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What is Choline?
Choline is a vital nutrient present in various forms within humans and animals, playing key roles in metabolic pathways and cellular functions. It can be obtained from dietary sources as well as synthesized endogenously in the liver through the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. Despite the significance of the PEMT pathway, dietary choline intake remains crucial, as endogenous synthesis alone cannot meet the cellular, tissue, and organ requirements for proper function.
In ruminants, dietary choline is extensively degraded by rumen bacteria, making the PEMT pathway a critical source of endogenous choline. For lactating dairy cows, rumen-protected choline supplementation has shown to mitigate hepatic fatty infiltration and enhance VLDL transport during periods of high metabolic demand, such as the periparturient period.
For humans, milk and dairy products serve as significant sources of dietary choline. Interestingly, the choline content and metabolite proportions in milk and infant formula can vary considerably, influencing the bioavailability of these compounds in neonates. Such variations can arise from differences in milk sourced from cows and potential post-harvest processing effects.
During lactation, physiological adaptations result in altered choline metabolism and metabolite concentrations in milk. While these changes have been studied in human and porcine milk, systematic assessments in dairy cows remain less explored.
At Creative Proteomics, liquid chromatography-tandem mass spectrometry (LC-MS/MS) have been developed and validated to identify and quantify major choline-containing compounds, enhancing the understanding and analysis of these crucial metabolites across biological contexts.
Choline and Choline Metabolites Analysis Offered by Creative Proteomics
Choline and Metabolite Quantitation: Our service offers precise measurement of choline and its metabolites across various biological matrices, supporting research into their roles in physiological and pathological processes.
Choline Metabolite Profiling: We provide detailed profiling of both water-soluble and lipid-soluble choline metabolites, revealing their distribution and abundance across sample types. This service aids in exploring metabolic pathways and identifying potential biomarkers.
Structural and Functional Analysis of Choline Metabolites: Using advanced mass spectrometry, we offer structural characterization of choline metabolites, including complex phospholipids, to understand their roles in cellular membranes.
Data Analysis and Interpretation: In addition to data generation, we provide comprehensive analysis and interpretation, helping researchers draw meaningful insights from their results.
Research Consulting and Method Development: Our team assists with experimental design, methodology selection, and method development to address specific analytical challenges.
List of Choline and Choline Metabolites We Can Detect
| Choline and Choline Metabolites Quantified in This Service | |||
|---|---|---|---|
| Choline | Acetylcholine (ACh) | Phosphocholine (PCho) | Glycerophosphocholine (GPC) |
| Cytidine Diphosphate Choline (CDP-Choline) | Phosphatidylcholine (PC) | Lysophosphatidylcholine (LPC) | Betaine |
| Sphingomyelin | Dimethylglycine (DMG) | Homocysteine | Trimethylamine N-oxide (TMAO) |
| Choline-Phosphate | Ethanolamine Phosphate (Etn-P) | ||
Brochures
Metabolomics Services
We provide unbiased non-targeted metabolomics and precise targeted metabolomics services to unravel the secrets of biological processes.
Our untargeted approach identifies and screens for differential metabolites, which are confirmed by standard methods. Follow-up targeted metabolomics studies validate important findings and support biomarker development.
Download our brochure to learn more about our solutions.
Technology Platforms for Choline Analysis
Liquid Chromatography-Mass Spectrometry (LC-MS): Offers high sensitivity and specificity for the separation and identification of choline and its metabolites.
Nuclear Magnetic Resonance (NMR) Spectroscopy: Provides detailed structural information, allowing for comprehensive metabolite characterization.
Gas Chromatography-Mass Spectrometry (GC-MS): Ideal for the analysis of volatile metabolites, ensuring precise quantification.
Electrospray Ionization (ESI) and Tandem Mass Spectrometry (MS/MS): Advanced platforms for enhancing detection capabilities and improving quantitation accuracy.
