Biometals (2015) 28:143–150 DOI 10.1007/s10534-014-9810-z
Measurement of total iron in Helicobacter pylori-infected gastric epithelial cells Sebastian E. Flores • Andrew S. Day Jacqueline I. Keenan
•
Received: 1 October 2014 / Accepted: 19 November 2014 / Published online: 27 November 2014 Ó Springer Science+Business Media New York 2014
Abstract Despite the evidence suggesting a role for Helicobacter pylori in the induction of systemic iron deficiency anaemia, little is known about the possibility of infection-associated changes in cellular iron homeostasis at the gastric epithelium. In this study we compared four different techniques for measuring iron in AGS cells, a gastric epithelial cell line that is widely used to model to H. pylori infection in vitro. Inductively coupled plasma-mass spectrometry proved to be an efficient method, but only when large numbers of cells were used. Two colorimetric assays that included the use of concentrated hydrochloric acid with or without potassium ferrocyanide detected iron in the micromolar but not the nanomolar range in cell-free standards. However, the third colorimetric assay that incorporated ferrozine proved to be highly accurate at detecting iron in the nanomolar range, and was able to detect iron in AGS cells, Moreover, using this assay, we were able to show that the level of iron in H. pyloriinfected AGS cells is significantly increased when compared to uninfected cells.
S. E. Flores J. I. Keenan (&) Department of Surgery, University of Otago Christchurch, PO Box 4345, Christchurch, New Zealand e-mail: [email protected] A. S. Day Department of Paediatrics, University of Otago Christchurch, Christchurch, New Zealand
Keywords Iron measurement Gastric epithelial cells Helicobacter pylori
Introduction Numerous clinical studies describe a link between Helicobacter pylori and iron deficiency (ID) or iron deficiency anaemia (IDA) (Choe et al. 1999; Valiyaveettil et al. 2005; Gessner et al. 2006) and the inference from these studies is that H. pylori accesses host iron in order to survive (and persist) in the human stomach. This iron needs to come from the bacterium’s immediate surroundings. Thus, any effect that H. pylori might have on host iron stores is likely to occur first at the gastric epithelium, where these bacteria persist for a lifetime in an untreated host (Kusters et al. 2006). Support for this hypothesis comes from several unrelated studies. Gastric epithelial cells from H. pyloriinfected mice demonstrate up-regulated transcription of lactoferrin (Lf) and ferritin (Mueller et al. 2004). The role of Lf is to sequester iron at mucosal surfaces, thus reducing its availability to bacteria (Alexander et al. 2012), whereas ferritin is an iron-storage protein whose expression is increased in response to high levels of iron and/or inflammation (Alkhateeb et al. 2013). There is evidence of Lf-associated internalisation of H. pylori into gastric epithelial cells (Coray et al. 2012) and increased Lf expression is seen in gastric tissue from patients infected with H. pylori (Dogan et al. 2012). To
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date, there are no reports of an H. pylori-associated change in ferritin levels in the human stomach, but an increase in cytosolic ferric iron is observed when a human gastric epithelial cell line is exposed to outer membrane vesicles constitutively shed from the surface of these bacteria (Chitcholtan et al. 2008). Collectively, these studies suggest that H. pylori may affect iron uptake, storage and/or distribution in gastric epithelial cells. The co-culture of AGS cells and H. pylori is widely used as an in vitro model to study this gastric infection, allowing the delineation of host-pathogen interactions without the confounders present in clinical or animal studies. Using this reductionist system, H. pylori is shown to adhere to the surface of these cells (Nilius et al. 1994) and induce effects that include nuclear factor (NF)-jB activation (Keates et al. 1997) and the up-regulation of epidermal growth factor receptor (EGFR) (Keates et al. 2007), two known inducers of H-ferritin mRNA expression that may contribute to increased iron storage in gastric epithelial cells (Konijn et al. 1999). A recent study found evidence of increased transferrin internalisation in H. pylori-infected polarized Madin Darby Canine Kidney (MDCK) cells (Tan et al. 2011) but to date any effect of H. pylori infection on gastric epithelial cell iron homeostasis has not been determined. We speculate that this may, in part, reflect the reportedly low levels of iron in gastric tissue (Yaman et al. 2007). Accordingly, the aim of our study was to evaluate different methods to measure total iron in AGS cells, and to use these method(s) to determine if H. pylori infection can alter the level of total intracellular iron.
