Measurement of internal body time by blood metabolomics

Abstract

Detection of internal body time (BT) via a few-time-point assay has been a longstanding challenge in medicine, because BT information can be exploited to maximize potency and minimize toxicity during drug administration and thus will enable highly optimized medication. To address this challenge, we previously developed the concept, "molecular-timetable method," which was originally inspired by Linné's flower clock. In Linné's flower clock, one can estimate the time of the day by watching the opening and closing pattern of various flowers. Similarly, in the molecular-timetable method, one can measure the BT of the day by profiling the up and down patterns of substances in the molecular timetable. To make this method clinically feasible, we now performed blood metabolome analysis and here report the successful quantification of hundreds of clock-controlled metabolites in mouse plasma. Based on circadian blood metabolomics, we can detect individual BT under various conditions, demonstrating its robustness against genetic background, sex, age, and feeding differences. The power of this method is also demonstrated by the sensitive and accurate detection of circadian rhythm disorder in jet-lagged mice. These results suggest the potential for metabolomics-based detection of BT ("metabolite-timetable method"), which will lead to the realization of chronotherapy and personalized medicine.

Fig. 1.

Circadian patterns of metabolites in mouse plasma. (A) Circadian changes in corticosterone levels in the plasma of CBA/N mice under LD (Left) and DD, Center) conditions. All values are mean ± SEM. The white bars above the graph indicate day, gray bars indicate subjective day, and black bars indicate night/subjective night. ZT0 is the time of light on, and CT0 is the time the light used to be turned on. (B) Circadian oscillatory metabolites in the plasma of CBA/N mice [negative ions (up); positive ions (down)]. 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 (molecular peak times are indicated as colors). (C and D) Identified oscillatory peaks measured by negative ion mode (C) and positive ion mode (D). Mean value was set to 1.0.

Fig. 2.

BT estimation. BT measurements of mice kept under LD (A) and DD (B) conditions. Colors of the dots indicate the molecular peak times of each substance (Table S1). Peak time of the red cosine curves indicates estimated BT and peak time of the blue indicates the time the sample was taken (“environmental time”). The greater the degree of overlap of the red and blue curves, the greater the accuracy of the measurement. The dashed vertical lines show the BT (red) or environmental time (ZT/CT, blue). See Table S2 for statistics.

Fig. 3.

Genetic background. BT measurement using C57BL/6 mice plasma collected under LD (A) and DD (B) conditions. Colors of the dots indicate the molecular peak times of each substance (Table S1). Peak time of the red cosine curves indicate estimated BT and peak time of the blue indicate the environmental time. The dashed vertical lines show the BT (red) or environmental time (ZT/CT, blue). See Table S2 for statistics.

Fig. 4.

Age, sex differences and feeding condition. (A) BT measurement of young male (Top), aged male (Middle), and young female mice plasma (Bottom) harvested at ZT0 (Left) and ZT12 (Right). (B) BT measurement of young male mice kept under food-deprivation conditions. Colors of the dots indicate the molecular peak times of each substance (Table S1). Peak time of the red cosine curves indicates estimated BT, and peak time of the blue indicates the environmental time. The dashed vertical lines show the BT (red) or environmental time (ZT, blue). Results for young male mice (A) are replotted from Fig. 2A for comparison. See also Table S2 for statistics.

Fig. 5.

Detection of jet lag. (A) Schematic view of lighting conditions. White bars indicate light on, and black bars indicate light off. On day 1, the light was turned off 8 h earlier. Samples were collected at 2 time points on days 1, 5, and 14 after the LD shift (red triangles). (B) The actogram of a single mouse, showing that it was experiencing “jet lag” induced by the LD shift. Yellow shading indicates periods of light on, and gray shading indicates periods of light off. The red triangles indicate days 1 (Top), 5 (Middle), and 14 (Bottom). (C) BT measurement from mouse plasma collected before (day 1, Top), during (day 5, Middle), and after entrainment to the new LD cycle (day 14, Lower). Colors of the dots indicate the molecular peak time of each substance (Table S1). The red cosine curve is the estimation, the blue curve is the environmental time (pre LD condition shift), and the brown cosine curve is the environmental time (post shift). See also Table S2 for statistics.