Date of Award
Doctor of Philosophy (PhD)
Dr. G.D. Sweeney
In recent years attention has been drawn to the possible role of iron in a number pathological conditions including ischemia/reperfusion, halogenated aryl hydrocarbon toxicity as well as hereditary and transfusion-dependent iron overload. Although several different mechanisms may be operating in each of these situations, one of the chemical processes thought to be involved is lipid peroxidation (LP). LP is the free radical-mediated, oxidation of polyunsaturated fatty acids which has the potential to cause membrane, protein and nucleic acid damage. Using the non-invasive technique of measuring whole-body ethane and pentane production as an index of in vivo LP in mice, it is shown here that the ferric-NTA complex (Fe³⁺-NTA) is the most potent stimulus of this process yet described. Fe³⁺-NTA was found to be lethal to mice at doses above 5 mg Fe³⁺/kg body weight with a dose of 7.5 mg Fe³⁺/kg, giving rise to a greater than 750-fold rise in the rate of ethane and pentane production 30 min following treatment of mice.
Liver and kidney were identified as likely sites of alkane origination. Radioactive iron (presented as ⁵⁹Fe³⁺-NTA) was concentrated primarily in the liver, and, to a lesser extent, in the kidney. In addition, estimation of products of lipid peroxidation with the 2-thiobarbituric acid (TBA) test confirmed liver and kidney as locations where Fe³⁺-NTA-stimulated LP had occurred in mice treated with the iron complex. Further work, therefore, examined the mechanism by which Fe³⁺-NTA promotes LP in liver tissue.
Isolated rat hepatocytes were found to be susceptible to Fe³⁺-NTA-dependent LP when the process was monitored by measuring the formation of ethane and TBA reacting compounds or emission of low level chemiluminescence. LP and iron association with cells was found to be temperature dependent, both processes being inhibited by incubation at 4°. Subcellular fractionation of rat liver indicated that Fe³⁺-NTA was capable of promoting LP in both mitochondrial and microsomal (primarily, endoplasmic reticulum) only in the presence of reduced pyridine nucleotides (NADH or NADPH). In this, Fe³⁺-NTA was similar to previous reports regarding the ferric-adenosine diphosphate complex (Fe³⁺-ADP).
In depth comparison of the promotion of microsomal LP by Fe³⁺-NTA and Fe³⁺-ADP suggested a common biochemical mechanism, central to which is the reduction of the ferric complex to ferrous. This led to an examination of the initiation of microsomal peroxidation by "free" ferrous ions. In contrast to NADPH/Fe³⁺-NTA-dependent LP which is very rapid, Fe²⁺ addition to microsomal suspensions caused LP only after a variable lag period. This delay could be reduced or abolished by simultaneous addition of Fe³⁺ or by conditions which would accelerate the oxidation of Fe²⁺ to Fe³⁺. In contrast, the delay Iength was increased by antioxidants which acted by hydrogen donation or one electron transfer indicating that the delay period represents time required for the formation of a species capable of initiating microsomal LP. Furthermore, additional Fe²⁺ was also found to increase the delay length. It is proposed that the mechanism behind the delayed initiation of LP is one electron transfer from excess Fe²⁺ to an as yet unidentified initiating species (possibly and Fe²⁺-O₂-Fe³⁺ complex) thus quenching it. Lipid peroxidation is then initiated following consumption of the surplus Fe²⁺. The lack of a delay in NADPH/Fe³⁺-complex dependent LP would be due to the rapid formation of an optimal ratio of Fe²⁺ : Fe³⁺; however, the mechanism of initiation of LP would appear to be similar.
The work presented in this thesis demonstrates the remarkable potential of small amounts of iron, when presented appropriately, to stimulate in vivo lipid peroxidation on a massive scale and with apparently lethal consequences. Furthermore the common mechanism of initiation of LP by iron complexes and the observation that one electron reduction prevents the initial step(s) in LP may prove invaluable in the development of antioxidant drugs for the prevention of iron-dependent cellular damage.
Goddard, John Graham, "In Vivo and In Vitro Studies on The Mechanism of Iron-Dependent Lipid Peroxidation in Liver" (1989). Open Access Dissertations and Theses. Paper 1896.