Materials and methods Materials Nitric acid, hydrochloric acid and ammonium acetate were purchased from VWR International (Radnor, PA, USA), potassium hexacyanoferrate (II) trihydrate (potassium ferrocyanide), thiourea and 3-(2-Pyridyl)-5,6diphenyl-1,2,4-triazine-40 ,400 -disulfonic acid sodium salt (ferrozine) were obtained from Sigma, while potassium permanganate was obtained from Fisons Scientific Apparatus (Ipswich, UK). L-ascorbic acid was purchased from Duchefa Biochemie (Haarlem, The Netherlands) and the Multielement calibration standard used for ICP-
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MS was obtained from Agilent technologies (Santa Clara, CA, USA). Dulbecco’s PBS (D-PBS) and Trypsin–EDTA were from Life Technologies (Mulgrave, VIC, Australia), RPMI-1640 medium and fetal bovine serum (FBS) were from Invitrogen, (Auckland, NZ). Cell culture and treatment AGS cells, a human gastric adenocarcinoma cell line (CRL 1739; American Type Culture Collection, Manassas, VA), were cultured in RPMI-1640 medium supplemented with 10 % (v/v) heat-inactivated FBS. Cells (1 9 105) were grown (in six well plates) at 37 °C in air containing 5 % CO2, with cells cultured until they reached 80 % confluence (approximately 72 h). Fresh medium was added to each well before the addition (or not) of H. pylori for a further 15 h. Bacteria culture and enumeration H. pylori strain 60190 (ATCC 49503) is a well characterized clinical isolate that is cagA positive (Tummuru et al. 1993) and has a vacA s1m1 genotype (Atherton et al. 1995). The bacteria were routinely cultured on Columbia sheep blood agar plates (Fort Richard, Auckland, NZ) in a microaerophilic atmosphere at 37 °C for 2–3 days. Prior to co-culture with AGS cells, H. pylori were resuspended in RPMI-1640 medium supplemented with 10 % (v/v) heat-inactivated fetal bovine serum and bacterial numbers were quantitated by density, based on a standard curve measured at an absorbance of 620 nm (SpectraMaxÒ 190, Molecular devices, Sunnyvale, CA, USA). An absorbance of 0.2 was determined to be approximately 1 9 108 colony forming units (CFUs) per ml. H. pylori were added to cell cultures at a multiplicity of infection (MOI) of 10:1. Inductively coupled plasma-mass spectrometry (ICP-MS) AGS cells (*1.5 9 106) were transferred to 15 ml tubes and centrifuged at 1,500 g for 10 min. Cell pellets were washed twice with D-PBS and resuspended in 5 ml of D-PBS. Two 10 ll aliquots were removed from each tube and trypan blue-stained cells (0.25 % w/v in PBS) were counted using a haemocytometer. The remaining sample was centrifuged
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(1,500 g for 10 min) and the pelleted cells were digested overnight in 3 ml of 60 % nitric acid (HNO3) at 65 °C before being diluted to 10 ml with ultrapure water. The concentration of iron in each sample was determined using an Agilent 7700 ICP-mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). A multi-element calibration standard containing 10 mg of Fe/l was used as a reference sample (Agilent Technologies).
UV/Visible spectrometry with hydrochloric acid and potassium ferrocyanide A 10 mM iron standard stock was generated by dissolving 0.278 g of FeSO47H2O (equivalent to 1 mmol) in 100 ml of ultrapure water, with one drop of 10 M HCl added to the solution to prevent iron precipitation. The concentration of iron was confirmed by ICP-MS analysis, and the solution was used to generate two standard curves that ranged over micromolar (lmol) and nanomolar (nmol) concentrations of iron, respectively. Each standard (in duplicate) was dried overnight at 110 °C. 1 ml of 5 M hydrochloric acid (HCl) was added to every tube and the sample was digested for 4 h at 60 °C (with the lids on to avoid evaporation of the acid.) Each sample was then split into two 500 ll aliquots. An equal volume of 5 M HCl was added to one aliquot, and 500 ll of freshly prepared 5 % w/v potassium ferrocyanide was added to the second aliquot to generate iron blue and Prussian blue, respectively. The HCl-treated standards were added to the wells of a flat-bottomed clear 96 well plate and absorbance was read at 351 nm whereas the standards with added hydrochloric acid and potassium ferrocyanide were incubated for 35 min in the dark before absorbance was read at 700 nm (Rad, Janic et al. 2007). Each assay was read using a SpectraMaxÒ 190 absorbance microplate reader (Molecular devices, Sunnyvale, CA, USA).
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(Thermo Fisher Scientific, Waltham, MA, USA), as detailed by (Fish 1988) before 200 ll of a freshly prepared digestion reagent (a 1:1 solution of 1.4 M HCl and 4.5 % (w/v) potassium permanganate) was added to each tube. The samples were digested for 2 h at 60 °C, then cooled to room temperature before the addition of 50 ll of a detection reagent (6.5 mM ferrozine, 2.5 M ammonium acetate, 1 M ascorbic acid and 2 mM thiourea). Each tube was vortexed and incubated for 30 min before the samples were transferred to a clear 96 well plate. Absorbances were read at 564 nm with a SpectraMaxÒ 190 absorbance microplate reader (Davis et al. 1986; Riemer et al. 2004).
Measurement of iron in AGS cells Total AGS cell intracellular iron levels were measured by the ferrozine assay (Riemer et al. 2004) with the modification proposed by (Fish 1988). AGS cells (1 9 105) were cultured for 72 h in six-well plates, washed with D-PBS and fresh medium was added before the addition (or not) of H. pylori for 15 h. After extensive washing, the cells were lifted with trypsin– EDTA. The pelleted cells were lysed by incubation with 200 ll NaOH (50 mM) for 2 h at 37 °C, and two 10 ll aliquots were taken to measure protein concentration (Lowry, Rosebrough et al. 1951). The remaining lysate was dried at 45 °C using a SpeedVacTM SPD111 V vacuum concentrator (Thermo Fisher Scientific, Waltham, MA) before addition of 200 ll of digestion reagent (see above) for 2 h at 60 °C. Samples were then cooled to room temperature, before the addition of 50 ll of a detection reagent (as detailed above). Absorbances were read at 564 nm and iron values were calculated against a standard curve. For the dose–response curve, AGS cells were supplemented with 0, 50, 100 or 200 lM FeSO4 for 24 h before the ferrozine assay was used to measure cellular iron levels.
