Development of the Caco-2 Model for Assessment of Iron Absorption and Utilisation at Supplemental Levels

Caco-2 cells may be typically used as a first step to investigate the bioavailability of different dietary and fortificant forms of iron (Fe) at low levels (< 10 µM) in tissue culture medium (TCM). Whether this model is suitable with supplemental levels of Fe (ca. 200 µM in TCM) is not clear and neither, therefore, is the choice of reference iron compound under those conditions as a ‘positive control’. Here we show that with 200 ?M iron in TCM (serum-free MEM), Fe(II) sulphate precipitates and while high levels of ascorbic acid can prevent this, it is to the detriment of the Caco-2 cell monolayer and/or it adversely affects the pH of the TCM. Adjusting the pH of TCM to account for this issue again leads to Fe precipitation, which is detectable as both a true precipitate (~ 50%) and a nano-precipitate in suspension (~20%). In contrast, Fe(III) maltol which, clinically, appears less toxic to the intestinal mucosa than Fe(II) sulphate, retains solubility at supplemental levels in cell culture medium, without adversely affecting pH or the Caco-2 cell monolayer. Moreover, the iron is also well utilized by the cells as assessed through ferritin formation. Thus Caco-2 cells may also provide a model for screening iron uptake and utilisation at supplemental levels through the cellular generation of ferritin although care must be taken in ensuring (i) appropriate TCM conditions (e.g. pH and chemical speciation of the iron) (ii) monolayer integrity (i.e. the assay response is not an artefact of toxicity) and (iii) that an appropriate reference material is used (e.g. Fe:maltol at 1:5 ratio)

Development of the Caco-2 Model for Assessment of Iron Absorption and Utilisation at Supplemental Levels

Caco-2 cells may be typically used as a first step to investigate the bioavailability of different dietary and fortificant forms of iron (Fe) at low levels (< 10 µM) in tissue culture medium (TCM). Whether this model is suitable with supplemental levels of Fe (ca. 200 µM in TCM) is not clear and neither, therefore, is the choice of reference iron compound under those conditions as a ‘positive control’. Here we show that with 200 ?M iron in TCM (serum-free MEM), Fe(II) sulphate precipitates and while high levels of ascorbic acid can prevent this, it is to the detriment of the Caco-2 cell monolayer and/or it adversely affects the pH of the TCM. Adjusting the pH of TCM to account for this issue again leads to Fe precipitation, which is detectable as both a true precipitate (~ 50%) and a nano-precipitate in suspension (~20%). In contrast, Fe(III) maltol which, clinically, appears less toxic to the intestinal mucosa than Fe(II) sulphate, retains solubility at supplemental levels in cell culture medium, without adversely affecting pH or the Caco-2 cell monolayer. Moreover, the iron is also well utilized by the cells as assessed through ferritin formation. Thus Caco-2 cells may also provide a model for screening iron uptake and utilisation at supplemental levels through the cellular generation of ferritin although care must be taken in ensuring (i) appropriate TCM conditions (e.g. pH and chemical speciation of the iron) (ii) monolayer integrity (i.e. the assay response is not an artefact of toxicity) and (iii) that an appropriate reference material is used (e.g. Fe:maltol at 1:5 ratio)

Ferritin-protein levels in Caco-2 cells following exposure to LM Fe(III) poly oxo-hydroxide (nano Fe), Fe(III) maltol (FeM) or Fe(II) sulphate-ascorbate (FeSO₄ + AA).

<p><b>A</b>, Ferritin-protein regulation in differentiated and undifferentiated cells. ***, <i>p</i>=0.0003. Cells were incubated for 1 h with 200 μM Fe plus a further 23 h in fresh, non-supplemented MEM to allow for ferritin formation. <b>B</b>, Phase distribution of Fe in the BSS uptake medium: i.e. fractional percentage of microparticulate (black bars), nanoparticulate (red bars) and soluble Fe (open bars) for each Fe material. Values are mean ± s.d. of 3 independent experiments. <b>C</b>, Effect of LM Fe(III) poly oxo-hydroxide particle dispersion (in BSS medium, closed bars) or agglomeration (in MEM medium, open bars) on ferritin-protein levels in differentiated cells: the LM Fe(III) poly oxo-hydroxide was dispersed in its nano-form (99 ± 2% nano) using BSS or agglomerated (97 ± 2% microparticulate) using MEM. Data are mean of 3 independent experiments (each experiment with 3 replicate wells). FeM: soluble iron control, Fe(III) maltol. ***, <i>p</i>=0.0002 for the comparison between BSS and MEM. <b>D</b>, TEER changes in differentiated Caco-2 cell monolayer at different time points during incubation with BSS supplemented with LM Fe(III) poly oxo-hydroxide (open circles) or non-supplemented BSS control (closed inverted triangles). Incubations were for 3 h with 200 μM Fe (measurements at 1, 2 & 3 h) plus a further 21 h in fresh, non-supplemented MEM (24-h). Values are expressed as a percentage of the initial measurement and are shown as mean ± s.d. of 3 independent experiments (each experiment with 3 replicate wells). Experimental points are connected with a solid line to aid visualization and not because a linear relationship is assumed between time and TEER measurement. Detailed methodology is available in the Methods Section and in Methods S1.</p

Lysososmal dissolution of LM Fe(III) poly oxo-hydroxide.