Sample Requirements for Choline Analysis
| Sample Type | Recommended Amount | Storage Conditions | Additional Notes |
|---|---|---|---|
| Plasma/Serum | 100-200 µL | Store at -80°C | Avoid repeated freeze-thaw cycles |
| Urine | 500 µL | Store at -80°C | No preservatives; filter if needed |
| Tissue (e.g., liver, brain) | 50-100 mg | Flash freeze, store at -80°C | Homogenize in appropriate buffer |
| Cells (cultured) | ~1-5 million cells | Store at -80°C | Wash cells to remove media before freezing |
| Cerebrospinal Fluid (CSF) | 100-200 µL | Store at -80°C | Ensure no hemolysis in the sample |
| Saliva | 200-500 µL | Store at -80°C | Collect using clean, sterile containers |
| Milk | 500 µL | Store at -80°C | Avoid lipid separation by freezing quickly |
PCA chart
PLS-DA point cloud diagram
Plot of multiplicative change volcanoes
Metabolite variation box plot
Pearson correlation heat map
Quantification of choline in serum and plasma using a clinical nuclear magnetic resonance analyzer
Journal: Clinica Chimica Acta
Published: 2022
Background
Choline is recognized as a gut microbiome metabolite associated with cardiovascular risk and various chronic diseases. The study aimed to develop a high-throughput nuclear magnetic resonance (NMR) assay for measuring choline concentrations using the Vantera® Clinical Analyzer. The assay was designed to address the growing interest in gut microbiome-related metabolites and their implications for health and disease, specifically in relation to cardiometabolic conditions. The analytical characteristics of the choline assay were evaluated to facilitate its clinical application.
Materials & Methods
1) Specimens:
De-identified clinical specimens were pooled at Labcorp, with additional blood draws from volunteers who provided informed consent. Samples were collected in Greiner Bio-One® serum tubes or EDTA plasma tubes.
2) Sample Preparation and NMR Acquisition:
Serum or plasma was diluted with citrate/phosphate buffer (3:1 v/v) to pH 5.3. One-dimensional proton (1H) NMR spectra were acquired using the CPMG technique on a 400 MHz Agilent spectrometer.
3) Choline Quantification:
Choline was quantified via a multi-step deconvolution algorithm, and concentrations were derived from a standard curve of known choline amounts.
4) Method Comparison:
Choline levels from NMR were compared to those from liquid chromatography tandem mass spectrometry (LC-MS/MS) using serum from multiple donors.
5) Analytical Validation:
The NMR assay was validated for linearity, imprecision, and method comparison following CLSI guidelines.
6) Reference Intervals:
Reference intervals for choline were determined using samples from healthy adults based on the 2.5th and 97.5th percentiles.
7) Interference Evaluation:
The potential interference of various substances was assessed to identify significant effects on choline test results.
Results
Choline Quantification via NMR
Choline concentration was quantified by modeling the methyl proton signal peak in CPMG spectra of plasma or serum. The signal peak integral was converted to concentration (µmol/L) using a standard curve for known choline concentrations.
Linearity of NMR-Based Choline Assay
Linearity was evaluated through regression analysis of NMR results against expected choline concentrations. The polynomial fit for serum was Y=0.0004X2+0.8967X−0.3302Y = 0.0004X^2 + 0.8967X - 0.3302Y=0.0004X2+0.8967X−0.3302 (R² = 0.999), while for plasma, it was linear: Y=0.996X+0.165Y = 0.996X + 0.165Y=0.996X+0.165 (R² = 1.000), demonstrating linearity across a broad range of choline levels.
Method Comparison: NMR vs. Mass Spectrometry
A comparative study between NMR and LC-MS/MS showed a high correlation (R = 0.998) with a slope of 1.057 and an intercept of −9.216. The Bland-Altman plot indicated a lower bias in NMR, especially at lower concentrations. A recovery study confirmed that the NMR assay accurately measured free choline concentrations.
Assay Performance and Stability
Specimen type evaluation showed no significant bias between serum from different tube types, but choline concentrations in EDTA plasma were 38% lower than in serum. Intra-assay precision (%CV) ranged from 6.2% to 14.8% in serum and 5.4% to 11.3% in EDTA plasma. Stability tests indicated that choline remained stable in serum for up to 15 days frozen and in plasma for up to 14 days refrigerated.
Reference Intervals
Reference intervals were established from samples of 529 healthy adults: mean serum choline was 11.7 ± 4.0 µmol/L and plasma choline was 7.3 ± 2.7 µmol/L. Significant differences between male and female choline levels were observed in both serum and plasma.
In Vitro Testing for Interference
Nineteen substances (both endogenous and exogenous) were tested for interference with choline assay results. No interference was observed at tested concentrations, confirming the assay's reliability against common substances.
1D 1H CPMG spectrum showing how the choline peak is modeled for quantification.
Linearity of expected versus NMR-measured choline in serum (left) and in plasma (right). **No 2nd/3rd order polynomial fit is statistically better than a linear fit at the 5% significance level.
Deming regression (A) comparison between LC/MS/MS and NMR measured choline. Bland-Altman plots of the residuals (B). The limits of agreement (LOAs) are depicted as dotted blue lines and 0% bias is a solid grey line.