Ferrozine assay Statistical analysis A standard curve ranging from 0 to 24 nmol of iron was generated from a 1:100 dilution of the iron stock solution (see above) in 1.6 ml Eppendorf tubes (in duplicate). The aliquots were fully dried at 45 °C using a SpeedVacTM SPD111 V vacuum concentrator
Results are presented as the ±SE of the means of at least three independent experiments. Data were analysed by Pearson correlation, ANOVA followed by Tukey’s post hoc test or non-parametric t test, as
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Fig. 1 Total iron in AGS cells measured by ICP-MS. Suspensions of a 1.5 9 106 cells digested in 3 ml of HNO3 or b 8 9 106 cells digested in 1 ml of HNO3, were diluted to 10 or 3 ml, respectively with ultrapure water before measuring iron by
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ICP-MS. Results are ±SEM of three independent experiments. ****, results are statistically different from those of cell-free blank (p \ 0.0001 as determined by t test)
appropriate. If p \ 0.05, the differences were considered to be statistically significant. Statistics were calculated with GraphPad Prism software (version 6.00).
Results Inductively coupled plasma-mass spectrometry (ICP-MS) measurement of iron in AGS cells This method lacked the sensitivity to discriminate between the level of iron in a pellet of 1.5 9 106 AGS cells (0.12–0.14 lmol Fe/l, mean 0.13 ± 0.003 lmol Fe/l) and a control blank containing only nitric acid diluted with ultrapure water (0.13 ± 0.003 lmol Fe/l) (Fig. 1a). However, when the number of AGS cells was increased to 8 9 106 digested in 1 ml of HNO3 (instead of 3 ml), and the resultant digest diluted to 3 ml instead of 10 ml, ICP-MS detected 1.46 ± 0.07 lmol Fe/l in the cells. This was significantly more (p \ 0.0001) than the level of iron detected in the blank (0.35 ± 0.003 lmol Fe/l; Fig. 1b). No iron was detected in the ultrapure water used in both assays (not shown). The small standard error of the means between these experiments suggested that ICP-MS is an extremely precise method for estimating iron concentration in AGS cells (Fig. 1). However, the need to standardise cell numbers prior to pelleting and digestion was associated with poor assay reproducibility that, in turn, meant no significant correlation was found between the number of AGS cells and total iron content (Fig. 2).
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Fig. 2 Poor correlation between cell numbers and iron concentration measured by ICP-MS. AGS cells were counted using a haemocytometer and digested prior to measurement of total iron levels by ICP-MS. The figure show results of three independent experiments done in triplicate. R2 and p value were obtained with the Pearson correlation test
The ferrozine assay is a sensitive colorimetric assay for measuring iron The measurement of iron standards was used to determine the relative sensitivity of each of the three colorimetric assays investigated as a means to measure iron levels in AGS cells. First, identical sets of standards that encompassed high (lmol) and low (nmol) concentrations of iron were treated with HCl or HCl/KFeCN to determine which of these two protocols demonstrated the greatest sensitivity at detecting iron over these ranges. As shown in Fig. 3a, the use of HCl to detect soluble iron was highly reproducible when the metal was present in the range of 0–6 lmol (p \ 0.0001). Moreover, HCl appeared a more accurate means of measuring iron when compared to
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Fig. 3 Detection of iron by concentrated hydrochloric acid with or without potassium ferrocyanide. Concentrated hydrochloric acid with (b, d) or without potassium ferrocyanide (a, c) was used to detect iron over lmol (a, b) and nmol (c, d) ranges. The correlation between iron content and absorbance is represented by R2 and p values (Pearson correlation test). Figures are representative of three independent experiments
duplicate sets of the higher range of standards treated with HCl/KFeCN method (p = 0.034; Fig. 3b). However, these methods proved to be less sensitive when the iron content of the standards was reduced to the range of nmol. In these experiments, the correlation between known iron content and absorbance was poor when the samples were treated with HCl (p = 0.081; Fig. 3c) and non-existent when the samples were treated with HCl/KFeCN (Fig. 3d). The third colorimetric assay investigated involved the use of ferrozine, which is a compound that binds ferrous (but not ferric) iron to produce a magentacoloured compound that absorbs light at a wavelength of 550 nm (Riemer et al. 2004). A standard curve between 0 and 24 nmol of iron was generated based on the measurement of total iron levels in AGS cells using ICPMS (Fig. 1) and UV/Visible spectrometry with HCl and HCl/KFeCN (Fig. 3). As shown in Fig. 4, there was a highly significant correlation between the concentration and detection of iron over the range tested (0–24 nmol; p \ 0.0001; Fig. 4a), including the lower end of the scale (0–1.5 nmol; p \ 0.0001; Fig. 4b). Using the ferrozine assay we found that the level of total iron in AGS cells was 2.170 ± 0.260 nmol of iron/mg protein. Moreover, iron levels in these cells increased dose-dependently in response to iron supplementation during culture (Fig. 5). We also
observed that the level of iron in H. pylori-infected AGS cells was significantly increased (p \ 0.05) when compared to uninfected cells (3.033 ± 0.281 and 2.170 ± 0.260 nmol of iron/mg protein, respectively; Fig. 6). This increase did not reflect the number of cell-associated bacteria remaining after washing (not shown). Accordingly, it was considered to signal a perturbation in cellular iron homeostasis in response to H. pylori infection that was independent of the extracellular iron concentration.
