The use of the Ussing chamber system to investigate iron absorption by the duodenum, jejunum and ileum in the mouse : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Physiology at Massey University, Palmerston North, New Zealand

Abstract

Iron deficiency anaemia is found in approximately 30% of the worlds population and is particularly prevalent in developing countries. The majority of these deficiencies are due to insufficient absorption of iron from the diet. Iron is absorbed primarily by the proximal small intestine, however, there is evidence for a gradient of absorption along the full length of the small intestine. In 1951 Ussing and Zerahn developed a bicameral method for studying iron transport by in vitro epithelia. This method has been used previously to investigate iron transport mechanisms in the proximal small intestine. In the present study Ussing chambers were used to investigate iron absorption by the full length of the mouse small intestine. Consistently high levels of iron were removed from the mucosal compartment by all regions of the small intestine. This iron removal was due to the physiological actions of the tissue and was not caused by iron adhering to the interior of the Ussing chamber apparatus. There was no change in iron uptake when large intestine or caecum was used in place of small intestine. Ferrous gluconate was chosen as the reference test chemical as it is a readily bioavailable form of iron which has been used previously to investigate iron absorption with the Ussing chamber model. There was a consistently high level of iron uptake when 27.9 mg/L or 9.3 mg/L was added to the mucosal compartment, with no significant differences between results for either concentration. When 9.15 mg/L manganese sulphate was combined with 9.3 mg/L ferrous gluconate in the mucosal compartment, iron removal was significantly lower in the proximal than the mid small intestine. This was presumably due to competition between the iron and the manganese for transport by the DCT1 protein. When 200 mg/L calcium chloride and 9.3 mg/L ferrous gluconate were added to the mucosal compartment, there was no significant difference to results compared to ferrous gluconate alone. The addition of glucose to the intestinal lumen has been shown previously to increase the passive transport of solutes across the intestinal mucosa. However, in the present experiments when glucose was added to the mucosal Ringer's solution in place of mannitol there was a significant decrease in iron removed from the mucosal compartment by all intestinal regions. There was evidence that the gluconate portion of ferrous gluconate increased iron absorption in the distal small intestine. This was supported by a significant decrease in iron uptake by the distal small intestine when ferrous sulphate was used in place of ferrous gluconate. Ferric chloride was unsuitable for use in this system as it precipitated out of the Ringer's solution. Histological examination of jejunal samples after a typical Ussing chamber experiment found there was no damage to the tissue and the epithelial layer remained intact. There were significant levels of iron found in both the intestinal tissue and secreted mucus for all intestinal segments. The binding of iron to secreted mucus appears to involve a significant proportion of iron and should be measured in all future Ussing chamber studies