Effect of acute total sleep deprivation on plasma melatonin, cortisol and metabolite rhythms in females.

Disruption to sleep and circadian rhythms can impact on metabolism. The study aimed to investigate the effect of acute sleep deprivation on plasma melatonin, cortisol and metabolites, to increase understanding of the metabolic pathways involved in sleep/wake regulation processes. Twelve healthy young female participants remained in controlled laboratory conditions for ~92 hr with respect to posture, meals and environmental light (18:00-23:00 hr and 07:00-09:00 hr <8 lux; 23:00-07:00 hr 0 lux (sleep opportunity) or <8 lux (continuous wakefulness); 09:00-18:00 hr ~90 lux). Regular blood samples were collected for 70 hr for plasma melatonin and cortisol, and targeted liquid chromatography-mass spectrometry metabolomics. Timepoints between 00:00 and 06:00 hr for day 1 (baseline sleep), day 2 (sleep deprivation) and day 3 (recovery sleep) were analysed. Cosinor analysis and MetaCycle analysis were performed for detection of rhythmicity. Night-time melatonin levels were significantly increased during sleep deprivation and returned to baseline levels during recovery sleep. No significant differences were observed in cortisol levels. Of 130 plasma metabolites quantified, 41 metabolites were significantly altered across the study nights, with the majority decreasing during sleep deprivation, most notably phosphatidylcholines. In cosinor analysis, 58 metabolites maintained their rhythmicity across the study days, with the majority showing a phase advance during acute sleep deprivation. This observation differs to that previously reported for males. Our study is the first of metabolic profiling in females during sleep deprivation and recovery sleep, and offers a novel view of human sleep/wake regulation and sex differences.

Investigation of metabolites for estimating blood deposition time.

Trace deposition timing reflects a novel concept in forensic molecular biology involving the use of rhythmic biomarkers for estimating the time within a 24-h day/night cycle a human biological sample was left at the crime scene, which in principle allows verifying a sample donor's alibi. Previously, we introduced two circadian hormones for trace deposition timing and recently demonstrated that messenger RNA (mRNA) biomarkers significantly improve time prediction accuracy. Here, we investigate the suitability of metabolites measured using a targeted metabolomics approach, for trace deposition timing. Analysis of 171 plasma metabolites collected around the clock at 2-h intervals for 36 h from 12 male participants under controlled laboratory conditions identified 56 metabolites showing statistically significant oscillations, with peak times falling into three day/night time categories: morning/noon, afternoon/evening and night/early morning. Time prediction modelling identified 10 independently contributing metabolite biomarkers, which together achieved prediction accuracies expressed as AUC of 0.81, 0.86 and 0.90 for these three time categories respectively. Combining metabolites with previously established hormone and mRNA biomarkers in time prediction modelling resulted in an improved prediction accuracy reaching AUCs of 0.85, 0.89 and 0.96 respectively. The additional impact of metabolite biomarkers, however, was rather minor as the previously established model with melatonin, cortisol and three mRNA biomarkers achieved AUC values of 0.88, 0.88 and 0.95 for the same three time categories respectively. Nevertheless, the selected metabolites could become practically useful in scenarios where RNA marker information is unavailable such as due to RNA degradation. This is the first metabolomics study investigating circulating metabolites for trace deposition timing, and more work is needed to fully establish their usefulness for this forensic purpose.

Effect of sleep deprivation on the human metabolome.

Sleep restriction and circadian clock disruption are associated with metabolic disorders such as obesity, insulin resistance, and diabetes. The metabolic pathways involved in human sleep, however, have yet to be investigated with the use of a metabolomics approach. Here we have used untargeted and targeted liquid chromatography (LC)/MS metabolomics to examine the effect of acute sleep deprivation on plasma metabolite rhythms. Twelve healthy young male subjects remained in controlled laboratory conditions with respect to environmental light, sleep, meals, and posture during a 24-h wake/sleep cycle, followed by 24 h of wakefulness. Two-hourly plasma samples collected over the 48 h period were analyzed by LC/MS. Principal component analysis revealed a clear time of day variation with a significant cosine fit during the wake/sleep cycle and during 24 h of wakefulness in untargeted and targeted analysis. Of 171 metabolites quantified, daily rhythms were observed in the majority (n = 109), with 78 of these maintaining their rhythmicity during 24 h of wakefulness, most with reduced amplitude (n = 66). During sleep deprivation, 27 metabolites (tryptophan, serotonin, taurine, 8 acylcarnitines, 13 glycerophospholipids, and 3 sphingolipids) exhibited significantly increased levels compared with during sleep. The increased levels of serotonin, tryptophan, and taurine may explain the antidepressive effect of acute sleep deprivation and deserve further study. This report, to our knowledge the first of metabolic profiling during sleep and sleep deprivation and characterization of 24 h rhythms under these conditions, offers a novel view of human sleep/wake regulation.

