Effect of the circadian clock time of day and sleep on the human metabolome: identification of metabolite rhythms

Lead Research Organisation: University of Surrey
Department Name: Biochemistry & Physiology

Abstract

The study of metabolomics is a relatively new technique to identify metabolites that are produced by body processes and are involved in regulatory metabolic pathways. It is highly likely that certain metabolites will be altered in disease or during drug therapy and thus, being able to measure and identify these (biomarkers) will be critical for future health and disease diagnostics. For metabolomic profiling to be of value for clinicians in the diagnosis of disease, however, it is essential to establish accurate baseline data from healthy controls. Correct interpretation of metabolomic data will require a thorough knowledge of the impact of time of day as well as the effect of a person's internal biological (circadian) timing system on the metabolomic profile. Biological circadian rhythms and time of day variation occur in most physiological markers e.g. melatonin, cortisol, glucose; metabolites identified in plasma and urine will be no exception. However, to date there has been no systematic study of circadian variation in the human metabolome using established circadian protocols. In addition how a typical living environment (light/dark cycle, sleep/wake cycle, meals) affects metabolomic profiles needs to be determined. Using strictly controlled laboratory studies in healthy volunteers and cutting edge metabolomic technology we thus aim to characterise the effect of the circadian clock, the time of day, the light/dark environment, meals and sleep on rhythmic and non-rhythmic metabolites identified in plasma and urine. Metabolites that show rhythmic circadian and time-of-day variation (cycling) and those that do not (non-cycling metabolites), as well as metabolic processes affected by sleep and sleep deprivation, will be identified through the use of cutting edge, highly sensitive, liquid chromatography-mass spectrometric (LC-MS) techniques. At Surrey we have proven expertise in conducting circadian and sleep deprivation experiments. Using our recently established LC-MS methodology (Surrey and ICR) we have pilot data in healthy volunteers kept in controlled conditions similar to the proposed studies. Significant time of day variation has been observed in at least 20 plasma and 20 urine metabolites, based on Orthogonal Projections to Latent Structures (OPLS) analysis (Simca Software, Waters). Therefore in terms of expertise, clinical and analytical facilities and technical skills the proposal is feasible. Identification of metabolite rhythms and how these are affected by external factors (time of day, wakefulness, sleep, environmental lighting, regular meals) will provide reliable baseline data which will be crucial for the future use, and correct interpretation, of metabolomics in the detection and treatment of human disease. In addition, our Project Partner (Erasmus MC University Medical Center (EUMC), Rotterdam) will perform proteome and transcriptome analysis on selected samples across the 24 h day from both studies with a view to combining the data. The biological samples, metabolomic database and research findings will be shared and disseminated for the benefit of a wide range of professionals involved in disease diagnosis and treatment (e.g. clinicians, clinical biochemists) which will ultimately benefit society.

Technical Summary

Metabolomics is a relatively recent technology that has the potential to identify new biomarkers that may be modified in health, disease or during drug therapy. However, the biological variation in the human metabolome due to the internal circadian timing system, sleep/wakefulness, light/dark cycle and time of day variation has not been taken into account. Biological (circadian) rhythms and time-of-day variation occurs in most physiological markers (proteins, mRNA, hormones, metabolites). Disregarding this fundamental aspect of physiology may lead to the incorrect interpretation of metabolomic, transcriptomic and proteomic data. The proposed studies will test the primary hypothesis that there is rhythmic expression of certain metabolites that are linked to a person's internal circadian clock. Secondly the studies will test the hypothesis that these metabolic rhythms will be different in 'real life' conditions (light/dark cycle, meals, sleep). Laboratory studies will be performed in healthy volunteers, firstly under strictly controlled 'constant routine' conditions to minimise the effect of exogenous confounding factors (e.g. light, sleep, posture, activity, meals). Circadian-controlled and non-circadian controlled metabolites will be identified in plasma and urine extracts using highly sensitive liquid chromatography-mass spectrometric (LC-MS) techniques. In addition gene expression, proteome and peptidome profiles will be assessed in blood and urine samples taken under the same conditions (collaboration with EUMC) which will provide substantial added value. This project will generate reliable baseline data across the 24 hour day that will be crucial for the future use of metabolomics, proteomics and genomics in the detection and treatment of human disease. In addition, the findings will provide new insights into the metabolic processes and pathways linked to the circadian timing system and sleep processing.