<p><b>A</b>, Solubility in simulated lysosomal conditions at pH 5.0 with 10 mM citric acid and 0.15 M NaCl. Soluble Fe was measured by ICP-OES following 5 min ultrafiltration (3000 Da MWCO) for the LM Fe(III) poly oxo-hydroxide (black) and for un-modified Fe(III) poly oxo-hydroxide (solid blue). Nanoparticulate Fe was obtained from the Fe in the supernatant following centrifugation excluding the soluble (ultrafilterable) Fe, and is shown for LM Fe(III) poly oxo-hydroxide (red) and for un-modified Fe(III) poly oxo-hydroxide (dotted blue). Values are plotted as mean ± s.d. of 3 independent experiments (each experiment with 3 replicates). <b>B</b>, Effect of inhibition of lysosomal acidification using monensin on Fe utilization by differentiated Caco-2 cells. Data are shown as a percentage of the control (without monensin) at 24 h: i.e. 1 h exposure to 200 µM nanoparticulate LM Fe(III) poly oxo-hydroxide (open circles) or Fe(III) maltol (closed squares) ± 5-30 µM monensin followed by 23 h in non-supplemented MEM. Results are means ± s.d. of 3 independent experiments (each experiment with 3 replicate wells). **, <i>p</i><0.005; ***, <i>p</i><0.001 in relation to the soluble Fe control (Fe(III)maltol). <b>C</b>, Change in TEER in the Caco-2 cell monolayer following 1 h exposure to 10 μM monensin (closed squares), 30 μM monensin (open diamonds) or non-supplemented BSS control (closed inverted triangles) and with 23 h further incubation in fresh MEM (24 h in total). Values are expressed as a percentage of the initial measurement at the start of the exposure time (corresponding to 0 h) and are shown as mean ± s.d. of 2 independent experiments (each experiment with 3 replicate wells). Experimental points are connected with a solid line to aid visualization and not because a linear relationship is assumed between time and TEER measurement. ***, <i>p</i>=0.0003; ****, <i>p</i><0.0001 in relation to the non-supplemented BSS control.</p

Characterisation of hydrolysed Fe(III) with simulated digestion and of aquated LM Fe(III) poly oxo-hydroxide.

<p><b>A</b>, Transmission Electron Microscopy (TEM) images collected from a drop of suspension after simulated digestion of 1 mM Fe(III) chloride in the presence of 2 g/L mucin and low molecular weight ligands. The boxed regions are shown magnified below and highlight the presence of fine, poorly crystalline nanoparticles dispersed in an amorphous gel. Crystallinity is indicated by the spots in the inset diffractograms (fast Fourier transforms) in the boxed regions and lattice spacings are discussed in the main text. Scale bar represents 5 nm. <b>B</b>, Whole area EDX analysis of a particle agglomerate similar to those in ‘A’ shows elemental compositions (the specimen support film and grid produce the background C and Cu signals respectively). <b>C</b>, Hydrodynamic size distribution of nanoparticulate 500 µM LM Fe(III) poly oxo-hydroxide in balanced salt solution (BSS) measured by Dynamic Light Scattering (DLS). Values are expressed as mean diameter ± s.d. (3 independent measurements) on a log₁₀ scale.</p

Caco-2 cell acquisition of dietary iron(III) invokes a nanoparticulate endocytic pathway

Dietary non-heme iron contains ferrous [Fe(II)] and ferric [Fe(III)] iron fractions and the latter should hydrolyze, forming Fe(III) oxo-hydroxide particles, on passing from the acidic stomach to less acidic duodenum. Using conditions to mimic the in vivo hydrolytic environment we confirmed the formation of nanodisperse fine ferrihydrite- like particles. Synthetic analogues of these (~ 10 nm hydrodynamic diameter) were readily adherent to the cell membrane of differentiated Caco-2 cells and internalization was visualized using transmission electron microscopy. Moreover, Caco-2 exposure to these nanoparticles led to ferritin formation (i.e., iron utilization) by the cells, which, unlike for soluble forms of iron, was reduced ( p =0.02) by inhibition of clathrin-mediated endocytosis. Simulated lysosomal digestion indicated that the nanoparticles are readily dissolved under mildly acidic conditions with the lysosomal ligand, citrate. This was confirmed in cell culture as monensin inhibited Caco-2 utilization of iron from this source in a dose dependent fashion ( p <0.05) whilet soluble iron was again unaffected. Our findings reveal the possibility of an endocytic pathway for acquisition of dietary Fe(III) by the small intestinal epithelium, which would complement the established DMT-1 pathway for soluble Fe(II

