to the relatively small sample size. A reduction in vWF-Ag was observed possibly reflecting diminished endothelial activation, due to a decrease in intravascular hemolysis.

EPID-14. ASSOCIATION OF ELEMENTAL IRON WITH GLIOBLASTOMA PATIENT OUTCOMES

Abstract Very few studies have examined the role of trace metals, such as iron, in glioblastoma biology and patient outcomes. Here we directly measured iron content in matched glioblastoma tumor and plasma samples using inductively coupled plasma mass spectroscopy (ICP-MS) or inductively coupled plasma atomic emission spectroscopy (ICP-AES), and then studied associations of iron with clinical parameters such as median overall survival (mOS). Elemental iron analysis was performed on a total of 31 glioblastoma samples, 21 of which had matched plasma samples that were analyzed together. Plasma samples from age-matched healthy volunteers were used as a control for comparisons against plasma from glioblastoma patients. Plasma samples from glioblastoma patients had higher levels of iron compared to age-matched healthy control samples (1307.2 ng/mL vs. 575.2 ng/mL, P = .000013). Among glioblastoma patients, higher levels of plasma iron were associated with prolonged survival (mOS: 20.7 months vs. 12.4 months, P = 0.0063). Plasma iron levels, however, were not significantly correlated with tumoral iron levels (R = 0.095, P = 0.70), indicative of regulation between plasma and brain iron. Unsurprisingly, high tumor iron content failed to significantly associate with survival. To gain insights into whether iron may be associated with immune function, we analyzed plasma samples with multiplexed immunoassays. Glioblastoma plasma samples that contained higher levels of iron were associated with increased IFN-β (10.91 pg/mL vs 7.43 pg/mL, P = 0.004) and PDGF-AB/BB (1275.3 pg/mL vs 492.8 pg/mL, P = 0.009), whereas no such association was observed in healthy plasma samples. Our results demonstrate that plasma iron is associated with clinical outcomes and potentially with alterations in IFN-β and/or PDGF-AB/BB signaling in glioblastoma patients. These findings illustrate the importance of iron in glioblastoma biology and demonstrate its potential prognostic value.

ALK2 and JAK2 Inhibition for Improved Treatment of Anemia in Myelofibrosis Patients: Preclinical Profile of an ALK2 Inhibitor Zilurgisertib in Combination with Ruxolitinib

Introduction: Anemia is a common occurrence in patients with myelofibrosis (MF) and is associated with the need for red blood cell transfusion and poor clinical prognosis. JAK inhibitors such as ruxolitinib are used extensively to treat symptoms of MF and improve quality of life and overall survival. However, JAK inhibitors may also contribute to myelosuppression. Therapeutic interventions that allow for optimization of JAK inhibition with no concern of anemia would benefit patients with MF and potentially lower the rate of treatment discontinuation or suboptimal dosing. Recent studies have demonstrated that inhibition of ACVR1/ALK2, a bone morphogenetic protein receptor that is upstream of the transcriptional regulation of hepcidin, could reduce serum hepcidin levels in patients with MF and improve anemia (Verstovsek S et al. Lancet 2023; Oh S et al. Clin Lymphoma Myeloma Leuk 2022). Reducing levels of hepcidin, a key regulator of plasma iron levels, and restoring erythropoiesis would broadly benefit patients with MF. Based on these recent data, we developed zilurgisertib, a potent and selective ALK2 inhibitor that could be administered in combination with ruxolitinib at doses titrated to the needs of patients with MF. Methods and Results: In biochemical and cellular assays, zilurgisertib inhibited ALK2 kinase activity and SMAD1/5 phosphorylation with IC 50 values of 15 nM and 63 nM, respectively. In Huh-7 cells stimulated with BMP-6, zilurgisertib inhibited hepcidin production with an IC 50 of 20 nM, demonstrating the compound is a potent ALK2 inhibitor capable of regulating iron homeostasis via hepcidin. To assess possible off-target effects of zilurgisertib, kinome profiling at 10 μM ATP was performed at Reaction Biology (Malvern, PA) to determine the overall specificity across 356 kinases (see Figure). At 200 nM, zilurgisertib only inhibited ALK2, ALK1 (to 50%), and ALK6 (to 48%). In addition, zilurgisertib at 20 μM did not affect viability of HEK293 cells, a human cell line commonly used to assess general cell health and compound toxicity. Similarly, zilurgisertib did not affect viability of human fibroblasts or endothelial cells at concentrations up to 5 μM. To test whether the combination of ruxolitinib and zilurgisertib could suppress hepcidin and restore erythropoiesis in an in vivo mouse model of cancer-induced anemia, B16F10 cells were injected intraperitoneally, mimicking a metastatic tumor that leads to anemia 1 week after injection. In a dose-dependent manner, zilurgisertib improved hemoglobin by 2 to 3 g/dL and red blood cell counts, while reducing both liver pSMAD and circulating hepcidin levels by 50% or more compared with vehicle control. The combination of zilurgisertib with ruxolitinib had no effect on this activity, suggesting that inhibition of JAK2 does not inhibit erythropoiesis restored following ALK2 inhibition. Conclusion: Taken together, the potent and selective on-target activity of zilurgisertib suggests that ALK2 inhibition could reduce hepcidin and improve anemia, and that the combination of zilurgisertib with ruxolitinib is a rational and attractive approach to mitigate anemia in patients with MF. The combination of ruxolitinib and zilurgisertib is currently being evaluated in a phase 1 clinical trial in patients with anemia due to myeloproliferative disorders (NCT04455841).

