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. 2012 Sep 11;109(37):15036-41.
doi: 10.1073/pnas.1207768109. Epub 2012 Aug 27.

Human Blood Metabolite Timetable Indicates Internal Body Time

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Free PMC article

Human Blood Metabolite Timetable Indicates Internal Body Time

Takeya Kasukawa et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

A convenient way to estimate internal body time (BT) is essential for chronotherapy and time-restricted feeding, both of which use body-time information to maximize potency and minimize toxicity during drug administration and feeding, respectively. Previously, we proposed a molecular timetable based on circadian-oscillating substances in multiple mouse organs or blood to estimate internal body time from samples taken at only a few time points. Here we applied this molecular-timetable concept to estimate and evaluate internal body time in humans. We constructed a 1.5-d reference timetable of oscillating metabolites in human blood samples with 2-h sampling frequency while simultaneously controlling for the confounding effects of activity level, light, temperature, sleep, and food intake. By using this metabolite timetable as a reference, we accurately determined internal body time within 3 h from just two anti-phase blood samples. Our minimally invasive, molecular-timetable method with human blood enables highly optimized and personalized medicine.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Experimental condition. (A) The concept of body-time detection with conventional and molecular timetable methods illustrated by Linné’s flower clock. In the conventional method a single indicator monitored over a few days detects internal body time; in the molecular timetable method, multiple metabolic “flowers” are simultaneously measured at a few time points, which reduce efforts in sampling. (B) Metabolite timetable construction. We collected time-course blood samples, isolated plasma, measured with LC–MS, and selected circadian-oscillating metabolites as indicators. In body-time detection, we collected blood samples at a few time points, isolated plasma, measured with LC–MS, and estimated body time. (C) Image of the constant routine (CR) experiments. During CR, subjects stayed in chairs while various measurements were performed. Note: the man in this picture is demonstrating the set-up and is not an actual subject in this study. (D) Sampling schedule for each subject. Black circles indicate the time points when blood samples were taken and when subjects ate during CR. White boxes indicate the time when subjects were awake, and black boxes indicate when they were asleep. The blood samples of three subjects during CR1 were used for timetable construction, and other samples were used for body-time estimation. (E) Measured melatonin (Upper) and cortisol (Lower) rhythms in the collected blood samples during CR1 (Left) and CR2 (Right). The cortisol and melatonin levels show that some subjects (e.g., subject E in CR2) have shifted internal body time against sampled time. CR, constant routine; SCR, semiconstant routine; BT, body time; LC–MS, liquid chromatography mass spectrometry.
Fig. 2.
Fig. 2.
Heat maps of circadian-oscillating metabolites in the human timetable. Circadian-oscillating metabolite peaks in the plasma samples [positive ions (A); negative ions (B)] are shown. Some metabolites in this heat map were identified and shown in Fig. 4. On the heat maps, magenta tiles indicate a high quantity of substances and green tiles indicate a low quantity in plasma. Metabolites are sorted according to their molecular peak time.
Fig. 3.
Fig. 3.
Estimation of human body time. (A) Difference between expected body time and sampled time. Nine sample sets for body-time estimation were classified into three groups by the magnitude of the difference: small (black), moderate (gray), and large (white). (BD) Body-time estimation at 12 h on the first day for nine sample sets in small (B), moderate (C), and large (D) body-time difference groups. Colors of the dots indicate the molecular peak times of each metabolite. Peak time of the red cosine curves indicates estimated body time and peak time of the blue indicates the time of the expected body time. The smaller the distance between red and blue curves, the greater the accuracy of the measurement. Dashed vertical lines show the estimated body time (red), expected body time (blue), or the time the sample was taken (black). In all subjects, expected body time (based on cortisol rhythm) and estimated body time (based on molecular timetable) were similar, indicated by the close proximity of the blue and red dashed lines. (E) Summary of body-time estimation. Difference between expected and estimated body time for all estimated samples is shown. The leftmost three samples were ones in the small difference group, the middle five samples were in the moderate difference group, and the rightmost sample was in the large difference group. All samples estimated internal body time within or around 3-h differences between expected and estimated body time. BT, body time.
Fig. 4.
Fig. 4.
Identified circadian-oscillating metabolites. Abundance is shown of cortisol (A), cortisone (B), urocortisone-3-glucronide (C), pregnanolone sulfate-like metabolite (D), (allo)urocortisol-3-glucronide (E), l-phenylalanine (F), tryptophan (G), and l-leucine (H) in the blood sampled from three subjects (subject A, subject B, and subject C) whose CR1 samples were used in the metabolite timetable construction. X axes show the sampled time from 12 h on day 2 to 20 h on day 3 in CR1, and y axes show area of the corresponding peak in the LC–MS analysis.

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