Discussion The AGS gastric epithelial cell line is widely used to model H. pylori infection in vitro and our goal was to determine if infection is able to perturb cellular iron homeostasis in these cells. To date, there have been no studies into the effect H. pylori infection has on iron metabolism in AGS cells however it is likely, given evidence that these bacteria reportedly affect iron homeostasis in a canine kidney (MDCK) cell line by increasing transferrin internalization (Tan et al. 2011). Accordingly, our objective was to identify an assay that would allow us to measure total iron content in AGS cells, and to use this assay to measure any infection-associated change.
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Fig. 4 Detection of iron by the ferrozine assay. Iron was measured over the range of 0–24 nmol by the ferrozine assay (a). The lower end of the curve is also shown (b). Results are
Fig. 5 Measurement of iron in AGS cells. AGS cells were supplemented with 0, 50, 100 or 200 lM FeSO4 for 24 h before iron levels were measured using the ferrozine assay. Results are ±SEM of three independent experiments. **, ****, results are statistically significantly different (p \ 0.01 and 0.0001 respectively; One-way ANOVA with Tukey’s multiple comparisons)
We evaluated four different methods to measure iron levels in AGS cells. These included inductively coupled plasma-mass spectrometry (ICP-MS), which involves spraying a solution containing iron into argon heated by electromagnetic fields to around 6,000 °C (inductively coupled plasma) that generates ions (that are then introduced to a quadrupole mass spectrometer) whose frequency is subsequently measured by an electron multiplier (Perkin-Elmer 2011). This technique is widely used to measure iron in biological samples (Li et al. 2012; Rodushkin et al. 2013) but in this setting, ICP-MS lacked the sensitivity to detect intracellular iron levels in AGS cells when cell numbers were low. In contrast, when cell numbers were increased (and total volume decreased), the assay proved to be extremely precise. However, the large
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representative of three independent experiments. R2 and p values are shown (Pearson correlation test)
Fig. 6 Measurement of iron in H. pylori-infected AGS cells. The ferrozine assay was used to measure total intracellular iron in uninfected AGS cells (Ctrl) and cells infected with H. pylori (Hp). Protein concentration was used to standardize cell numbers. Results are ±SEM of seven independent experiments carried out in duplicate or triplicate (p \ 0.05; t-test)
number of cells required to discriminate cellular iron from the background signal associated with sample digestion, coupled with poor assay reproducibility (that may reflect variable cell loss over the course of the assay) led us to consider another approach. Three colorimetric methods based on the generation of compounds that relate to the concentration of iron in a solution (LeVine et al. 1998; Riemer et al. 2004; Rad et al. 2007) were also investigated to measure iron levels in AGS cells. We first determined which of the three assays was capable of detecting low levels of iron because of evidence (albeit limited) that iron levels in gastric tissue are low in comparison to other cell types (Yaman et al. 2007; House et al. 2012). The use of HCl appeared more accurate than the potassium ferrocyanide method of measuring iron levels in AGS cells over the micromolar range.
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However, this accuracy was not sustained when the iron content of the standards was reduced to nmol. In contrast, the ferrozine assay was able to detect iron levels between 0.2 and 24 nmol, a finding that may in part reflect our use of a modified version of the original protocol. In the modified assay thiourea was added instead of neocuproin to reduce interference from copper (Smith et al. 1984; Paller and Hedlund 1994) and a vacuum concentrator was used to dry the samples at negative pressure while at the same time spinning them to avoid the loss of solutes (Fish 1988). An unrelated study reports that iron levels in astrocytes are approximately 10 nmol of iron per mg of protein (Hoepken et al. 2004) and using atomic absorption spectrometry, the levels of iron in brain (House et al. 2012) are reported to be between 4 and 12 times higher than those detected in gastric tissue (Yaman et al. 2007). Accordingly, our finding that AGS cells have 2.28 ± 0.28 nmol of iron per mg of AGS cell protein suggests this is likely to be within the expected range. Moreover, our observation of a concentration-dependent increase in intracellular iron levels in these cells in response to iron-supplementation during growth suggests our assay is specific as well as sensitive. H. pylori-infected cells were found to have notably higher levels of total iron than uninfected cells. This is intriguing because in the co-culture system used for these experiments the amount of iron available to cells may be reduced by the presence of bacteria but not increased. Thus, any changes in cellular iron levels that are independent of extracellular concentrations of iron must be explained by alterations in the control mechanism of cell iron homeostasis that include promoting iron uptake from the culture medium. This increase in total iron correlated with an increase in lysosomal labile iron, evidenced by the observation of a significant increase in iron deposits within intracellular compartments (not shown). A similar pattern of iron deposition was also observed in the iron-loaded (but uninfected) AGS cells. In summary, we compared the ability of four assays to measure iron levels in cultured gastric epithelial cells and determined that of the four, only the ferrozine assay was able to accurately measure iron at the low levels reportedly found in gastric tissue. Using this assay, we were also able to demonstrate that H. pylori infection increases total cellular iron in AGS cells. This supports the hypothesis that these bacteria affect
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the control of cellular iron metabolism. Furthermore, it may prove to be linked to the observation that H. pylori are able to persist for a lifetime in an untreated host. Acknowledgments
None
Conflict of interest The authors declare they have no conflict of interest.
