The procedure devised by the authors consisted of two parts. The first part involved analyzing blood from 13 patients. The plasma from the cells was isolated via centrifugation and then stored at -80⁰C within an hour of drawing blood. Using stable-isotope dilution liquid chromatograhy- electrospray ionization (ESI)- tandem mass spectrometry as described in a previous paper, SAM, SAH, methionine, cytathionine, choline and betaine were determined in human plasma. Then, as suggested in another paper, LC-MS/MS was used and total plasma homocysteine (tHcy) was measured. The examiners were kept blind to the disease diagnoses as they analyzed the samples. Statistical significance was established between control and experimental mice using a two-tailed T-test. Mice were used as test subjects. All the steps concerning mice, were approved by the animal care committee at Kent State University. Three controls (PBS injected) and four experimental (L-methionine injected) mice were examined. Mice were perfused with PBS after 24 hours of the final methionine dose to eradicate any contaminants in blood followed by removal of the cerebral cortex. From one half of the cerebral cortex, methionine and SAM (the only methyl group donor) concentrations were measured using LC-MS/MS as suggested in a previous paper. From the other half, H3K4me3 and DNMT3A proteins were taken and examined using western blotting. Increase in methionine levels caused a moderate increase in levels of SAM, H3K4me3 and DNMT3A as well as mitochondrial electron transport proteins. This procedure was performed for experimental and control mice. Proteins were then separated by gel electrophoresis and western blotted with antibodies to DNMT3A and H3K4me3 for nuclear parts and different electron transport chain complexes for mitochondrial parts. The program Imagej was used to perform densitometry. Relative protein levels were normalized to histone H3 for nuclear parts and aralar for mitochondrial parts. Statistical significance was established using a two tailed T-test. The authors of this paper measured the levels of methionine in patients at the RRMS stage and compared them to normal levels and found a reduction in levels of methionine in RRMS patients. The results of this paper suggest that levels of H3K4me3, DNMT3A and mitochondrial proteins are increased as methionine is injected within mice.Keeping examiners bind to the disease diagnosis was an excellent strategy adopted by the authors to overcome the experimenter-expectancy effect. This experiment is supported by a later study that also reports a decrease in methionine levels in MS patients and further suggests using methionine as a biomarker. Another study used a methionine based drug to enhance the expression of some genes that cause remyelination. Since the cause of multiple sclerosis is unknown but it is known that demyelination takes place in MS, this drug could potentially be used to treat MS or at least alleviate its symptoms. A study regarding mitochondrial proteins stated that methionine levels affect levels of H3K4me3 and the experiment was performed using liquid chromatography- high resolution mass spectromety. This is supported by a later paper that showed the concentration of SAM in association with DNMT3A, another mitochondrial protein, is crucial for the receptiveness of dentate neurons to environmental stimuli. So there’s a possible correlation between mitochondrial proteins and multiple sclerosis. Along with methionine, an increase in erythropoietin has also been reported to increase the levels of H3K4me3. This experimental design is a between subjects design- control and experimental groups are separate. If there are any individual differences or problems with any of the individual mice, then due to the small sample size (7 mice in total), the results might get skewed. Thus, the sample size needs to be increased greatly in order to disregard any differences that the individual mice may have. The experimenters would also have to ensure that they randomly select mice for experimental and control groups.
Methionine is an essential amino acid that has to be supplied via diet. It is also the start codon of all proteins/ RNA sequences. Thus, methionine is essential for proteins to be synthesized and perform functions. It is known that multiple sclerosis results in demyelination of neurons. It has been found that prolonged deprivation of methionine leads to apoptosis. Thus, it could be possible that decrease in circulating methionine levels would cause schwann cells to undergo apoptosis and thus, lead to MS. A number of disorders are related to problems with methionine metabolism including Alzheimer’s, multiple sclerosis, SACD, HIV related neuropathies, atherosclerosis, etc. It has been shown that a change in the pattern of methionine metabolism can affect histone H3 and experimental autoimmune encephalomyelitis (EAE). Future studies can look into changes in methionine metabolism and methionine deficiency as potential causes of Multiple Sclerosis. This can be done by conducting further research on methionine pathway- using trackers in live cells, for example, using GFP attached to a marker to track down methionine in a healthy individual and one in the RRMS stage. Potential immunotherapies dealing with supplementing methionine in case of deficiency and decreasing the amount of circulating methionine in case of methionine toxicity can be developed based on these studies, thereby, developing a treatment for MS.