Metabolic Youth in Middle Age: Predicting Aging in Caenorhabditis elegans Using Metabolomics.

Many mutations and allelic variants are known that influence the rate at which animals age, but when in life do such variants diverge from normal patterns of aging? Is this divergence visible in their physiologies? To investigate these questions, we have used (1)H NMR spectroscopy to study how the metabolome of the nematode Caenorhabditis elegans changes as it grows older. We identify a series of metabolic changes that, collectively, predict the age of wild-type worms. We then show that long-lived mutant daf-2(m41) worms are metabolically youthful compared to wild-type worms, but that this relative youth only appears in middle age. Finally, we show that metabolic age predicts the timing and magnitude of differences in age-specific mortality between these strains. Thus, the future mortality of these two genotypes can be predicted long before most of the worms die.

A metabolic signature of long life in Caenorhabditis elegans.

BACKGROUND: Many Caenorhabditis elegans mutations increase longevity and much evidence suggests that they do so at least partly via changes in metabolism. However, up until now there has been no systematic investigation of how the metabolic networks of long-lived mutants differ from those of normal worms. Metabolomic technologies, that permit the analysis of many untargeted metabolites in parallel, now make this possible. Here we use one of these, 1H nuclear magnetic resonance spectroscopy, to investigate what makes long-lived worms metabolically distinctive. RESULTS: We examined three classes of long-lived worms: dauer larvae, adult Insulin/IGF-1 signalling (IIS)-defective mutants, and a translation-defective mutant. Surprisingly, these ostensibly different long-lived worms share a common metabolic signature, dominated by shifts in carbohydrate and amino acid metabolism. In addition the dauer larvae, uniquely, had elevated levels of modified amino acids (hydroxyproline and phosphoserine). We interrogated existing gene expression data in order to integrate functional (metabolite-level) changes with transcriptional changes at a pathway level. CONCLUSIONS: The observed metabolic responses could be explained to a large degree by upregulation of gluconeogenesis and the glyoxylate shunt as well as changes in amino acid catabolism. These responses point to new possible mechanisms of longevity assurance in worms. The metabolic changes observed in dauer larvae can be explained by the existence of high levels of autophagy leading to recycling of cellular components.See associated minireview: http://jbiol.com/content/9/1/7.

Network Architecture and Mutational Sensitivity of the C. elegans Metabolome.

A fundamental issue in evolutionary systems biology is understanding the relationship between the topological architecture of a biological network, such as a metabolic network, and the evolution of the network. The rate at which an element in a metabolic network accumulates genetic variation via new mutations depends on both the size of the mutational target it presents and its robustness to mutational perturbation. Quantifying the relationship between topological properties of network elements and the mutability of those elements will facilitate understanding the variation in and evolution of networks at the level of populations and higher taxa. We report an investigation into the relationship between two topological properties of 29 metabolites in the C. elegans metabolic network and the sensitivity of those metabolites to the cumulative effects of spontaneous mutation. The correlations between measures of network centrality and mutability are not statistically significant, but several trends point toward a weak positive association between network centrality and mutational sensitivity. There is a small but significant negative association between the mutational correlation of a pair of metabolites (rM ) and the shortest path length between those metabolites. Positive association between the centrality of a metabolite and its mutational heritability is consistent with centrally-positioned metabolites presenting a larger mutational target than peripheral ones, and is inconsistent with centrality conferring mutational robustness, at least in toto. The weakness of the correlation between rM and the shortest path length between pairs of metabolites suggests that network locality is an important but not overwhelming factor governing mutational pleiotropy. These findings provide necessary background against which the effects of other evolutionary forces, most importantly natural selection, can be interpreted.

Visualizing Antimicrobials in Bacterial Biofilms: Three-Dimensional Biochemical Imaging Using TOF-SIMS.

