Simultaneous detection of five one-carbon metabolites in plasma using stable isotope dilution liquid chromatography tandem mass spectrometry

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2016-02, Vol.1012-1013, p.186-192
• Main Authors: Adaikalakoteswari, Antonysunil, Webster, Craig, Goljan, Ilona, Saravanan, Ponnusamy
• Format: Article
• Language: English
• Subjects: Dilution; Epigenetic modifications; Female; Gestation; Homocysteine; Homocysteine - blood; Homocysteine - chemistry; Homocysteine - metabolism; Humans; Isotope Labeling - methods; Isotopes; Limit of Detection; Linear Models; Liquid chromatography; Metabolites; Methionine; Methionine - blood; Methionine - chemistry; Methionine - metabolism; One-carbon metabolism; Polymethyl methacrylates; Pregnancy; Reproducibility of Results; Tandem Mass Spectrometry - methods; Vitamin B 12 Deficiency; Vitamin B12 deficiency
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2016.01.026

•Nutritional deficiencies disturbs 1-carbon cycle altering 1-C metabolite levels.•This changes methylation and epigenetic modifications of DNA resulting in adverse metabolic phenotype.•Pregnant women with low B12 levels had significantly altered levels of 1-C metabolites.•Our novel method measures five 1-C metabolites in a single run at low-cost by LC–MS/MS.•This offers the opportunity for measuring these in routine clinical practice. Disturbance in one-carbon (1-C) cycle occurs due to nutritional deficiencies (vitamin B12/folate) or specific genetic polymorphisms. This leads to altered levels of key 1-C metabolites such as SAM (s-adenosyl methionine), SAH (s-adenosyl homocysteine), methionine, homocysteine and MMA (methyl malonic acid). These 1-C metabolites are determinants of cellular methylation potential and epigenetic modifications of DNA which impairs metabolic pathways in several pathological diseases and developmental programming. Though methods were able to measure these analytes only independently, none of the methods detect simultaneously. Therefore we developed a method to measure these five 1-C metabolites in a single run using liquid chromatography tandem mass spectrometry (LC–MS/MS). We used stable isotopes dilution LC–MS/MS to measure the 1-C metabolites in human plasma. Blood samples were collected from pregnant women (n=30) at early gestation in the ongoing, multicentre, prospective PRiDE study. Linearity exhibited across the calibration range for all the analytes with the limit of detection (LOD) of 1.005nmol/l for SAM, 0.081nmol/l for SAH, 0.002μmol/l for methionine, 0.046μmol/l for homocysteine and 3.920nmol/l for MMA. The average recovery for SAM was 108%, SAH—110%, methionine—97%, homocysteine—91% and MMA—102%. The inter-assay CV for SAM was 7.3, SAH—5.6%, methionine—3.5%, homocysteine—7.0% and MMA—4.0%. The intra-assay CV for SAM was 8.7%, SAH—4.7%, methionine—5.4%, homocysteine—8.1% and MMA—6.1%. Pregnant women at early gestation with low B12 levels had significantly higher homocysteine, MMA, lower levels of methionine, SAM and SAM:SAH ratio and higher triglycerides. We developed a simple and rapid method to simultaneously quantify 1-C metabolites such as SAM, SAH, methionine, homocysteine and MMA in plasma by stable isotope dilution LC–MS/MS which would be useful to elucidate the epigenetic mechanisms related in the gene–nutrient interactions.