Quantifying Homocysteine Methionine Cycle Metabolites in Plasma and CSF

Title: High-throughput and Simultaneous Quantitative Analysis of Homocysteine–Methionine Cycle Metabolites and Co-factors in Blood Plasma and Cerebrospinal Fluid by Isotope Dilution LC–MS/MS

Journal: Analytical and Bioanalytical Chemistry

Published: October 18, 2016

Background

The methionine cycle is a crucial metabolic pathway involved in cardiovascular health and cognitive function. Dysregulation of this cycle has been linked to conditions such as mild cognitive decline, vascular dementia, and Alzheimer's disease. However, existing analytical methods for monitoring methionine cycle metabolites and co-factors are limited, requiring multiple separate approaches. This study introduces a novel high-throughput and simultaneous quantification method for 17 key metabolites using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). The method enables precise and accurate quantitation in both plasma and cerebrospinal fluid, facilitating large-scale studies on nutrition, health monitoring, and disease risk management.

Materials & Methods

Materials and Reagents

Chemicals and Solvents: LC-MS-grade acetonitrile (ACN), LC-grade methanol (MeOH), formic acid, perfluoroheptanoic acid (PFHA), ascorbic acid, sodium hydroxide (NaOH), hydrochloric acid (HCl), tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), and ammonium acetate (NH4OAc) were purchased from Sigma-Aldrich Chemie GmbH (Switzerland). Deionized water (R > 18 MΩ/cm, TOC < 10 ppb) was generated using a Millipore-Q system (Millipore, USA).

Analytes and Standards: HA, taurine, serine, cystine, glycine, homocystine (HCy2), riboflavin, methionine, pyridoxine, cystathionine, SAH, pyridoxamine, SAM, DMG, choline, betaine, and 5-MTHF, along with their stable isotope-labeled internal standards (IS) (e.g., taurine-13C2, glycine-d2, HA-d4, SAM-d4), were sourced from Sigma-Aldrich, CDN Isotopes (Canada), Cambridge Isotopes Laboratories (USA), Cayman Chemical (USA), and Merck (Switzerland).

Standard and Internal Standard Preparation

Stock Solutions: Individual standards and IS were dissolved in 0.1 M HCl, 10 mmol/L NH4OAc, 0.1 M NaOH, or MeOH/H2O (depending on solubility). 5-MTHF was stabilized in 10 mmol/L NH4OAC containing 10% ascorbic acid and 2% DTT to prevent oxidation. Solutions were stored at −20°C for ≤3 months.

Working Solutions: Calibrants (1–7) and IS working solutions were prepared in ACN/H2O (5:95, v/v), covering micromolar (HA, amino acids) and nanomolar (vitamins, cofactors) ranges (Tables S1–S3, ESM).

LC-MS/MS Instrumentation

System: A Thermo Fisher Accela UHPLC 1250 Pump coupled to a TSQ Quantum Vantage triple quadrupole mass spectrometer with a heated electrospray ionization (H-ESI) source.

Chromatography: Separation was achieved on a Waters UPLC XSelect HSST3 column (100 × 2.1 mm, 2.5 μm) using gradient elution (details in ESM Tables S4–S5). Total run time: 13 min; injection volume: 10 μL.

Sample Collection and Preparation

Human Cohort: Plasma and CSF samples from 12 individuals (6 Alzheimer's disease patients and 6 controls, aged ≥50 years) were provided by PrecisionMed, Inc. (USA, Protocol 8009).

Processing:

Plasma/CSF: Thawed samples (50 μL) were mixed with 10 μL IS, 50 μL TCEP (100 mg/mL), and 140 μL methanol + 1% FA. Vortexed (15 min, 1350 rpm, 4°C), centrifuged (14,500 rpm, 5 min), and filtered (0.22 μm) before analysis.
Calibration and QC: Calibration samples (50 μL calibrant + 10 μL IS + 50 μL TCEP + 140 μL ACN/H2O) and QC samples (low/high levels) were prepared with each batch.

Method Validation

Matrix Effects: Evaluated via post-extraction spiking. Matrix effect (%) = (post-extraction peak area / solvent peak area) × 100.

Specificity/Selectivity: Verified by comparing retention times and mass spectra of standards vs. unspiked matrices.

Linearity, LOD, and LOQ: Calibration curves (analyte/IS peak area ratio) were linear (R² > 0.99). LOD (S/N ≥ 3) and LOQ (S/N ≥ 10) were determined using spiked samples.

Trueness and Precision: Assessed via spiked recovery tests (low/medium/high levels, 6 replicates) with repeatability (intra-day) and intermediate precision (inter-day, multi-operator).

Statistical Analysis

Tests: Two-sample Kolmogorov-Smirnov (KS) tests (α = 0.05) compared metabolite distributions between groups.

Software: R v3.2.2 and ggplot2 were used for analysis and visualization.

Results

The developed LC–MS/MS method successfully quantified 17 metabolites involved in the homocysteine–methionine cycle in plasma and cerebrospinal fluid (CSF), expanding coverage to include betaine, dimethylglycine (DMG), choline, and 5-methyltetrahydrofolic acid (5-MTHF). This allows for a more comprehensive assessment of methionine metabolism, particularly through the betaine homocysteine methyltransferase (BHMT) pathway.

Analytical Performance: The method demonstrated high specificity, sensitivity, and throughput, with a short run time (13 minutes). A simplified sample preparation was optimized for plasma and applied to CSF, including protein precipitation and chemical reduction for total cysteine and homocysteine quantification.
Extraction Optimization: TCEP was identified as the most efficient reducing agent for disulfide bond reduction, ensuring stability and high extraction efficiency. Methanol with 1% formic acid provided the best peak intensities and shapes for protein precipitation.
Chromatography & Validation: The method showed strong linearity (R² > 0.99) across seven calibration levels, with stable isotope-labeled internal standards effectively compensating for matrix effects. Precision and accuracy were within recommended limits, with recoveries between 90–109%.
Application to Alzheimer's Disease: In biological samples from healthy and Alzheimer's disease (AD) patients, AD subjects had significantly higher S-adenosylhomocysteine (SAH) levels and lower S-adenosylmethionine (SAM) levels, consistent with previous studies. A notable increase in glycine levels in CSF and a trend toward elevated homocysteine levels in plasma were also observed.

Typical SRM chromatograms obtained for spiked CSF samples at the medium level

Plots in plasma samples. Concentrations for glycine and total homocysteine are in micromolars and for SAH and SAM in nanomolars.

Plots in CSF samples. Concentrations for glycine are in micromolars and for SAH and SAM in nanomolars.

Reference

Guiraud, Seu Ping, et al. "High-throughput and simultaneous quantitative analysis of homocysteine–methionine cycle metabolites and co-factors in blood plasma and cerebrospinal fluid by isotope dilution LC–MS/MS." Analytical and bioanalytical chemistry 409 (2017): 295-305. http://dx.doi.org/10.1007/s00216-016-0003-1

* For Research Use Only. Not for use in diagnostic procedures.

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