Bacterial biofilms are groups of bacteria that exist within a self-produced extracellular matrix, adhering to each other and usually to a surface. They grow on medical equipment and inserts such as catheters and are responsible for many persistent infections throughout the body, as they can have high resistance to many antimicrobials. Pseudomonas aeruginosa is an opportunistic pathogen that can cause both acute and chronic infections and is used as a model for research into biofilms. Direct biochemical methods of imaging of molecules in bacterial biofilms are of high value in gaining a better understanding of the fundamental biology of biofilms and biochemical gradients within them. Time of flight-secondary-ion mass spectrometry (TOF-SIMS) is one approach, which combines relatively high spatial resolution and sensitivity and can perform depth profiling analysis. It has been used to analyze bacterial biofilms but has not yet been used to study the distribution of antimicrobials (including antibiotics and the antimicrobial metal gallium) within biofilms. Here we compared two methods of imaging of the interior structure of P. aeruginosa in biological samples using TOF-SIMS, looking at both antimicrobials and endogenous biochemicals: cryosectioning of tissue samples and depth profiling to give pseudo-three-dimensional (pseudo-3D) images. The sample types included both simple biofilms grown on glass slides and bacteria growing in tissues in an ex vivo pig lung model. The two techniques for the 3D imaging of biofilms are potentially valuable complementary tools for analyzing bacterial infection. IMPORTANCE Modern analytical techniques are becoming increasingly important in the life sciences; imaging mass spectrometry offers the opportunity to gain unprecedented amounts of information on the distribution of chemicals in samples-both xenobiotics and endogenous compounds. In particular, simultaneous imaging of antibiotics (and other antimicrobial compounds) and bacterium-derived metabolites in complex biological samples could be very important in the future for helping to understand how sample matrices impact the survival of bacteria under antibiotic challenge. We have shown that an imaging mass spectrometric technique, TOF-SIMS, will be potentially extremely valuable for this kind of research in the future.

Sex differences in the circadian profiles of melatonin and cortisol in plasma and urine matrices under constant routine conditions.

Conflicting evidence exists as to whether there are differences between males and females in circadian timing. The aim of the current study was to assess whether sex differences are present in the circadian regulation of melatonin and cortisol in plasma and urine matrices during a constant routine protocol. Thirty-two healthy individuals (16 females taking the oral contraceptive pill (OCP)), aged 23.8 ± 3.7 (mean ± SD) years, participated. Blood (hourly) and urine (4-hourly) samples were collected for measurement of plasma melatonin and cortisol, and urinary 6-sulfatoxymelatonin (aMT6s) and cortisol, respectively. Data from 28 individuals (14 females) showed no significant differences in the timing of plasma and urinary circadian phase markers between sexes. Females, however, exhibited significantly greater levels of plasma melatonin and cortisol than males (AUC melatonin: 937 ± 104 (mean ± SEM) vs. 642 ± 47 pg/ml.h; AUC cortisol: 13581 ± 1313 vs. 7340 ± 368 mmol/L.h). Females also exhibited a significantly higher amplitude rhythm in both hormones (melatonin: 43.8 ± 5.8 vs. 29.9 ± 2.3 pg/ml; cortisol: 241.7 ± 23.1 vs. 161.8 ± 15.9 mmol/L). Males excreted significantly more urinary cortisol than females during the CR (519.5 ± 63.8 vs. 349.2 ± 39.3 mol) but aMT6s levels did not differ between sexes. It was not possible to distinguish whether the elevated plasma melatonin and cortisol levels observed in females resulted from innate sex differences or the OCP affecting the synthetic and metabolic pathways of these hormones. The fact that the sex differences observed in total plasma concentrations for melatonin and cortisol were not reproduced in the urinary markers challenges their use as a proxy for plasma levels in circadian research, especially in OCP users.

The mutational structure of metabolism in Caenorhabditis elegans.

A properly functioning organism must maintain metabolic homeostasis. Deleterious mutations degrade organismal function, presumably at least in part via effects on metabolic function. Here we present an initial investigation into the mutational structure of the Caenorhabditis elegans metabolome by means of a mutation accumulation experiment. We find that pool sizes of 29 metabolites vary greatly in their vulnerability to mutation, both in terms of the rate of accumulation of genetic variance (the mutational variance, VM) and the rate of change of the trait mean (the mutational bias, ΔM). Strikingly, some metabolites are much more vulnerable to mutation than any other trait previously studied in the same way. Although we cannot statistically assess the strength of mutational correlations between individual metabolites, principal component analysis provides strong evidence that some metabolite pools are genetically correlated, but also that there is substantial scope for independent evolution of different groups of metabolites. Averaged over mutation accumulation lines, PC3 is positively correlated with relative fitness, but a model in which metabolites are uncorrelated with fitness is nearly as good by Akaike's Information Criterion.

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

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.

Fluorodeoxyuridine affects the identification of metabolic responses to daf-2 status in Caenorhabditis elegans.

Fluorodeoxyuridine (FUdR) is often used to help maintain synchronous populations of Caenorhabditis elegans adults, for instance as would typically be the case in studying age-related effects. However, given that FUdR inhibits DNA synthesis and therefore reproduction, it will clearly have significant wide-ranging biological effects. It is often assumed that these can be compensated for using appropriate controls. We show here that this is not the case for a metabolomic analysis of a long-lived daf-2 mutant strain: not only were the effects of FUdR much greater than the effects of the mutation, there were clear interactions between FUdR and genotype, such that identification of daf-2-dependent metabolites would have been compromised on FUdR plates. This indicates that FUdR should only be used with caution for C. elegans ageing experiments, and should not be assumed to be independent of other factors being studied.