CHAPTER THREE - LIPID PEROXIDA TION
3.2 EFFECT OF METRIFONATE ON QUINOLINIC ACID INDUCED CHANGES IN RAT BRAIN HOMOGENATE
3.2.1 INTRODUCTION
The kynurenine pathway (KP) is a major route of L-tryptophan catabolism leading to production of a number of biologically active molecules. Quinolinic acid (2, 3-pyridine dicarboxylic acid) (QA) is a heterocyclic amino acid (Wolfensberger et aI., 1983) and is a neurotoxic metabolite of the tryptophan - kynurenine pathway (Heyes and Morrison, 1997). It is present in the human and rat brain in nanomolar concentrations (Stone, 1993).
This excitotoxin is synthesized in the liver and CNS (Flanagan et at., 1995) and has been reported to be present in the brain in an uneven distribution pattern. Quinolinic acid is considered to be involved in the pathogenesis of a number of inflammatory neurological diseases. Quinolinic acid induced neurotoxicity closely mimics the patterns of selective nerve cell loss obseverd in Huntington's disease and temporal lobe epilepsy (Martin and Beal, 1992) A major aspect of QA toxicity is lipid peroxidation its markers in the brain, found in Alzheimer's disease, (Guilleman and Brew, 2002). The following experiment was conducted to investigate the effect of QA on lipid peroxidation in rat brain homogenate. QA induced neurotoxicity results from the activation of ion channels through which sodium, potassium and calcium flood into the cell (Stone, 1993). The increased intracellular calcium concentration sets of a cascade of events that culminate in the generation of free radicals. Free radicals are especially toxic because these initiate lipid destroying chain reactions (Halliwell and Chirico, 1993).
Metrifonate is an acetylcholinesterase inhibitor used in the symptomatic treatment of Alzheimer's disease. This experiment aimed to investigate whether metrifonate has any neuroprotective properties, i.e. if it can protect rat brain homogenate from QA induced neurotoxicity and free radical damage.
THE THIOBARBITURIC (TBA) - MALONDIALDEHYDE (MDA) ASSAY
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Lipid peroxidation often occurs in response to oxidative stress, and a great diversity of aldehydes is formed when lipid hydroperoxides break down in biological systems. Some of these aldehydes are highly reactive and may be considered as second toxic messengers which disseminate and augment initial free radical events (Esterbaur, 1991). The aldehydes most intensively studied so far are 4-hydroxynonenal, 4-hydroxyhexenal, and malondialdehyde (Esterbaur, 1991). Malondialdehyde is a reliable indicator of free radical·induced damage (Reiter et ai, 1995; Yamamoto, 1990; Wang et al., 1996).
A widely used technique in the determination ofperoxidation of tissue, is the thiobarbituric assay (TBA). Malondialdehyde (MDA), is an end product of lipid
peroxidation (Wang el al., 1996), and is the most abundant individual aldehyde resulting from lipid peroxidation and it has been shown to be capable of altering proteins, DNA, RNA and other biomolecules in vitro (Schaunenstein, 1977).
This test is based on the reaction of one molecule of MDA with two molecules of TBA, resulting in the formation of a pink chromogen (shown in fig. 3.2. above ). This complex has an absorption maximum at 532nm. The reaction takes place in acidic conditions (pH of2 - 3) at 90 - 100°C for one hour.
Chapter 3 - Lipid Peroxidation
Butylated hydroxytoluene (BHT) in methanol (0.5% w/v) is added to stop oxidative processes during the assay. Trichloroacetic acid (TCA) in Milli-Q water (10% w/v) is added to the tissue sample. TCA is also used to precipitate protein, which is pelleted by centrifugation. An aliquot of the supernatant is allowed to react with TBA in Milli-Q water (0.33% w/v) in a boiling water bath for one hour. After cooling, and extracting with butanol, the absorbance is read at 532nm and the concentration ofMDA determined from a standard curve generated from 1, 1,3,3-tetramethoxypropane.
3.2.2 MATERIALS AND METHODS
3.2.2.1 Animals
Adult male Wistar rats weighing between 250 and 300 g were used in this experiment, and were housed and maintained under the conditions described in section 2.2.2.1.
3.2.2.2 Chemicals and Reagents
1,1,3,3-Tetramethoxypropane (MDA) was obtained from Fluka AG, Switzerland.