Planned Impact

Scientific advancement and knowledge: The discovery of novel biomarkers for disease detection and progression relies on the ability to identify significant differences between healthy and diseased individuals, and high-quality, accurate baseline data. However, in order for such biomarkers to be identified and have clinical use, it is necessary to establish a reliable set of baseline data as well as a thorough knowledge of how they are influenced by both endogenous and exogenous factors across the day and night. Firstly we will characterise the impact of the internal biological circadian clock on metabolomic profiles in plasma and urine. Secondly we will determine the effect of normal conditions (of light/dark, sleep/wake and meals) on these metabolite rhythms. Key benefits of this study will be: 1. Profiles of an array of metabolite, genetic, protein and peptide biomarkers obtained under strictly controlled conditions (i.e. controlled lighting, wakefulness, isocaloric meals, and physical activity). 2. Baseline profiles of metabolites that do display a circadian rhythm, as well as those that do not (non-cycling). The Institute of Cancer Research (ICR; Raynaud) has a particular interest in identifying non-cycling metabolites that could be used diagnostically and to monitor drug efficacy. 3. Identification of where and how metabolite, protein and peptide pathways interact, thus providing a starting point for future studies with a more in-depth Systems Biology approach. 4. A better appreciation of how time-of-sampling affects metabolomic profiles which will improve the treatment and diagnosis of disease. 5. Determining the effect of sleep per se and sleep deprivation (e.g. as experienced by night shift workers) on the human metabolome. 6. A potential forensic application (thus our collaboration with EUMC) in the accurate determination of sample deposition time at a crime scene, as well as establishment of time-of-death and, in addition, whether the deceased was awake or asleep during the night. 7. The potential discovery of cycling metabolites that can be used as novel biomarkers of peripheral clocks. 8. The potential discovery of novel sleep-controlled metabolites and pathways. Public health: The findings and how these are used by clinicians, associated professions (e.g. clinical biochemists; pathologists; pharmacologists) to improve disease diagnosis and optimise its treatment will ultimately benefit the public. Improved knowledge of the complex processes underlying circadian timing and sleep will lead to more targeted treatment of sleep and circadian rhythm disorders (e.g. as experienced by night shift workers).The detrimental health effects of shift work and disrupted daily biological rhythms and sleep are now well known (demonstrated by the Surrey group and others). However, the biological basis of these detrimental effects is not well understood. Characterising the impact of sleep deprivation as experienced by night shift workers on metabolic profiles will add to our understanding of these damaging effects and ultimately direct changes that will benefit health. Increased production and output: Both the immediate and long-term effects of shift work are generally accepted to negatively affect the health and performance of the worker which has consequences for economic productivity and safety. It is recognised that current shift schedules may not be optimal for the health of the worker or industrial productivity. The ability to non-invasively monitor circadian timing in shift workers in the field using metabolomic profiling will ultimately help in optimising the design of shift schedules.

Publications

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publication icon
Davies SK (2014) Effect of sleep deprivation on the human metabolome. in Proceedings of the National Academy of Sciences of the United States of America

 
Description We have characterised time of day variation in the human metabolome and have shown that a large number of metabolite exhibit 24 h rhythmicity. In addition we have identified metabolites that vary during sleep and are affected by sleep deprivation in both males and females. The contribution of the endogenous circadian timing system to the metabolite rhythms has also been characterised.
Exploitation Route Our findings have been published and these have created a lot of interest and a number of collaborations (e.g. University of Cambridge; Imperial College; University of Lausanne; Washington State University; University of Helsinki, University of Padova). These have now been established to perform targeted metabolomics in different sleep and circadian protocols in humans and other species. Most recently we have shown that metabolomics is a powerful tool to measure peripheral clock function during simulated shift work. This will have important future applications in studies of shift workers in trying to establish the underlying mechanisms involved in the adverse metabolic effects of working shifts.
Sectors Aerospace, Defence and Marine,Communities and Social Services/Policy,Education,Environment,Healthcare,Leisure Activities, including Sports, Recreation and Tourism,Pharmaceuticals and Medical Biotechnology,Retail,Transport

 
Description Recognition that the time of day impacts on all blood diagnostic tests. Our findings have also led to a growing awareness of the importance of metabolomics as a tool in sleep and circadian rhythm research, opening up the prospect of sleep and circadian metabolic biomarkers. This had led to a series of International Biomarkers Workshops in the US and EU where all the international sleep and circadian societies have contributed. There is now an international effort of which we are part to standardise sleep and circadian biomarker studies to ensure high quality reproducible data that can be shared with groups across the globe.
First Year Of Impact 2016
Sector Education,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology,Retail,Transport
Impact Types Societal,Policy & public services

 
Description Genone scale analysis guidelines
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact The published guidelines is a multi-author paper and will act as a blue print for large scale omics analysis of time-series data.
 