Estrogens and Prolactin Do Not Regulate Maternal and Embryo Iron Homeostasis during Pregnancy

Iron is essential for maternal and fetal health but the molecular mechanisms ensuring increased iron availability during pregnancy are not well understood. Hepcidin is the key iron-regulatory hormone and functions by blocking iron absorption, recycling and mobilization from stores, effectively decreasing plasma iron levels. In healthy human and rodent pregnancies, maternal hepcidin decreases starting in the second trimester and is nearly undetectable by late pregnancy. This decrease in hepcidin allows for greater iron availability for transfer to the fetus. If hepcidin is not appropriately suppressed during pregnancy, it results in fetal anemia, intrauterine growth restriction, and even fetal demise in severe cases 1. Unrelated to pregnancy, hormones estrogen and prolactin have been previously implicated in hepcidin regulation 2,3, although with conflicting results on the direction of hepcidin change 4-6. The levels of both hormones increase dramatically over the course of pregnancy, thus we hypothesized that they may modulate iron homeostasis during pregnancy. Studying the role of estrogens and prolactin during pregnancy is challenging as both hormones are required for pregnancy. Additionally, mice with global knockout (KO) of either estrogen receptor alpha or prolactin receptor are infertile. Thus, to determine the contribution of estrogens and prolactin on maternal hepcidin suppression during pregnancy, we generated liver-specific knockouts of either estrogen receptor alpha ( Er- α f/f;Alb-Cre +) or prolactin receptor ( Prlr f/f;Alb-Cre +), here abbreviated as ER-α KO and PRLR KO. Female ER-α KO, PRLR KO and WT mice were set up for timed pregnancy, and liver hepcidin mRNA, as well as maternal and embryo iron and hematological parameters were measured at E18.5 . Hepcidin suppression in ER-α KO and PRLR KO dams was not different from that in WT dams, suggesting that estrogens and prolactin are not major mediators of maternal hepcidin suppression. Additionally, maternal and embryo hematological parameters and liver iron concentrations were similar between WT, ER-α KO, PRLR KO pregnancies, further supporting our observation that estrogens and prolactin do not regulate iron homeostasis in pregnancy. The mechanisms by which maternal hepcidin is suppressed during pregnancy remain to be determined. 1Sangkhae V et al, Blood, 2020 2Yang Q et al, Endocrinology, 2012 3Wang J et al, Nanomedicine: nanotechnology, biology and medicine, 2015 4Zhen et al, Hepatology, 2013 5Ikeda Y et al, PloS one, 2012 6Lehtihet M et al, PloS one, 2016

Differences in iron balance observed with dietary cholic acid supplementation and marginal iron deficiency in rats.

We investigated whether a cholic acid (CA)-supplemented diet and marginal iron deficiency (MID) diet influence hepatic lipid accumulation and iron balance in rats for 2 weeks. The CA diet enhanced hepatic lipid accumulation and modulated iron metabolism such as enhancement of fecal iron excretion, reduction in iron absorption, and no alteration in plasma iron levels. The MID diet did not alter hepatic lipid concentrations with reduced iron concentration in the liver and plasma. In combination, influence of the CA supplementation on the hepatic iron concentration was opposite between iron-sufficient and MID conditions. In the liver, the CA diet enhanced lipocalin 2 expression, whereas the MID diet enhanced transferrin receptor 1 expression and reduced hepcidin expression. This study revealed an involvement of 12-hydroxylated bile acids in regulation of hepatic iron concentration under MID condition.

Weak response of bovine hepcidin induction to iron through decreased expression of Smad4.

Hepcidin negatively regulates systemic iron levels by inhibiting iron entry into the circulation. Hepcidin production is increased in response to an increase in systemic iron via the activation of the bone morphogenetic protein (BMP) pathway. Regulation of hepcidin expression by iron status has been proposed on the basis of evidence mainly from rodents and humans. We evaluated the effect of iron administration on plasma hepcidin concentrations in calves and the expression of bovine hepcidin by the BMP pathway in a cell culture study. Hematocrit as well as levels of blood hemoglobin and plasma iron were lower than the reference level in calves aged 1-4 weeks. Although intramuscular administration of iron increased iron-related parameters, plasma hepcidin concentrations were unaffected. Treatment with BMP6 increased hepcidin expression in human liver-derived cells but not in bovine liver-derived cells. A luciferase-based reporter assay revealed that Smad4 was required for hepcidin reporter transcription induced by Smad1. The reporter activity of hepcidin was lower in the cells transfected with bovine Smad4 than in those transfected with murine Smad4. The lower expression levels of bovine Smad4 were responsible for the lower activity of the hepcidin reporter, which might be due to the instability of bovine Smad4 mRNA. In fact, the endogenous Smad4 protein levels were lower in bovine cells than in human and murine cells. Smad4 also confers TGF-β/activin-mediated signaling. Induction of TGF-β-responsive genes was also lower after treatment with TGF-β1 in bovine hepatocytes than in human hepatoma cells. We revealed the unique regulation of bovine hepcidin expression and the characteristic TGF-β family signaling mediated by bovine Smad4. The present study suggests that knowledge of the regulatory expression of hepcidin as well as TGF-β family signaling obtained in murine and human cells is not always applicable to bovine cells.