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. pylori-infected AGS cells is significantly increased when compared to uninfected cells.
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Alexander DB, Iigo M, Yamauchi K, Suzui M, Tsuda H (2012) Lactoferrin: an alternative view of its role in human biological fluids. Biochem Cell Biol 90(3):279–306
Alkhateeb AA, Connor JR (2013) The significance of ferritin in cancer: anti-oxidation, inflammation and tumorigenesis. Biochim Biophys Acta 1836(2):245–254
Atherton JC, Cao P, Peek RM Jr, Tummuru MK, Blaser MJ, Cover TL (1995) Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem 270(30):17771–17777
Chitcholtan K, Hampton MB, Keenan JI (2008) Outer membrane vesicles enhance the carcinogenic potential of Helicobacter pylori. Carcinogenesis 29(12):2400–2405
Choe YH, Kim SK, Son BK, Lee DH, Hong YC, Pai SH (1999) Randomized placebo-controlled trial of Helicobacter pylori eradication for iron-deficiency anemia in preadolescent children and adolescents. Helicobacter 4(2):135–139
Coray DS, Heinemann JA, Tyrer PC, Keenan JI (2012) Human lactoferrin increases Helicobacter pylori internalisation into AGS cells. World J Microbiol Biotechnol 28(5):1871–1880
Davis MD, Kaufman S, Milstien S (1986) A modified ferrozine method for the measurement of enzyme-bound iron. J Biochem Biophys Methods 13(1):39–45
Dogan Y, Erkan T, Onal Z, Usta M, Dogusoy G, Cokugras FC, Kutlu T (2012) Lactoferrin levels in the gastric tissue of Helicobacter pylori-positive and -negative patients and its effect on anemia. Mediators Inflamm 2012:214581
Fish WW (1988) Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples. Methods Enzymol 158:357–364
Gessner BD, Baggett HC, Muth PT, Dunaway E, Gold BD, Feng Z, Parkinson AJ (2006) A controlled, household-randomized, open-label trial of the effect that treatment of Helicobacter pylori infection has on iron deficiency in children in rural Alaska. J Infect Dis 193(4):537–546
Hoepken HH, Korten T, Robinson SR, Dringen R (2004) Iron accumulation, iron-mediated toxicity and altered levels of ferritin and transferrin receptor in cultured astrocytes during incubation with ferric ammonium citrate. J Neurochem 88(5):1194–1202
House E, Esiri M, Forster G, Ince PG, Exley C (2012) Aluminium, iron and copper in human brain tissues donated to the Medical Research Council’s Cognitive Function and Ageing Study. Metallomics 4(1):56–65
Keates S, Hitti YS, Upton M, Kelly CP (1997) Helicobacter pylori infection activates NF-kappa B in gastric epithelial cells. Gastroenterology 113(4):1099–1109
Keates S, Keates AC, Katchar K, Peek RM Jr, Kelly CP (2007) Helicobacter pylori induces up-regulation of the epidermal growth factor receptor in AGS gastric epithelial cells. J Infect Dis 196(1):95–103
Konijn AM, Glickstein H, Vaisman B, Meyron-Holtz EG, Slotki IN, Cabantchik ZI (1999) The cellular labile iron pool and intracellular ferritin in K562 cells. Blood 94(6):2128–2134
Koorts AM, Viljoen M (2007) Ferritin and ferritin isoforms I: structure-function relationships, synthesis, degradation and secretion. Arch Physiol Biochem 113(1):30–54
Kusters JG, van Vliet AH, Kuipers EJ (2006) Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 19(3):449–490
LeVine SM, Wulser MJ, Lynch SG (1998) Iron quantification in cerebrospinal fluid. Anal Biochem 265(1):74–78
Li G, Brockman J, Lin S, Schell L, Robertson J (2012) Measurement of the trace elements Cu, Zn, Fe, and Mg and the ultratrace elements Cd Co, Mn, and Pb in limited quantity human plasma and serum samples by inductively coupled plasma-mass Spectrometry. Am J Anal Chem 3(9):5
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275
Mueller A, Merrell DS, Grimm J, Falkow S (2004) Profiling of microdissected gastric epithelial cells reveals a cell type-specific response to Helicobacter pylori infection. Gastroenterology 127(5):1446–1462
Muhsen K, Cohen D (2008) Helicobacter pylori infection and iron stores: a systematic review and meta-analysis. Helicobacter 13(5):323–340
Nilius M, Bode G, Buchler M, Malfertheiner P (1994) Adhesion of Helicobacter pylori and Escherichia coli to human and bovine surface mucus cells in vitro. Eur J Clin Invest 24(7):454–459
Paller MS, Hedlund BE (1994) Extracellular iron chelators protect kidney cells from hypoxia/reoxygenation. Free Radic Biol Med 17(6):597–603
Perkin-Elmer (2011) The 30 minute guide to ICP-MS. Technical note: mass spectrometry. www.perkinelmer.com
Rad AM, Janic B, Iskander AS, Soltanian-Zadeh H and Arbab AS (2007). Measurement of quantity of iron in magnetically labeled cells: comparison among different UV/Vis spectrometric methods. Biotechniques 43(5): 627–628, 630, 632
Riemer J, Hoepken HH, Czerwinska H, Robinson SR, Dringen R (2004) Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells. Anal Biochem 331(2):370–375
Rodushkin I, Engstrom E, Baxter DC (2013) Isotopic analyses by ICP-MS in clinical samples. Anal Bioanal Chem 405(9):2785–2797
Smith FE, Herbert J, Gaudin J, Hennessy DJ, Reid GR (1984) Serum iron determination using ferene triazine. Clin Biochem 17(5):306–310
Tan S, Noto JM, Romero-Gallo J, Peek RM Jr, Amieva MR (2011) Helicobacter pylori perturbs iron trafficking in the epithelium to grow on the cell surface. PLoS Pathog 7(5):e1002050
Tummuru MK, Cover TL, Blaser MJ (1993) Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production. Infect Immun 61(5):1799–1809
Valiyaveettil AN, Hamide A, Bobby Z, Krishnan R (2005) Effect of anti-Helicobacter pylori therapy on outcome of iron-deficiency anemia: a randomized, controlled study. Indian J Gastroenterol 24(4):155–157
Yaman M, Kaya G, Yekeler H (2007) Distribution of trace metal concentrations in paired cancerous and non-cancerous human stomach tissues. World J Gastroenterol 13(4):612–618
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Flores, S.E., Day, A.S. & Keenan, J.I. Measurement of total iron in Helicobacter pylori-infected gastric epithelial cells. Biometals 28, 143–150 (2015). https://doi.org/10.1007/s10534-014-9810-z
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DOI: https://doi.org/10.1007/s10534-014-9810-z