Identification of human plasma metabolites exhibiting time-of-day variation using an untargeted liquid chromatography-mass spectrometry metabolomic approach

Abstract

Although daily rhythms regulate multiple aspects of human physiology, rhythmic control of the metabolome remains poorly understood. The primary objective of this proof-of-concept study was identification of metabolites in human plasma that exhibit significant 24-h variation. This was assessed via an untargeted metabolomic approach using liquid chromatography-mass spectrometry (LC-MS). Eight lean, healthy, and unmedicated men, mean age 53.6 (SD ± 6.0) yrs, maintained a fixed sleep/wake schedule and dietary regime for 1 wk at home prior to an adaptation night and followed by a 25-h experimental session in the laboratory where the light/dark cycle, sleep/wake, posture, and calorific intake were strictly controlled. Plasma samples from each individual at selected time points were prepared using liquid-phase extraction followed by reverse-phase LC coupled to quadrupole time-of-flight MS analysis in positive ionization mode. Time-of-day variation in the metabolites was screened for using orthogonal partial least square discrimination between selected time points of 10:00 vs. 22:00 h, 16:00 vs. 04:00 h, and 07:00 (d 1) vs. 16:00 h, as well as repeated-measures analysis of variance with time as an independent variable. Subsequently, cosinor analysis was performed on all the sampled time points across the 24-h day to assess for significant daily variation. In this study, analytical variability, assessed using known internal standards, was low with coefficients of variation <10%. A total of 1069 metabolite features were detected and 203 (19%) showed significant time-of-day variation. Of these, 34 metabolites were identified using a combination of accurate mass, tandem MS, and online database searches. These metabolites include corticosteroids, bilirubin, amino acids, acylcarnitines, and phospholipids; of note, the magnitude of the 24-h variation of these identified metabolites was large, with the mean ratio of oscillation range over MESOR (24-h time series mean) of 65% (95% confidence interval [CI]: 49-81%). Importantly, several of these human plasma metabolites, including specific acylcarnitines and phospholipids, were hitherto not known to be 24-h variant. These findings represent an important baseline and will be useful in guiding the design and interpretation of future metabolite-based studies.

FIGURE 1

Flowchart summarizing data analysis workflow.

FIGURE 2

Time profiles of four plasma metabolites with different acrophases: (a) acetylcarnitine, (b) LysoPE(18:1), (c) proline, and (d) cortisol. On the horizontal axis, black bar indicates lights-off (0 lux) and white bar lights-on (440–825 lux). Internal standard (IS) shows the analytical variation of each ion in the pooled, replicate samples analyzed throughout the LC-MS run.

FIGURE 3

Plasma acetylcarnitine profiles for eight individuals maintained under controlled light/dark, sleep/wake, posture, and calorific intake conditions. Black bar indicates lights-off (0 lux) and white bar lights-on (440–825 lux).

FIGURE 4

(Top panel) The temporal profile of palmitoylcarnitine in human plasma. (Bottom panel) CPT1a gene expression in a mouse model from an independent study (Hughes et al., 2009). Black/gray bars indicate lights-off and the white bar lights-on.

SUPPLEMENTARY FIGURE 1

Venn diagram showing overlapping sets of metabolite features detected by OPLS-DA of paired time points and repeated-measures ANOVA across all time points.

SUPPLEMENTARY FIGURE 2

Plasma profiles of metabolite feature with mass/charge 455.19 Da and retention time 5.7 min for eight individuals detected by repeated-measures ANOVA but not pairwise OPLS-DA comparisons, illustrating higher intersubject variability relative to intrasubject time-of-day variation. All participants were maintained under controlled light/dark, sleep/wake, posture, and calorific intake conditions. Black bar indicates lights-off (0 lux) and white bar lights-on (440–825 lux).