Butylated hydroxytoluene (BHT), 2-thiobarbituric acid (TBA) and QA and Metrifonate were purchased from Sigma Chemical Co, SI. Louis, USA. Trichloroacetic acid and butanol were purchased from Saarchem (pTY) Ltd., Krugersdorp, South Africa.
3.2.2.3 Brain Removal
The brains were surgically removed and either used immediately or stored at -70 0 C as described earlier in section 2.2.2.3.
3.2.2.4 Preparation of the homogenate
The rats were sacrificed by cervical dislocation and the brains were surgically removed and homogenised in the same manner described in section 2.2.2.4.
3.2.2.5 Preparation of the standard curve
A series of standards (0-20 nmol/ml with 5nmol/ml intervals) was prepared using 1,1,3,3- tetramethoxypropane (MDA) and a standard curve generated by measuring the absorbance at 532nm using a GBC UVNIS 916 spectrophotometer and plotted against the molar equivalent weight ofMDA in the complex assayed (appendix 3).
3.2.2.6 Lipid Peroxidation
A modification of the method of Placer et ai., (1966), was used in this experiment. Rat brain homogenate was prepared as described earlier and incubated in an oscillating water bath for one hour at 37° C with varying concentrations of metrifonate and ImM QA, to make a final volume of I ml after which 0.5ml of BHT (0.5% w/v in methanol) was added to each tube after incubation, followed by the addition of I rnl of TCA (lO%w/v). The tubes were then centrifuged at 2000rpm for 20 minutes to remove insoluble proteins. Two rnillilitres of the supernatant was transferred to a clean set of tubes and 0.5rnl TBA (0.33g1100ml Milli-Q water) was added. The tubes were boiled for one hour at 95°C, and then cooled on ice. Thereafter the TBA-MDA complex was extracted with butanol and read at 532nrn. MDA levels were determined from the standard curve generated from
1,1,3,3 -tetramethoxypropane.
Chapter 3 - Lipid Peroxidation
3.2.2.7 In vitro exposure of rat brain homogenate to Quinolinic Acid and Metrifonate
Quinolinic acid and MET were dissolved in Milli-Q and to dilute to yield the following concentrations in the final volume of Iml of the reaction mixture. For the in vitro assay of MET and QA, homogenate (lmL) containing varying concentrations of metrifonate (0, 0.25, 0.5, and I mM) was incubated in an oscillating water bath for I hr at 37°C, to which I mM QA was added and incubated for a further I hour. At the end of the incubation period, 0.5mL BHT (0.5mg/ml in methanol) and ImL TCA (15% in water) were added to the mixture. The samples were centrifuged at 2000x g for 20min at 4°C to remove insoluble proteins. Following centrifugation, 2mL of protein free supernatant was removed from each tube and a 0.5mL aliquot ofTBA (0.33% in water) was added to this fraction. The tubes were sealed and heated for an hour at 95°C in a water bath. After cooling, the TBA-MDA complexes were extracted with 2mL of butanol. The absorbance was read at 532nm and the MDA levels were determined from a standard curve generated from I, I, 3, 3-tetramethoxypropane. Final results were represented as nmol MDAlmg tissue.
3.2.3 RESULTS
The final results were expressed as nanomoles of MDA produced per mg tissue. The in vitro exposure of brain homogenate to I mM QA alone produced a significant increase in lipid peroxidation when compared to the control. There was also a significant increase in lipid peroxidation of the varying concentrations of MET with I mM QA compared to the control and I mM QA alone.
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Effect of Metrifonate and Quinolinic Acid in vitro
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Fig 3.3. Concentration-dependent effect of MET and QA on Lipid peroxidation in rat brain homogenate. Each Bar represents the mean
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SEM, n=5; (**' p<O.OOl in comparison to control, ** p<O.Ol in comparison to control, * p<O.05 in comparison to control and@@ P<O.Ol in comparison to lmM QA only).3.2.4 DISCUSSION
The graph and data imply that MET does not protect the brain homogenate from the lipid peroxidation damage induced by QA. In the presence of MET with QA, there is an increase in the levels ofMDA produced. The level ofMDA produced at ImM MET with ImM QA is higher than the MDA level produced in the presence of ImM QA only.
Therefore, the presence of MET increases lipid peroxidation. Thus MET does not protect the brain homogenate from QA-induced damage, but instead it causes additional peroxidation.
Chapter 3 - Lipid Peroxidation
3.3 EFFECT OF METRIFONATE ON LIPID PEROXIDATION IN RAT BRAIN