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Measurement of total iron in Helicobacter pylori-infected gastric epithelial cells Sebastian E. Flores • Andrew S. Day Jacqueline I. Keenan
•
Received: 1 October 2014 / Accepted: 19 November 2014 / Published online: 27 November 2014 Ó Springer Science+Business Media New York 2014
Abstract Despite the evidence suggesting a role for Helicobacter pylori in the induction of systemic iron deficiency anaemia, little is known about the possibility of infection-associated changes in cellular iron homeostasis at the gastric epithelium. In this study we compared four different techniques for measuring iron in AGS cells, a gastric epithelial cell line that is widely used to model to H. pylori infection in vitro. Inductively coupled plasma-mass spectrometry proved to be an efficient method, but only when large numbers of cells were used. Two colorimetric assays that included the use of concentrated hydrochloric acid with or without potassium ferrocyanide detected iron in the micromolar but not the nanomolar range in cell-free standards. However, the third colorimetric assay that incorporated ferrozine proved to be highly accurate at detecting iron in the nanomolar range, and was able to detect iron in AGS cells, Moreover, using this assay, we were able to show that the level of iron in H. pyloriinfected AGS cells is significantly increased when compared to uninfected cells.
S. E. Flores J. I. Keenan (&) Department of Surgery, University of Otago Christchurch, PO Box 4345, Christchurch, New Zealand e-mail: [email protected] A. S. Day Department of Paediatrics, University of Otago Christchurch, Christchurch, New Zealand
Keywords Iron measurement Gastric epithelial cells Helicobacter pylori
Introduction Numerous clinical studies describe a link between Helicobacter pylori and iron deficiency (ID) or iron deficiency anaemia (IDA) (Choe et al. 1999; Valiyaveettil et al. 2005; Gessner et al. 2006) and the inference from these studies is that H. pylori accesses host iron in order to survive (and persist) in the human stomach. This iron needs to come from the bacterium’s immediate surroundings. Thus, any effect that H. pylori might have on host iron stores is likely to occur first at the gastric epithelium, where these bacteria persist for a lifetime in an untreated host (Kusters et al. 2006). Support for this hypothesis comes from several unrelated studies. Gastric epithelial cells from H. pyloriinfected mice demonstrate up-regulated transcription of lactoferrin (Lf) and ferritin (Mueller et al. 2004). The role of Lf is to sequester iron at mucosal surfaces, thus reducing its availability to bacteria (Alexander et al. 2012), whereas ferritin is an iron-storage protein whose expression is increased in response to high levels of iron and/or inflammation (Alkhateeb et al. 2013). There is evidence of Lf-associated internalisation of H. pylori into gastric epithelial cells (Coray et al. 2012) and increased Lf expression is seen in gastric tissue from patients infected with H. pylori (Dogan et al. 2012). To
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date, there are no reports of an H. pylori-associated change in ferritin levels in the human stomach, but an increase in cytosolic ferric iron is observed when a human gastric epithelial cell line is exposed to outer membrane vesicles constitutively shed from the surface of these bacteria (Chitcholtan et al. 2008). Collectively, these studies suggest that H. pylori may affect iron uptake, storage and/or distribution in gastric epithelial cells. The co-culture of AGS cells and H. pylori is widely used as an in vitro model to study this gastric infection, allowing the delineation of host-pathogen interactions without the confounders present in clinical or animal studies. Using this reductionist system, H. pylori is shown to adhere to the surface of these cells (Nilius et al. 1994) and induce effects that include nuclear factor (NF)-jB activation (Keates et al. 1997) and the up-regulation of epidermal growth factor receptor (EGFR) (Keates et al. 2007), two known inducers of H-ferritin mRNA expression that may contribute to increased iron storage in gastric epithelial cells (Konijn et al. 1999). A recent study found evidence of increased transferrin internalisation in H. pylori-infected polarized Madin Darby Canine Kidney (MDCK) cells (Tan et al. 2011) but to date any effect of H. pylori infection on gastric epithelial cell iron homeostasis has not been determined. We speculate that this may, in part, reflect the reportedly low levels of iron in gastric tissue (Yaman et al. 2007). Accordingly, the aim of our study was to evaluate different methods to measure total iron in AGS cells, and to use these method(s) to determine if H. pylori infection can alter the level of total intracellular iron.
