Baylor Research Institute

Arning, E. and T. Bottiglieri (2016). “Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.” Methods Mol Biol 1378: 175-182.

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We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folic acid and is involved in regulation of homocysteine and DNA synthesis. Measurement of 5-MTHF in CSF provides diagnostic information regarding diseases affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism. Determination of 5-MTHF in CSF (50 muL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF-(13)C5) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400 nM) and has an analytical measurement range of 3-1000 nM.

Arning, E. and T. Bottiglieri (2016). “Quantification of gamma-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.” Methods Mol Biol 1378: 109-118.

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We describe a simple stable isotope dilution method for accurate and precise measurement of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of GABA in CSF (50 muL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directly onto the HPLC-ESI-MS/MS system. Determination of total GABA in CSF requires additional sample preparation in order to hydrolyze all the bound GABA in the sample to the free form. This requires hydrolyzing the sample by boiling in acidic conditions (hydrochloric acid) for 4 h. The sample is then further diluted 1:10 with a 90 % acetonitrile/0.1 % formic acid solution and injected into the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve and is linear from 6 nM to 1000 nM and 0.63 muM to 80 muM for free and total GABA, respectively.

Gomez-Hoyos, J., R. Schroder, M. Reddy, I. J. Palmer and H. D. Martin (2016). “Femoral Neck Anteversion and Lesser Trochanteric Retroversion in Patients With Ischiofemoral Impingement: A Case-Control Magnetic Resonance Imaging Study.” Arthroscopy 32(1): 13-18.

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PURPOSE: To assess the relationship between the femoral neck version (FNV) and lesser trochanteric version (LTV) in symptomatic patients with ischiofemoral impingement (IFI) as compared with asymptomatic hips. METHODS: The FNV and LTV of patients with symptomatic IFI who underwent magnetic resonance imaging assessment including a standardized femoral version study protocol were compared with those of patients with asymptomatic hips in this retrospective, observational study. Patients with isolated intra-articular pathology, prior hip fracture, and lesser trochanter deformity were excluded. The FNV, LTV, ischiofemoral space, and quadratus femoris space were evaluated on axial magnetic resonance imaging, as well as the angle between the LTV and the FNV. Independent t-tests were used to determine differences between groups. RESULTS: Data from 11 out 15 symptomatic patients and 250 out of 320 asymptomatic patients were analyzed. The mean ischiofemoral space (11.9 v 22.9 mm; P < .001; 95% confidence interval [CI], 6.9 to 15.2) and mean quadratus femoris space (7.2 mm v 14.9 mm; P < .001; 95% CI, 5.4 to 8.6) were significantly smaller in symptomatic patients versus asymptomatic patients. There was no difference in mean LTV between groups (-23.6 degrees v -24.2 degrees ; P = .8; 95% CI, -7.5 to 6.4), however, the mean FNV (21.7 degrees v 14.1 degrees ; P = .02; 95% CI, -14.2 to -1.1) and the angle between the FNV and LTV on average (45.4 degrees v 38.3 degrees ; P = .01; 95% CI, -12.9 to -1.3) were higher in symptomatic than in asymptomatic patients, with statistical significance. CONCLUSIONS: The femoral mean neck anteversion and the mean angle between the FNV and LTV are significantly higher in patients with symptomatic IFI. The mean LTV is not increased in patients with symptomatic ischiofemoral impingement as compared with those patients with asymptomatic hips. LEVEL OF EVIDENCE: Level III, diagnostic study.

Sweetman, L., P. Ashcraft and J. Bennett-Firmin (2016). “Quantitative Organic Acids in Urine by Two Dimensional Gas Chromatography-Time of Flight Mass Spectrometry (GCxGC-TOFMS).” Methods Mol Biol 1378: 183-197.

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Seventy-six organic acids in urine specimens are determined with quantitative two dimensional Gas Chromatography-Time of Flight Mass Spectrometry (GCxGC-TOFMS). The specimen is treated with urease to remove urea then derivatized to form pentafluorobenzyl oximes (PFBO) of oxoacids. The sample is then treated with ethyl alcohol to precipitate proteins and centrifuged. After drying the supernatant, the organic acids are derivatized to form volatile trimethylsilyl (TMS) derivatives for separation by capillary two dimensional Gas Chromatography (GCxGC) with temperature programming and modulation. Detection is by Time of Flight Mass Spectrometry (TOFMS) with identification of the organic acids by their mass spectra. Organic acids are quantitated by peak areas of reconstructed ion chromatograms with internal standards and calibration curves. Organic acids are quantified to determine abnormal patterns for the diagnosis of more than 100 inherited disorders of organic acid metabolism. Characteristic abnormal metabolites are quantified to monitor dietary and other modes of treatment for patients who are diagnosed with specific organic acid disorders.

Singhal, N. K., S. Li, E. Arning, K. Alkhayer, R. Clements, Z. Sarcyk, R. S. Dassanayake, N. E. Brasch, E. J. Freeman, T. Bottiglieri and J. McDonough (2015). “Changes in Methionine Metabolism and Histone H3 Trimethylation Are Linked to Mitochondrial Defects in Multiple Sclerosis.” Journal of Neuroscience 35(44): 15170-15186.

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Mitochondrial changes, including decreased expression of electron transport chain subunit genes and impaired energetic, have been reported in multiple sclerosis (MS), but the mechanisms involved in these changes are not clear. To determine whether epigenetic mechanisms are involved, we measured the concentrations of methionine metabolites by liquid chromatography tandem mass spectrometry, histone H3 methylation patterns, and markers of mitochondrial respiration in gray matter from postmortem MS and control cortical samples. We found decreases in respiratory markers as well as decreased concentrations of the methionine metabolites S-adenosylmethionine, betaine, and cystathionine in MS gray matter. We also found expression of the enzyme betaine homocysteine methyltransferase in cortical neurons. This enzyme catalyzes the remethylation of homocysteine to methionine, with betaine as the methyl donor, and has previously been thought to be restricted to liver and kidney in the adult human. Decreases in the concentration of the methyl donor betaine were correlated with decreases in histone H3 trimethylation (H3K4me3) in NeuN+ neuronal nuclei in MS cortex compared with controls. Mechanistic studies demonstrated that H3K4me3 levels and mitochondrial respiration were reduced in SH-SY5Y cells after exposure to the nitric oxide donor sodium nitroprusside, and betaine was able to rescue H3K4me3 levels and respiratory capacity in these cells. Chromatin immunoprecipitation experiments showed that betaine regulates metabolic genes in human SH-SY5Y neuroblastoma cells. These data suggest that changes to methionine metabolism may be mechanistically linked to changes in neuronal energetics in MS cortex.