Description IBRO Advanced Training
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact Delivery of recent research data to a group of African scientists attending the IBRO-UM5 Africa advanced Neuroscience School. Training of these scientists in circadian biology and sleep.
 
Description Sleep and Circadian Biomarkers
Geographic Reach Europe 
Policy Influence Type Membership of a guideline committee
 
Description Anticipation of meal time in humans
Amount £810,047 (GBP)
Funding ID BB/S01814X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 09/2022
 
Description EU FP7-HEALTH-2011
Amount € 5,997,889 (EUR)
Funding ID 278397 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 11/2011 
End 06/2017
 
Description FAPESP - CONFAP - UK Academies
Amount R$ 35,000 (BRL)
Funding ID 2016/50417-3 
Organisation São Paulo Research Foundation (FAPESP) 
Sector Public
Country Brazil
Start 05/2017 
End 04/2018
 
Description MRC Newton001
Amount £49,000 (GBP)
Funding ID MR/N006321/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2015 
End 10/2016
 
Description Santander Staff Mobility award
Amount £2,000 (GBP)
Organisation Santander Bank 
Sector Private
Country United States
Start 04/2014 
End 07/2014
 
Description Seasonal rhythms
Amount $2,025,000 (USD)
Funding ID NIH 
Organisation Columbia University 
Sector Academic/University
Country United States
Start 09/2016 
End 08/2021
 
Description Time is of the essence: Revealing the 'bone clock' in humans
Amount £32,604 (GBP)
Funding ID 420 
Organisation Royal Osteoporosis Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2019 
End 02/2021
 
Description UGPN Research Collaboration Fund
Amount £6,500 (GBP)
Organisation University of Surrey 
Sector Academic/University
Country United Kingdom
Start 08/2012 
End 07/2014
 
Title Metabolites in interstitial fluid 
Description We have validated a LC/MS targeted metabolomics method to measure metabolites in human interstitial fluid using high-resolution sampling 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? No  
Impact Our developed targeted metabolomics method will enable measurement of metabolites in human interstitial fluid collected via a unique ambulatory high resolution sampling device that was developed at University of Bristol. 
 
Title Targeted LC/MS metabolomics 
Description A targeted metabolomics method using liquid chromatography/mass spectrometry has been established in house at the University of Surrey. It can quantitatively measure between 130-180 metabolites in 10 ul plasma. Along with established assays to measure the circadian hormones, melatonin and cortisol, we can now characterise metabolite rhythms in health and disease. Establishing this technology has resulted in over 10 new research collaborations that have begun to produce joint publications. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact We are using targeted LC/MS metabolomics to characterise metabolite rhythms in health and disease. My team's findings have pioneered the area of sleep and circadian metabolomics. Following a large number of invited speaker symposiums at international sleep and circadian rhythms conferences (over 15 since 2014), I have been invited to a number of universities across the globe to present the technology and how it applies to sleep and circadian rhythm research. Establishing this technology as routine at Surrey has led to over 10 new research collaborations that are beginning to produce joint scientific publications. 
 
Title Circadian metabolomics 
Description Hourly blood samples from healthy young men and women kept in constant routine conditions for 40 h. A number of collaborations have arisen (Imperial College, University of Surrey, University of Sheffield and University of Birmingham) in which these human samples have been shared. These samples have been analysed by NMR, normal phase LC/MS and specific targeted analysis. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact The constant routine protocol allows endogenous circadian rhythms in the analytes measured to be observed. 
 
Title Human metabolomics time-series 
Description Time series samples have been collected from healthy volunteers undergoing controlled laboratory protocols designed to assess the effect of time of day, feeding/fasting; light/dark; sleep/wake on metabolite rhythms in plasma and urine. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Samples (plasma and urine) have been shared with collaborators at Imperial College. One joint publication showing the effect of sleep deprivation on NMR metabolites is published. Currently bile acids are being measured in the plasma samples to characterise daily and circadian rhythms in the bile acids. 
 