Reference
- Garcia, Erwin, et al. "Quantification of choline in serum and plasma using a clinical nuclear magnetic resonance analyzer." Clinica Chimica Acta 524 (2022): 106-112.
How do you ensure accurate quantitation of choline and its metabolites in complex biological matrices?
We employ multiple strategies to ensure the accuracy of quantitation. First, we use isotope-labeled internal standards to account for matrix effects, ion suppression, and extraction efficiency. These standards help correct for any losses during sample preparation or variations in instrument response. Second, our highly sensitive LC-MS/MS and other mass spectrometry platforms provide exceptional resolution and specificity, enabling clear separation of choline metabolites from other compounds in complex matrices. Finally, we validate each method rigorously with quality control samples to ensure consistent and accurate results.
Can you analyze choline and its metabolites in multiple biological matrices from the same study?
Yes, we are equipped to analyze choline and its metabolites in multiple matrices from the same study, such as plasma, urine, tissue, milk, and cerebrospinal fluid (CSF). Each matrix requires a tailored approach, including optimized extraction protocols and appropriate internal standards to account for matrix-specific factors. This multi-matrix capability allows for comprehensive studies that explore how choline metabolism is influenced across different tissues and fluids, providing a holistic understanding of metabolic processes.
How does post-harvest processing of milk or other biological samples affect choline metabolite analysis?
Post-harvest processing, such as heating, freezing, or homogenization, can alter the structure and concentration of choline and its metabolites, particularly in milk. For example, pasteurization may lead to the degradation of some metabolites, while freezing can cause lipid separation. To minimize these effects, we recommend rapid freezing of milk samples and avoiding excessive processing prior to analysis. When necessary, we can assess how specific processing methods may impact your sample and provide recommendations for optimizing sample integrity.
What is the typical turnaround time for choline and choline metabolite analysis?
The turnaround time for choline and metabolite analysis typically ranges from 2 to 4 weeks, depending on the complexity of the study and the number of samples being analyzed. More extensive studies involving multiple biological matrices or custom method development may require additional time. However, we strive to provide clear communication throughout the process and offer expedited services for urgent projects upon request.
How do you ensure data quality and reproducibility in choline metabolite profiling?
We maintain strict quality control procedures at every stage of the analysis. This includes the use of calibration curves for quantitation, replicate analyses, and quality control (QC) samples interspersed throughout the sample batch to monitor instrument performance. We also run both positive and negative control samples to ensure the robustness of the method. Our advanced data processing algorithms further help in eliminating noise and ensuring reproducibility. These measures allow us to consistently deliver high-quality, reproducible data that researchers can rely on for publication or further study.
Can you detect low-abundance choline metabolites, and what detection limits do you achieve?
Yes, we are capable of detecting low-abundance choline metabolites thanks to the high sensitivity of our LC-MS/MS and other mass spectrometry platforms. The detection limits vary depending on the specific metabolite and sample type, but we typically achieve detection in the low picomolar (pM) to nanomolar (nM) range for most choline-related compounds. Our use of advanced detection techniques, such as electrospray ionization (ESI), further enhances sensitivity, enabling accurate quantification of even trace-level metabolites.
Can your analysis distinguish between different choline metabolite forms, such as free choline, phosphocholine, and glycerophosphocholine?
Yes, our analytical methods can clearly distinguish between various choline forms, including free choline, phosphocholine (PCho), and glycerophosphocholine (GPC). Our LC-MS/MS platform provides excellent separation and quantitation of these metabolites based on their distinct mass-to-charge (m/z) ratios and retention times. This capability is particularly important in studies investigating choline metabolism, as different forms play unique roles in biological processes, and their relative abundance can vary across tissues and physiological conditions.
Quantification of choline in serum and plasma using a clinical nuclear magnetic resonance analyzer.
Garcia, Erwin, et al.
Journal: Clinica Chimica Acta
Year: 2022
https://doi.org/10.1016/j.cca.2021.11.031
Pharmacometabolomic Approach to Investigate the Response to Metformin in Patients with Type 2 Diabetes: A Cross-Sectional Study.
Naja, Khaled, et al.
Journal: Biomedicines
Year: 2023
https://doi.org/10.3390/biomedicines11082164
Aberrant lipid accumulation and retinal pigmental epithelium dysfunction in PRCD-deficient mice.
Motipally, Sree I., et al.
Journal: bioRxiv
Year: 2024
https://doi.org/10.1101/2024.03.08.584131