Thrombotic and hemorrhagic complications of COVID-19 in adults hospitalized in high-income countries compared with those in adults hospitalized in low- and middle-income countries in an international registry

Background: COVID-19 has been associated with a broad range of thromboembolic, ischemic, and hemorrhagic complications (coagulopathy complications). Most studies have focused on patients with severe disease from high-income countries (HICs). Objectives: The main aims were to compare the frequency of coagulopathy complications in developing countries (low- and middle-income countries [LMICs]) with those in HICs, delineate the frequency across a range of treatment levels, and determine associations with in-hospital mortality. Methods: Adult patients enrolled in an observational, multinational registry, the International Severe Acute Respiratory and Emerging Infections COVID-19 study, between January 1, 2020, and September 15, 2021, met inclusion criteria, including admission to a hospital for laboratory-confirmed, acute COVID-19 and data on complications and survival. The advanced-treatment cohort received care, such as admission to the intensive care unit, mechanical ventilation, or inotropes or vasopressors; the basic-treatment cohort did not receive any of these interventions. Results: The study population included 495,682 patients from 52 countries, with 63% from LMICs and 85% in the basic treatment cohort. The frequency of coagulopathy complications was higher in HICs (0.76%-3.4%) than in LMICs (0.09%-1.22%). Complications were more frequent in the advanced-treatment cohort than in the basic-treatment cohort. Coagulopathy complications were associated with increased in-hospital mortality (odds ratio, 1.58; 95% CI, 1.52-1.64). The increased mortality associated with these complications was higher in LMICs (58.5%) than in HICs (35.4%). After controlling for coagulopathy complications, treatment intensity, and multiple other factors, the mortality was higher among patients in LMICs than among patients in HICs (odds ratio, 1.45; 95% CI, 1.39-1.51). Conclusion: In a large, international registry of patients hospitalized for COVID-19, coagulopathy complications were more frequent in HICs than in LMICs (developing countries). Increased mortality associated with coagulopathy complications was of a greater magnitude among patients in LMICs. Additional research is needed regarding timely diagnosis of and intervention for coagulation derangements associated with COVID-19, particularly for limited-resource settings

The value of open-source clinical science in pandemic response: lessons from ISARIC

International audienc

Respiratory support in patients with severe COVID-19 in the International Severe Acute Respiratory and Emerging Infection (ISARIC) COVID-19 study: a prospective, multinational, observational study

Background: Up to 30% of hospitalised patients with COVID-19 require advanced respiratory support, including high-flow nasal cannulas (HFNC), non-invasive mechanical ventilation (NIV), or invasive mechanical ventilation (IMV). We aimed to describe the clinical characteristics, outcomes and risk factors for failing non-invasive respiratory support in patients treated with severe COVID-19 during the first two years of the pandemic in high-income countries (HICs) and low middle-income countries (LMICs). Methods: This is a multinational, multicentre, prospective cohort study embedded in the ISARIC-WHO COVID-19 Clinical Characterisation Protocol. Patients with laboratory-confirmed SARS-CoV-2 infection who required hospital admission were recruited prospectively. Patients treated with HFNC, NIV, or IMV within the first 24 h of hospital admission were included in this study. Descriptive statistics, random forest, and logistic regression analyses were used to describe clinical characteristics and compare clinical outcomes among patients treated with the different types of advanced respiratory support. Results: A total of 66,565 patients were included in this study. Overall, 82.6% of patients were treated in HIC, and 40.6% were admitted to the hospital during the first pandemic wave. During the first 24 h after hospital admission, patients in HICs were more frequently treated with HFNC (48.0%), followed by NIV (38.6%) and IMV (13.4%). In contrast, patients admitted in lower- and middle-income countries (LMICs) were less frequently treated with HFNC (16.1%) and the majority received IMV (59.1%). The failure rate of non-invasive respiratory support (i.e. HFNC or NIV) was 15.5%, of which 71.2% were from HIC and 28.8% from LMIC. The variables most strongly associated with non-invasive ventilation failure, defined as progression to IMV, were high leukocyte counts at hospital admission (OR [95%CI]; 5.86 [4.83-7.10]), treatment in an LMIC (OR [95%CI]; 2.04 [1.97-2.11]), and tachypnoea at hospital admission (OR [95%CI]; 1.16 [1.14-1.18]). Patients who failed HFNC/NIV had a higher 28-day fatality ratio (OR [95%CI]; 1.27 [1.25-1.30]). Conclusions: In the present international cohort, the most frequently used advanced respiratory support was the HFNC. However, IMV was used more often in LMIC. Higher leucocyte count, tachypnoea, and treatment in LMIC were risk factors for HFNC/NIV failure. HFNC/NIV failure was related to worse clinical outcomes, such as 28-day mortality. Trial registration This is a prospective observational study; therefore, no health care interventions were applied to participants, and trial registration is not applicable