Materials and methods Materials Nitric acid, hydrochloric acid and ammonium acetate were purchased from VWR International (Radnor, PA, USA), potassium hexacyanoferrate (II) trihydrate (potassium ferrocyanide), thiourea and 3-(2-Pyridyl)-5,6diphenyl-1,2,4-triazine-40 ,400 -disulfonic acid sodium salt (ferrozine) were obtained from Sigma, while potassium permanganate was obtained from Fisons Scientific Apparatus (Ipswich, UK). L-ascorbic acid was purchased from Duchefa Biochemie (Haarlem, The Netherlands) and the Multielement calibration standard used for ICP-
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MS was obtained from Agilent technologies (Santa Clara, CA, USA). Dulbecco’s PBS (D-PBS) and Trypsin–EDTA were from Life Technologies (Mulgrave, VIC, Australia), RPMI-1640 medium and fetal bovine serum (FBS) were from Invitrogen, (Auckland, NZ). Cell culture and treatment AGS cells, a human gastric adenocarcinoma cell line (CRL 1739; American Type Culture Collection, Manassas, VA), were cultured in RPMI-1640 medium supplemented with 10 % (v/v) heat-inactivated FBS. Cells (1 9 105) were grown (in six well plates) at 37 °C in air containing 5 % CO2, with cells cultured until they reached 80 % confluence (approximately 72 h). Fresh medium was added to each well before the addition (or not) of H. pylori for a further 15 h. Bacteria culture and enumeration H. pylori strain 60190 (ATCC 49503) is a well characterized clinical isolate that is cagA positive (Tummuru et al. 1993) and has a vacA s1m1 genotype (Atherton et al. 1995). The bacteria were routinely cultured on Columbia sheep blood agar plates (Fort Richard, Auckland, NZ) in a microaerophilic atmosphere at 37 °C for 2–3 days. Prior to co-culture with AGS cells, H. pylori were resuspended in RPMI-1640 medium supplemented with 10 % (v/v) heat-inactivated fetal bovine serum and bacterial numbers were quantitated by density, based on a standard curve measured at an absorbance of 620 nm (SpectraMaxÒ 190, Molecular devices, Sunnyvale, CA, USA). An absorbance of 0.2 was determined to be approximately 1 9 108 colony forming units (CFUs) per ml. H. pylori were added to cell cultures at a multiplicity of infection (MOI) of 10:1. Inductively coupled plasma-mass spectrometry (ICP-MS) AGS cells (*1.5 9 106) were transferred to 15 ml tubes and centrifuged at 1,500 g for 10 min. Cell pellets were washed twice with D-PBS and resuspended in 5 ml of D-PBS. Two 10 ll aliquots were removed from each tube and trypan blue-stained cells (0.25 % w/v in PBS) were counted using a haemocytometer. The remaining sample was centrifuged
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(1,500 g for 10 min) and the pelleted cells were digested overnight in 3 ml of 60 % nitric acid (HNO3) at 65 °C before being diluted to 10 ml with ultrapure water. The concentration of iron in each sample was determined using an Agilent 7700 ICP-mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). A multi-element calibration standard containing 10 mg of Fe/l was used as a reference sample (Agilent Technologies).
UV/Visible spectrometry with hydrochloric acid and potassium ferrocyanide A 10 mM iron standard stock was generated by dissolving 0.278 g of FeSO47H2O (equivalent to 1 mmol) in 100 ml of ultrapure water, with one drop of 10 M HCl added to the solution to prevent iron precipitation. The concentration of iron was confirmed by ICP-MS analysis, and the solution was used to generate two standard curves that ranged over micromolar (lmol) and nanomolar (nmol) concentrations of iron, respectively. Each standard (in duplicate) was dried overnight at 110 °C. 1 ml of 5 M hydrochloric acid (HCl) was added to every tube and the sample was digested for 4 h at 60 °C (with the lids on to avoid evaporation of the acid.) Each sample was then split into two 500 ll aliquots. An equal volume of 5 M HCl was added to one aliquot, and 500 ll of freshly prepared 5 % w/v potassium ferrocyanide was added to the second aliquot to generate iron blue and Prussian blue, respectively. The HCl-treated standards were added to the wells of a flat-bottomed clear 96 well plate and absorbance was read at 351 nm whereas the standards with added hydrochloric acid and potassium ferrocyanide were incubated for 35 min in the dark before absorbance was read at 700 nm (Rad, Janic et al. 2007). Each assay was read using a SpectraMaxÒ 190 absorbance microplate reader (Molecular devices, Sunnyvale, CA, USA).
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(Thermo Fisher Scientific, Waltham, MA, USA), as detailed by (Fish 1988) before 200 ll of a freshly prepared digestion reagent (a 1:1 solution of 1.4 M HCl and 4.5 % (w/v) potassium permanganate) was added to each tube. The samples were digested for 2 h at 60 °C, then cooled to room temperature before the addition of 50 ll of a detection reagent (6.5 mM ferrozine, 2.5 M ammonium acetate, 1 M ascorbic acid and 2 mM thiourea). Each tube was vortexed and incubated for 30 min before the samples were transferred to a clear 96 well plate. Absorbances were read at 564 nm with a SpectraMaxÒ 190 absorbance microplate reader (Davis et al. 1986; Riemer et al. 2004).
Measurement of iron in AGS cells Total AGS cell intracellular iron levels were measured by the ferrozine assay (Riemer et al. 2004) with the modification proposed by (Fish 1988). AGS cells (1 9 105) were cultured for 72 h in six-well plates, washed with D-PBS and fresh medium was added before the addition (or not) of H. pylori for 15 h. After extensive washing, the cells were lifted with trypsin– EDTA. The pelleted cells were lysed by incubation with 200 ll NaOH (50 mM) for 2 h at 37 °C, and two 10 ll aliquots were taken to measure protein concentration (Lowry, Rosebrough et al. 1951). The remaining lysate was dried at 45 °C using a SpeedVacTM SPD111 V vacuum concentrator (Thermo Fisher Scientific, Waltham, MA) before addition of 200 ll of digestion reagent (see above) for 2 h at 60 °C. Samples were then cooled to room temperature, before the addition of 50 ll of a detection reagent (as detailed above). Absorbances were read at 564 nm and iron values were calculated against a standard curve. For the dose–response curve, AGS cells were supplemented with 0, 50, 100 or 200 lM FeSO4 for 24 h before the ferrozine assay was used to measure cellular iron levels.
