Diurnal rhythms in the human urine metabolome during sleep and total sleep deprivation

Abstract

Understanding how metabolite levels change over the 24 hour day is of crucial importance for clinical and epidemiological studies. Additionally, the association between sleep deprivation and metabolic disorders such as diabetes and obesity requires investigation into the links between sleep and metabolism. Here, we characterise time-of-day variation and the effects of sleep deprivation on urinary metabolite profiles. Healthy male participants (n = 15) completed an in-laboratory study comprising one 24 h sleep/wake cycle prior to 24 h of continual wakefulness under highly controlled environmental conditions. Urine samples were collected over set 2-8 h intervals and analysed by (1)H NMR spectroscopy. Significant changes were observed with respect to both time of day and sleep deprivation. Of 32 identified metabolites, 7 (22%) exhibited cosine rhythmicity over at least one 24 h period; 5 exhibiting a cosine rhythm on both days. Eight metabolites significantly increased during sleep deprivation compared with sleep (taurine, formate, citrate, 3-indoxyl sulfate, carnitine, 3-hydroxyisobutyrate, TMAO and acetate) and 8 significantly decreased (dimethylamine, 4-DTA, creatinine, ascorbate, 2-hydroxyisobutyrate, allantoin, 4-DEA, 4-hydroxyphenylacetate). These data indicate that sampling time, the presence or absence of sleep and the response to sleep deprivation are highly relevant when identifying biomarkers in urinary metabolic profiling studies.

Figure 1. A schematic of the in-laboratory protocol.

The in-laboratory session consisted of an adaptation night (day 0) followed by a 24 h wake/sleep cycle and a 24 h wake/wake cycle. White sections indicate wake periods (100 lux, free to move); grey sections indicate wakefulness in a semi-recumbent position in dim light (<5 lux); black sections indicate sleep periods in a supine position in the dark (0 lux with eye masks). Standardized meals are represented by triangles; blue sections show the set urine collection time intervals. The midpoint time of each urine collection was used for statistical analyses. Modified from Ackermann et al. [16] with permission.

Figure 2. A typical urine ¹H NMR spectrum with identified metabolites.

The metabolites are numbered accordingly: 1: valine, 2: 3-hydroxyisobutyrate, 3: 4-deoxyerythronic acid (4-DEA), 4: 3-aminoisobutyrate, 5: 4-deoxythreonic acid (4-DTA), 6: 3-hydroxyisovalerate, 7: threonine/lactate, 8: 2/alfa-hydroxyisobutyrate, 9: alanine, 10: acetate, 11: phenylacetylglutamine, 12: p-cresol sulfate, 13: citrate, 14: dimethylamine, 15: creatine, 16: creatinine, 17: proline betaine, 18: carnitine, 19: trimethylamine-N-oxide (TMAO), 20: taurine, 21: glycine, 22: hippurate, 23: trigonelline, 24: ascorbate, 25: xylose, 26: allantoin, 27: urea*, 28: 4-hydroxyphenylacetate, 29: tyrosine, 30: 3-indoxyl sulfate, 31: histidine, 32: formate, 33: trigonelline, *urea peak was not quantified.

Figure 3. Metabolic differences during a night of sleep deprivation compared to a night of sleep (a) OPLS-DA scores and (b) loadings separating NMR spectra of urine samples from a night of sleep deprivation and a night of sleep.

The OPLS-DA model explains 54.1% and 89.5% of x-and y-variation, respectively. Car: carnitine, Cr: creatinine, DMA: dimethylamine, Gly: glycine, His: histidine, Tau: taurine.

Figure 4. Time-of-day variations in urinary metabolites.

(a) Mean (±SEM) PC1 scores (across all 15 individuals) from a PCA of all identified metabolites (n = 32), plotted against time, and (b) the corresponding PC1 loading. The individual time points are the midpoint times from intervals of pooled urine samples obtained sequentially (2–8 h) across the 60 h study protocol. Green areas, sleep period; yellow area, sleep deprivation night. 2-HIB: 2-hydroxyisobutyrate, 3-AIB: 3-aminoisobutyrate, 3-HIV: 3-hydroxyisovalerate, 3-IS: 3-indoxyl sulfate, 4-HPA: 4-hydroxyphenylacetate, DMA: dimethylamine, PAG: phenylacetylglutamine, p-CS: p-cresol sulfate, Pro-Bet: proline betaine.

Figure 5. Urinary metabolites with significant 24 h cosine rhythms.

(a) Metabolites exhibiting a significant 24 h cosine rhythm on both day 1 (wake/sleep) and day 2 (wake/wake). (b) Metabolites with a significant cosine rhythm on day 2 only. The midpoint of each urine collection interval was used for the analyses. Dashed line shows day 1/day 2 boundary. Green bars show sleep period (23:00–07:00 h); yellow bars show sleep deprivation period (23:00–07:00 h).

Figure 6. Correlations between urine metabolites measured in the study.

Hierarchically clustered heatmap showing the Spearman’s rank sum correlation between the identified urinary metabolites. 2-HIB: 2-hydroxyisobutyrate, 3-AIB: 3-aminoisobutyrate, 3-HIV: 3-hydroxyisovalerate, 3-IS: 3-indoxyl sulfate, 4-HPA: 4-hydroxyphenylacetate, DMA: dimethylamine, PAG: phenylacetylglutamine, p-CS: p-cresol sulfate, Pro-Bet: proline betaine.