Subhosmito Chakraborty, (2009) Effect of alcohol ingestion on proteins involved in iron transport in mouse duodenum. Masters thesis, Christian Medical College, Vellore.
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Abstract
HYPOTHESIS OF THE STUDY: Alcohol ingestion increases the expression and function of molecules that are involved in the absorption of non-haem iron from the gastrointestinal tract, thereby leading to iron overload. OBJECTIVES OF THE STUDY: The aim of this study was to determine the effect of alcohol ingestion on proteins in the mouse duodenum that are involved in transport of non-haem iron from the intestinal lumen into the systemic circulation. This was to be done by determining expression levels of 4 proteins of interest, namely, divalent metal transporter1 (DMT1), duodenal cytochrome reductase (dcytb), ferroportin and hephaestin. MATERIALS AND METHODOLOGY: MATERIALS : Equipment used: 1. Elix and Milli-Q ultrapure water systems (Millipore, USA) 2. pH meter (Systronics, India) 3. Refrigerated centrifuge (MPW R 350, MPW Poland) 4. Glass homogenizer and teflon pestle (1 ml capacity) (Kimble-Kontes, USA), 5. Mechanical homogenizer (REMI, Bombay, India), 6. Apparatus for SDS-PAGE and western blotting (Bio-Rad, USA), 7. Gel documentation system (Alpha Innotech, USA), 8. Real time thermocycler (Chromo4, Biorad, USA). Chemicals and reagents used: 1. Acrylamide, ammonium persulfate, -actin antibody, -mercaptoethanol, bovine serum albumin (BSA), chloroform, diethyl pyrocarbonate (DEPC), ethidium bromide, ferric chloride hexahydrate, guanidine hydrochloride, isopropanol, sodium lauryl sulphate (SDS) and TRI reagent were obtained from Sigma, India. 2. Casein (SRL, India) , 3. Disodium hydrogen phosphate, potassium chloride, potassium dihydrogen phosphate and sodium acetate were obtained from Sisco Research Laboratories, Pvt Ltd, Mumbai, India. 4. Absolute alcohol (99.9%) (Hayman, England), 5. Agarose (Genei, Bangalore, India), 6. 3 – Morpholinopropane sulfonic acid (MOPS) (Fluka Biochemika, Sigma, India) 7. Reverse transcription core kit, SYBR green PCR master mix kit and gene-specific primers were obtained from Eurogentec, Belgium. 8. Methanol (Qualigens), 9. Skimmed milk powder (Sagar, India), 10. Anti-DMT1 antibody (Santa Cruz, USA), 11. Anti-ferroportin antibody (Alpha Diagnostics International,USA) 12. Goat anti-mouse and anti-rabbit peroxidase-conjugated antibodies and chemiluminescent substrate were purchased from Pierce International, USA. All chemicals used were of analytical grade. METHODOLOGY: Animals used: Swiss albino male mice (weighing around 30 g), obtained from the animal house, Christian Medical College, Vellore, were used for all the experiments. They were divided into pairs of age and weight-matched control and test animals. The pairs were allotted for study periods of 1, 2 and 4 weeks. The experiments done were approved by Institutional Review Board (Approval No. 6589 dated 11th June, 2008) and the Institutional Animal Ethics Committee (IAEC) and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA), Government of India (Approval No.7/2009 dated 27th May, 2009). SUMMARY: The preliminary results of this study showed that administration of ethanol caused up-regulation of DMT1 (the apical iron transporter), in the mouse duodenum after 1 and 2 weeks of treatment. Ferroportin (the basolateral iron exporter), dcytb (the apical ferrireductase) and hephaestin (the basolateral ferroxidase) were found to be unaffected by the treatment. Alcohol administration for the periods of study did not cause significant alterations in serum iron levels at the time periods studied. LIMITATIONS OF THE STUDY: 1. It has not been possible, within the time frame of the current study, to determine the effect of alcohol on hepatic hepcidin expression. This will, however, be carried out shortly. 2. It has not been possible to measure serum hepcidin levels, due to non-availability of commercially available reagents to measure it in mouse serum. 3. It has, thus, not been possible to correlate the findings of this study with levels of hepcidin, which is known to be a central regulator of iron homeostasis.
| Item Type: | Thesis (Masters) |
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| Uncontrolled Keywords: | alcohol ingestion, proteins, in iron transport, mouse duodenum. |
| Subjects: | MEDICAL > Biochemistry |
| Depositing User: | Subramani R |
| Date Deposited: | 09 Jul 2020 16:25 |
| Last Modified: | 09 Jul 2020 16:25 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/12516 |
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