Ferrozine assay Statistical analysis A standard curve ranging from 0 to 24 nmol of iron was generated from a 1:100 dilution of the iron stock solution (see above) in 1.6 ml Eppendorf tubes (in duplicate). The aliquots were fully dried at 45 °C using a SpeedVacTM SPD111 V vacuum concentrator
Results are presented as the ±SE of the means of at least three independent experiments. Data were analysed by Pearson correlation, ANOVA followed by Tukey’s post hoc test or non-parametric t test, as
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Fig. 1 Total iron in AGS cells measured by ICP-MS. Suspensions of a 1.5 9 106 cells digested in 3 ml of HNO3 or b 8 9 106 cells digested in 1 ml of HNO3, were diluted to 10 or 3 ml, respectively with ultrapure water before measuring iron by
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ICP-MS. Results are ±SEM of three independent experiments. ****, results are statistically different from those of cell-free blank (p \ 0.0001 as determined by t test)
appropriate. If p \ 0.05, the differences were considered to be statistically significant. Statistics were calculated with GraphPad Prism software (version 6.00).
Results Inductively coupled plasma-mass spectrometry (ICP-MS) measurement of iron in AGS cells This method lacked the sensitivity to discriminate between the level of iron in a pellet of 1.5 9 106 AGS cells (0.12–0.14 lmol Fe/l, mean 0.13 ± 0.003 lmol Fe/l) and a control blank containing only nitric acid diluted with ultrapure water (0.13 ± 0.003 lmol Fe/l) (Fig. 1a). However, when the number of AGS cells was increased to 8 9 106 digested in 1 ml of HNO3 (instead of 3 ml), and the resultant digest diluted to 3 ml instead of 10 ml, ICP-MS detected 1.46 ± 0.07 lmol Fe/l in the cells. This was significantly more (p \ 0.0001) than the level of iron detected in the blank (0.35 ± 0.003 lmol Fe/l; Fig. 1b). No iron was detected in the ultrapure water used in both assays (not shown). The small standard error of the means between these experiments suggested that ICP-MS is an extremely precise method for estimating iron concentration in AGS cells (Fig. 1). However, the need to standardise cell numbers prior to pelleting and digestion was associated with poor assay reproducibility that, in turn, meant no significant correlation was found between the number of AGS cells and total iron content (Fig. 2).
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Fig. 2 Poor correlation between cell numbers and iron concentration measured by ICP-MS. AGS cells were counted using a haemocytometer and digested prior to measurement of total iron levels by ICP-MS. The figure show results of three independent experiments done in triplicate. R2 and p value were obtained with the Pearson correlation test
The ferrozine assay is a sensitive colorimetric assay for measuring iron The measurement of iron standards was used to determine the relative sensitivity of each of the three colorimetric assays investigated as a means to measure iron levels in AGS cells. First, identical sets of standards that encompassed high (lmol) and low (nmol) concentrations of iron were treated with HCl or HCl/KFeCN to determine which of these two protocols demonstrated the greatest sensitivity at detecting iron over these ranges. As shown in Fig. 3a, the use of HCl to detect soluble iron was highly reproducible when the metal was present in the range of 0–6 lmol (p \ 0.0001). Moreover, HCl appeared a more accurate means of measuring iron when compared to
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Fig. 3 Detection of iron by concentrated hydrochloric acid with or without potassium ferrocyanide. Concentrated hydrochloric acid with (b, d) or without potassium ferrocyanide (a, c) was used to detect iron over lmol (a, b) and nmol (c, d) ranges. The correlation between iron content and absorbance is represented by R2 and p values (Pearson correlation test). Figures are representative of three independent experiments
duplicate sets of the higher range of standards treated with HCl/KFeCN method (p = 0.034; Fig. 3b). However, these methods proved to be less sensitive when the iron content of the standards was reduced to the range of nmol. In these experiments, the correlation between known iron content and absorbance was poor when the samples were treated with HCl (p = 0.081; Fig. 3c) and non-existent when the samples were treated with HCl/KFeCN (Fig. 3d). The third colorimetric assay investigated involved the use of ferrozine, which is a compound that binds ferrous (but not ferric) iron to produce a magentacoloured compound that absorbs light at a wavelength of 550 nm (Riemer et al. 2004). A standard curve between 0 and 24 nmol of iron was generated based on the measurement of total iron levels in AGS cells using ICPMS (Fig. 1) and UV/Visible spectrometry with HCl and HCl/KFeCN (Fig. 3). As shown in Fig. 4, there was a highly significant correlation between the concentration and detection of iron over the range tested (0–24 nmol; p \ 0.0001; Fig. 4a), including the lower end of the scale (0–1.5 nmol; p \ 0.0001; Fig. 4b). Using the ferrozine assay we found that the level of total iron in AGS cells was 2.170 ± 0.260 nmol of iron/mg protein. Moreover, iron levels in these cells increased dose-dependently in response to iron supplementation during culture (Fig. 5). We also
observed that the level of iron in H. pylori-infected AGS cells was significantly increased (p \ 0.05) when compared to uninfected cells (3.033 ± 0.281 and 2.170 ± 0.260 nmol of iron/mg protein, respectively; Fig. 6). This increase did not reflect the number of cell-associated bacteria remaining after washing (not shown). Accordingly, it was considered to signal a perturbation in cellular iron homeostasis in response to H. pylori infection that was independent of the extracellular iron concentration.
