The metabolomic study of healthy ageing applying dried blood and urine spot collection

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

BACKGROUND AND SCIENTIFIC PROBLEM - The lifespan of the UK population is increasing at a greater rate than healthspan providing longer periods of ill health in later life and the associated economic strain on the NHS. There is a necessity to improve the healthy ageing of the UK population. The role of sedentary behaviour vs. exercise is being shown to have a strong influence on the healthy ageing-metabolism axis; exercise is a positive lifestyle intervention available to all. Metabolic changes can be studied applying nontargeted metabolomic analysis of thousands of metabolites. In these studies, biofluid samples are collected in a specialised centre which requires the subject to travel and expert staff and instruments to collect and process the samples; this process is costly and inefficient. However, sample collection away from specialised centres will reduce sample collection costs and allow longitudional studies to be performed cheaply and at minimal inconvenience to the subjects. The collection of dried blood or urine spots (DBS/DUS) away from specialised centres followed by metabolomic analysis offers a simple and robust solution. These devices are currently applied for targeted metabolite assays and we wish to develop this technology for non-targeted metabolomic studies of healthy ageing.
SCIENTIFIC OBJECTIVE - We propose the development and application of dried blood and urine spot collection with non-targeted metabolomics to be applied in the study of healthy ageing.
AIM 1 - Develop analytical methodologies for non-targeted metabolomic analysis of dried blood and urine spots. The small sample volumes (typically < 5microL) are analytically challenging in relation to sensitivity and range of metabolite classes detected. Here, two analytical techniques will be developed and assessed for non-targeted metabolomic analysis; (1) Liquid Extraction Surface Analysis (LESA) coupled to mass spectrometry for rapid and automated sample extraction and analysis and (2) capillary liquid
chromatography-mass spectrometry for analysis of lipid and hydrophilic classes of metabolites.
OUTPUT - analytical methods applicable for the non-targeted metabolomic analysis of dried blood and urine spots.
AIM 2 - Study the stability of dried blood and urine spots on currently available collection materials applying methods developed in aim 1; the metabolite stability is a concern in these studies. Here, the stability of dried blood and urine spots collected on a range of currently available papers and cards will be assessed for up to 18 months. At the same time new collection systems coated with enzyme and protease inhibitors will be developed and studied to assess their applicability. Both of these studies will assess stability and diversity of metabolite classes detectable.
OUTPUT - protocol and device for dried blood and urine spot collection.
AIM 3 - Apply the methods developed in (1) and (2) to study metabolism in sedentary behaviour and exercise in adults. With collaborators in healthy ageing and exercise at the University of Birmingham a longitudional metabolomic study of sedentary vs. exercise behaviour in adults applying DBS/DUS will be performed.
OUTPUT - mechanistic information on changes in metabolism in sedentary vs. exercising subjects at
different ages.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M015890/1 01/10/2015 30/09/2019
1653445 Studentship BB/M015890/1 01/10/2015 30/09/2019 Elliott Andrew Palmer
 
Title 3d printed template 
Description It is a 3d printed template to attach to the Advion Triversa Nanomate nESI instrument used in liquid extraction surface analysis mass spectrometry analysis. 
Type Of Art Artefact (including digital) 
Year Produced 2018 
Impact Allows the analysis of an increased number of dried blood spots at the same time. 
 
Description Key findings so far include:
- Providing samples are kept at reduced temperature (less than 4 degrees celcius), DBS and DUS are suitable for short to medium term (>1 month) storage of samples aimed for use within untargeted metabolomic studies.
- Assays providing comprehensive analysis of lipids also show increased stability than polar metabolites and therefore maybe suitable to even longer term storage at temperatures which would not be suitable for whole biolfuid.
- There is a high degree of overlap between features present in both Dried Blood Spots and Blood Plasma, as well as between Dried Urine spots and whole urine
- Small lower molecular weight compounds are able to be detected using Liquid Extraction Surface Analysis Mass Spectrometry, however the number of features detected when compared to Direct Infusion Mass Spectrometry and LCMS, is an order of magnitude lower.

A liquid extraction surface analysis method has been developed for the analysis of Dried Blood Spots which allows high throughput surface analysis of DBS possible. Using this method 20 samples per hour can be analysed and provide comparative results to Direct Infusion Mass Spectrometry without the need for manual sample extraction.
Additionally, as part of the project investigating hypervitaminosis A in young children, it was determined that Dried Serum Spots give the same biological conclusions as serum therefore they represent a potential suitable alternative to serum and plasma.
Exploitation Route DBS and DUS could be used to further the scope of metabolomic research within the wider community. Larger scale studies can be planned and performed at greatly reduced cost with less discomfort to patients. The volumes required will reduce storage costs as well as the larger number of samples collected benefiting statistical analysis of studies.
Sectors Healthcare,Security and Diplomacy

 
Description The ability to use dried blood spots (DBS) for untargeted metabolomic studies will greatly increase the number of potential patients who are able to give samples. Therefore increasing the number of samples able to be collected will drastically increase the statistical power of any experiment undertaken. Additionally, this will further widen the range of experiments that can be undertaken with regard to collecting samples either from patients who cannot make it into the clinic or collecting samples from remote locations before transport back to the laboratory. From current findings, the stability of DBS is suitable enough that (with the correct materials and procedures) patients are able to collect their own samples and send them either to the clinic or laboratory. Researchers are then able to move the samples into a preferred storage environment (such as a -80 degrees Celsius freezer) and be sure of the changes undergone to the metabolome during transport. Additionally, the ability to use liquid extraction surface analysis (LESA) techniques for the untargeted analysis of DBS will further allow mass spectrometry to be used as a screening tool for a wide range of conditions. With no sample preparation required of samples, high throughput of samples will be possible allowing for much faster analysis of samples than is currently possible.
First Year Of Impact 2018
 
Title Development of a LESA-MS method for the analysis of DBS 
Description Method to analyse dried blood spots applying liquid extraction surface analysis mass spectrometry. Method is able to extract thousands of metabolic features from a simple 5 second extraction from the surface of a dried blood spot with no prior sample preparation other than allowing the dried blood spot to dry for a couple of hours at room temperature. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact Method has similar performance characteristics as direct infusion mass spectrometry which required prior sample extraction. 
 
Description Study of Hypervitaminosis A in young children in Guatemala and Philippines 
Organisation Newcastle University
Department School of Agriculture Food and Rural Development Newcastle
Country United Kingdom 
Sector Academic/University 
PI Contribution As part of a wider project surrounding the study, I applied untargeted UHPLC-MS metabolomics methods looking at comparison between Dried Serum Spots and Serum.
Collaborator Contribution The partners involved in this collaboration were involved in the collection and preparation of samples from Guatemala and the Philippines.
Impact No outputs so far
Start Year 2017
 
Description Poster presented at the 1000 Elder's group Open Day at University of Birmingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Study participants or study members
Results and Impact Presented a poster at the 1000 Elder's group Open Day at University of Birmingham medical school. This was to advertise potential future studies to participants who are part of the 1000 elders group within Birmingham. This cohort is a group of members of the public who are aged 65 or above and interested in participating in research projects within the University of Birmingham.
Year(s) Of Engagement Activity 2016
 
Description Presentation for Institute of Metabolism and Systems Research group at the University of Birmingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Gave a presentation at the Institute of Metabolism and Systems Research away day regarding my PhD project and what it will involve. IMSR is a group of researchers from the University of Birmingham performing research surrounding metabolism and systems biology.
Year(s) Of Engagement Activity 2016