Title Sleep and ageing metabolomics data 
Description Biological samples taken during sleep, during sleep deprivation and following recovery sleep have been collected in both sexes. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact A number of collaborations have arisen (Imperial College and more recently University of Birmingham) in which the human samples collected during our sleep and circadian protocols have been shared. These samples have been analysed by NMR and normal phase LC/MS. One publication has already resulted. 
 
Description Bile acids 
Organisation Imperial College London
Department Imperial College Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Our expertise in chronobiology and sleep, access to biological samples, data and facilities
Collaborator Contribution Access to their facilities and expertise in metabolomics and bile acid measurment
Impact Research collaboration and research grant
Start Year 2015
 
Description Circadian and behaviour-driven metabolite rhythms 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We will share biological samples collected during our sleep and circadian studies as well as the previously analysed metabolomics data.
Collaborator Contribution Our partners have secured a Collaborative MIBTP PhD student to work on the project. The student will analyse our biological samples in their mass spectrometry facilities.
Impact Awarded a Collaborative MIBTP PhD studentship. The Midlands Integrative Biosciences Training Partnership 2 (MIBTP) is a BBSRC-funded doctoral training partnership between the University of Warwick, the University of Birmingham and the University of Leicester.
Start Year 2018
 
Description Circadian rhythms in bone markers 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution The constant routine protocol has generated plasma samples that are of value to assess circadian rhythms in bone in vivo by assessment of bone turnover markers.
Collaborator Contribution The partners will provide staff and facilities to measure bone turnover markers
Impact A grant has been obtained to fund this collaboration.
Start Year 2018
 
Description Circadian rhythms in bone markers 
Organisation University of Surrey
Department Department of Nutritional Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution The constant routine protocol has generated plasma samples that are of value to assess circadian rhythms in bone in vivo by assessment of bone turnover markers.
Collaborator Contribution The partners will provide staff and facilities to measure bone turnover markers
Impact A grant has been obtained to fund this collaboration.
Start Year 2018
 
Description Effect of shift work 
Organisation Washington State University
Country United States 
Sector Academic/University 
PI Contribution Performed targeted LC/MS metabolomics analysis
Collaborator Contribution Performed shift work study (day versus night work) in healthy volunteers
Impact Conference presentations
Start Year 2016
 
Description Gut microbiota 
Organisation Imperial College London
Department Department of Surgery and Cancer
Country United Kingdom 
Sector Academic/University 
PI Contribution Metabolomics LC/MS dataset; expert knowledge of circadian rhythms
Collaborator Contribution Expertise in gut microbiota analysis; training of research staff
Impact Collaboration is multi-disciplinary (physiology/pharmacology; analytical chemistry).
Start Year 2018
 
Description Metabolomics in HD 
Organisation University of Cambridge
Department School of Clinical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided metabolomics expertise; performed targeted LC/MS metabolomics on samples
Collaborator Contribution Knowledge of Huntington's disease; access to transgenic HD sheep and blood samples
Impact Publication (PMID:28223686)
Start Year 2014
 
Description Model of Hyperammonemia 
Organisation University of Helsinki
Country Finland 
Sector Academic/University 
PI Contribution Development of targeted LC/MS method to measure metabolites in rat brain tissue
Collaborator Contribution Designed and performed sleep deprivation experiments in a rat model of hyperammonemia; collected brain tissue samples
Impact Research publication and conference presentations
Start Year 2016
 
Description Model of Hyperammonemia 
Organisation University of Padova
Country Italy 
Sector Academic/University 
PI Contribution Development of targeted LC/MS method to measure metabolites in rat brain tissue
Collaborator Contribution Designed and performed sleep deprivation experiments in a rat model of hyperammonemia; collected brain tissue samples
Impact Research publication and conference presentations
Start Year 2016
 
Description NMR metabolomics 
Organisation Imperial College London
Department Imperial College Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Our expertise in chronobiology and sleep, access to biological samples, data and facilities
Collaborator Contribution Their expertise, access to NMR equipment and facilities
Impact Conference presentation Publication
Start Year 2013
 
Description Peripheral neuropathy metabolomics 
Organisation University of Milano-Bicocca
Country Italy 
Sector Academic/University 
PI Contribution We will perform targeted LC/MS metabolomics on samples collected by our partners
Collaborator Contribution Our partners will perform experiments (effect of chemotherapy on peripheral neuropathy in an animal model) and collect samples for our analysis
Impact Experiments are ongoing.
Start Year 2018
 