Discussion The AGS gastric epithelial cell line is widely used to model H. pylori infection in vitro and our goal was to determine if infection is able to perturb cellular iron homeostasis in these cells. To date, there have been no studies into the effect H. pylori infection has on iron metabolism in AGS cells however it is likely, given evidence that these bacteria reportedly affect iron homeostasis in a canine kidney (MDCK) cell line by increasing transferrin internalization (Tan et al. 2011). Accordingly, our objective was to identify an assay that would allow us to measure total iron content in AGS cells, and to use this assay to measure any infection-associated change.
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Fig. 4 Detection of iron by the ferrozine assay. Iron was measured over the range of 0–24 nmol by the ferrozine assay (a). The lower end of the curve is also shown (b). Results are
Fig. 5 Measurement of iron in AGS cells. AGS cells were supplemented with 0, 50, 100 or 200 lM FeSO4 for 24 h before iron levels were measured using the ferrozine assay. Results are ±SEM of three independent experiments. **, ****, results are statistically significantly different (p \ 0.01 and 0.0001 respectively; One-way ANOVA with Tukey’s multiple comparisons)
We evaluated four different methods to measure iron levels in AGS cells. These included inductively coupled plasma-mass spectrometry (ICP-MS), which involves spraying a solution containing iron into argon heated by electromagnetic fields to around 6,000 °C (inductively coupled plasma) that generates ions (that are then introduced to a quadrupole mass spectrometer) whose frequency is subsequently measured by an electron multiplier (Perkin-Elmer 2011). This technique is widely used to measure iron in biological samples (Li et al. 2012; Rodushkin et al. 2013) but in this setting, ICP-MS lacked the sensitivity to detect intracellular iron levels in AGS cells when cell numbers were low. In contrast, when cell numbers were increased (and total volume decreased), the assay proved to be extremely precise. However, the large
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representative of three independent experiments. R2 and p values are shown (Pearson correlation test)
Fig. 6 Measurement of iron in H. pylori-infected AGS cells. The ferrozine assay was used to measure total intracellular iron in uninfected AGS cells (Ctrl) and cells infected with H. pylori (Hp). Protein concentration was used to standardize cell numbers. Results are ±SEM of seven independent experiments carried out in duplicate or triplicate (p \ 0.05; t-test)
number of cells required to discriminate cellular iron from the background signal associated with sample digestion, coupled with poor assay reproducibility (that may reflect variable cell loss over the course of the assay) led us to consider another approach. Three colorimetric methods based on the generation of compounds that relate to the concentration of iron in a solution (LeVine et al. 1998; Riemer et al. 2004; Rad et al. 2007) were also investigated to measure iron levels in AGS cells. We first determined which of the three assays was capable of detecting low levels of iron because of evidence (albeit limited) that iron levels in gastric tissue are low in comparison to other cell types (Yaman et al. 2007; House et al. 2012). The use of HCl appeared more accurate than the potassium ferrocyanide method of measuring iron levels in AGS cells over the micromolar range.
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However, this accuracy was not sustained when the iron content of the standards was reduced to nmol. In contrast, the ferrozine assay was able to detect iron levels between 0.2 and 24 nmol, a finding that may in part reflect our use of a modified version of the original protocol. In the modified assay thiourea was added instead of neocuproin to reduce interference from copper (Smith et al. 1984; Paller and Hedlund 1994) and a vacuum concentrator was used to dry the samples at negative pressure while at the same time spinning them to avoid the loss of solutes (Fish 1988). An unrelated study reports that iron levels in astrocytes are approximately 10 nmol of iron per mg of protein (Hoepken et al. 2004) and using atomic absorption spectrometry, the levels of iron in brain (House et al. 2012) are reported to be between 4 and 12 times higher than those detected in gastric tissue (Yaman et al. 2007). Accordingly, our finding that AGS cells have 2.28 ± 0.28 nmol of iron per mg of AGS cell protein suggests this is likely to be within the expected range. Moreover, our observation of a concentration-dependent increase in intracellular iron levels in these cells in response to iron-supplementation during growth suggests our assay is specific as well as sensitive. H. pylori-infected cells were found to have notably higher levels of total iron than uninfected cells. This is intriguing because in the co-culture system used for these experiments the amount of iron available to cells may be reduced by the presence of bacteria but not increased. Thus, any changes in cellular iron levels that are independent of extracellular concentrations of iron must be explained by alterations in the control mechanism of cell iron homeostasis that include promoting iron uptake from the culture medium. This increase in total iron correlated with an increase in lysosomal labile iron, evidenced by the observation of a significant increase in iron deposits within intracellular compartments (not shown). A similar pattern of iron deposition was also observed in the iron-loaded (but uninfected) AGS cells. In summary, we compared the ability of four assays to measure iron levels in cultured gastric epithelial cells and determined that of the four, only the ferrozine assay was able to accurately measure iron at the low levels reportedly found in gastric tissue. Using this assay, we were also able to demonstrate that H. pylori infection increases total cellular iron in AGS cells. This supports the hypothesis that these bacteria affect
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the control of cellular iron metabolism. Furthermore, it may prove to be linked to the observation that H. pylori are able to persist for a lifetime in an untreated host. Acknowledgments
None
Conflict of interest The authors declare they have no conflict of interest.
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