Description Seasonal rhythms in the metabolome 
Organisation Columbia University
Country United States 
Sector Academic/University 
PI Contribution We have helped in the protocol design of this human seasonal experiment. The controlled laboratory study was conducted at the University of Surrey facilities. We will analyse both the melatonin and cortisol rhythms as well as perform targeted LC/MS metabolomics analysis on the samples across the day and across the seasons.
Collaborator Contribution Our partner secured NIH funding for this study to be conducted at Surrey.
Impact Preliminary work was presented as a poster at EBRS2019 Congress, Lyon, August, 2019. Collaboration is multi-disciplinary (chronobiology; immunology; evolutionary biology)
Start Year 2015
 
Description Tissue fluid sampling 
Organisation University of Bristol
Department School of Clinical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Developed targeted metabolomics method to measure metabolites in human interstitial fluid
Collaborator Contribution Performing healthy volunteer study to collect blood and interstitial fluid samples
Impact Experiments are ongoing. No outputs yet
Start Year 2016
 
Description BBC Horizon documentary 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Scientific advisor on BBC Horizon documentary addressing the human body clock and circadian rhythms in a time-free environment. Designed to educate the public about body clocks and circadian rhythms.
Year(s) Of Engagement Activity 2018
URL https://www.bbc.co.uk/programmes/b0bn5ys4
 
Description Design Museum Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Around 100 members of the public attended a day Workshop (Designing Time) at the London Design Museum. I gave a talk about Body Clocks. Other speakers addressed social and philosophical aspects of time and time keeping. The talks were interspersed with performance of music and dance.
Year(s) Of Engagement Activity 2019
URL https://designmuseum.org/whats-on/talks-courses-and-workshops/designing-time-circadian-dreams
 
Description International Biomarkers Workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Representatives from international Sleep and Circadian Rhythm Societies attended a day workshop (Boston, US) to discuss standardisation of methods to uncover sleep and circadian biomarkers. A white paper has been proposed.
Year(s) Of Engagement Activity 2017
 
Description Milano-Bicocca Workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Gave a talk at Neurobiology Workshop (University of Milan-Bicocca) emphasising the value of Metabolomics as a tool for neuroscience research. Developed a research collaboration with Prof Guido Cavaletti. We are actively seeking research funding.
Year(s) Of Engagement Activity 2017
 
Description Press release "New study reveals how shift work disrupts metabolism" 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Following publication of our PNAS paper, Washington State University and University of Surrey had a Press release "New study reveals how shift work disrupts metabolism". This had very wide coverage across the globe. Article picked up by over 150 new agencies, e.g. ANI (India) - July 10, 2018; Boston Globe - July 13, 2018; CBC Radio "Quirks & Quarks" (Canada) - July 13, 2018; Daily Mail (UK/US)- July 9, 2018; Deutschlandfunk Nova (Germany) - July 10, 2018; The Guardian (UK)- July 9, 2018; Le Scienze (Italy) - July 10, 2018; NIH Research Matters - July 31, 2018; Times of India (India) - July 10, 2018; XinhuaNet (China) - July 9, 2018.
Year(s) Of Engagement Activity 2018
URL https://www.surrey.ac.uk/news/new-study-reveals-how-shift-work-disrupts-metabolism
 
Description Royal Institution 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Public lecture at the Royal Institution along with Prof R Foster and Prof S. Lightman.
Year(s) Of Engagement Activity 2016
URL http://www.rigb.org/whats-on/events-2016/february/public-science-of-sleep
 
Description Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Gave a talk (How your body tells the time) at Winchester Science Festival, 20th July, 2013, audience comprised of children and adults from the general public
Year(s) Of Engagement Activity 2013
 
Description Somerset House 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Around 80 people attended a 24 h exhibition at Somerset House, London, 8th February (Event: 24 Hours in Uchronia with Helga Schmid). I gave a talk entitled Body Clock/s in a 24/7 World.
Year(s) Of Engagement Activity 2020
URL https://www.somersethouse.org.uk/whats-on/24-hours-uchronia-helga-schmid
 
Description WSU visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Gave a University lecture to WSU staff and researchers (Spokane, US) highlighting the potential of metabolomics as a tool in sleep and circadian rhythms research. A research collaboration with Prof. Hans van Dongen and his team has resulted.
Year(s) Of Engagement Activity 2016