The validation of biomarkers of metabolic efficacy in infant nutrition

Lead Research Organisation: MRC Centre Cambridge
Department Name: MRC Elsie Widdowson Laboratory

Abstract

Breastfeeding is the best start for a baby's life. The World Health Organisation (WHO) recommends that all women should exclusively breastfeed their babies until six months of age. However not all women have the opportunity to exclusively breastfeed their babies. In the UK it is reported that whilst 81% of mothers start with breast feeding only 17% of infants are exclusively breast-fed until 3 months, and 1% until 6 months of age. This does not have to be the mother's choice, already in the first weeks 13% of the breast feeding mothers get advice to provide the baby with extra feeding. Thus there is a need to design breast-milk alternatives that mimic the nutritional quality of breast milk as closely as possible.

Recent evidence suggests that non breast-fed infants have an increased risk of becoming obese and an increased risk of high blood pressure in later life. It is currently unclear why infant feeding practices have such a big impact on health outcomes later in life. However, breastfeeding seems to be metabolised differently by infants than formula milk; our research showed that babies that are breast fed have a significantly different blood lipid (fat) profile compared to bottle fed babies.

The aim of this project is to develop novel methods to help us understand the mechanism(s) of the differences in the metabolism of bottle fed babies and breast fed babies. We want to identify specific lipids that can be used as markers that reflect the benefits of breastfeeding. We will do this by exploring changes in the lipid profiles of babies in the UK during their first year of life. By comparing blood markers with patterns of infant growth we will be able to identify markers that can be used to assess the metabolic response to breast-milk alternatives. We will also include mothers and infants from rural and urban sub-Saharan Africa to explore between population differences in the association between mode of feeding, blood lipid levels, and infant health outcomes'.

One possible way in which mode of feeding may impact on the baby's metabolism is via bacteria in the gut (the gut microbiome). It is believed that, compounds in food, affect which bacteria live in the gut and that the different combinations of bacteria in the gut leads to different metabolic products that will be taken up into the blood stream. We do not know if these differences in the gut microbiome also affect the lipids in the blood. To explore this potential link, we will also study what the effect is of the gut microbiome of the babies on their metabolism.

Recently we developed a method using high resolution mass spectrometry to determine a detailed lipid profile covering over a 100 lipids from one dried blood spot. This is a blood sample obtained from a heel prick and only requires a drop of blood spotted on paper. This is the most suitable method to study the metabolism of babies. We will adapt this dried blood spot based method to captures both lipids and metabolites dependent on gut microbiome.

The identified markers, and the methods to measure these, will then be available for use by scientist and industry to study the effect of new formula or new feeding methods for infants who do not have the chance to be exclusively breast fed and give these babies the best possible start in life.

Technical Summary

The study of endogenous metabolism in healthy infants has remained largely unexplored. One of the main reasons is that repeated drawing of blood in healthy new-borns is regarded as over invasive. Dried blood spots (DBS) from heel pricks have long been established as the most appropriate sample format, to screen infants for inborn errors of metabolism. We recently developed and validated of a lipid profiling method using DBS from infants. This method provides data on approximately 100 endogenous lipids from a single 3.2 mm DBS disc. The process is automated, fast, robust, inexpensive and therefore suitable for studying large cohorts. We have already applied this method to a subset of DBS samples collected in the Cambridge Baby Growth Study and showed that lipid profiles of breast-fed infants are significantly different to formula-fed infants and that specific lipids are associated with growth rates. This work provides the basis for our hypothesis that the metabolism of breast-fed and formula-fed infants are differentially conditioned, which is reflected in clear differences in the lipid profiles at an early age. We hypothesize that these early detectable differences in metabolism are, at least partially, responsible for the phenomenon known as metabolic programming. This proposal aims to validate lipid biomarkers that reflect healthy metabolism associated with optimal neonatal growth. These biomarkers will deliver a robust indicator of metabolic efficacy of infant nutrition and will provide a practical tool that can assist the study of feeding strategies and development of new infant formulae for infants that cannot or should not be exclusively breastfed. We aim to make the research directly translational, through the delivery of standardised protocols, which allow for classification of a healthy or sub-optimal metabolic response. This can be implemented in studies of the role infant nutrition on metabolism and long term health.

Planned Impact

Exec Summary: The proposed project will provide: (a) a novel method, using a minimally-invasive sampling protocol, for the lipidomic assessment of the effects of early nutrition on metabolism; (b) the definitions of healthy and sub-optimal metabolic phenotypes; and (c) enable the development of formula milks that more closely mimic the metabolic responses observed in breast fed infants.

The market: According to the summary of the Euromonitor International report on baby food (http://www.euromonitor.com/baby-food-in-the-united-kingdom/report) in the UK the compound annual growth rate for baby food will remain constant at 5% over the next years rising to an estimated £1,046 million in 2018. The main reason for it is the rise in unit prices due to substantial product innovation. This clearly shows that companies involved in infant formula want to innovate and further develop better infant formula.

Need for innovation: It is essential for the development and innovation of new infant formula that there will be better tools to determine the metabolic effects of formula. Trials with new formula compositions are well designed but have to rely on gross phenotypic data like growth rate and BMI. The lack of biochemical markers that can help to assess the direct effect on metabolism and the lack of makers for aberrant growth, makes this research costly and slow.
Metabolic markers can therefore revolutionize study and trials of new formula. This can help to develop new feeding strategies for infants that cannot or should not be exclusively breastfed. Better feeding strategies for these infants will give them a healthier start of life and should reduce their risk of obesity and related disease in later life. This could have a significant impact on future healthcare costs throughout the life course by reducing the incidence of metabolic diseases.

Aims and outcomes: Our aim is to study the longitudinal changes in the metabolism of breastfed infants and compile an independent body of evidence on the metabolic effect of breast-feeding in optimal and suboptimal situation within the first year of life. It is not our aim to develop new formula or test current formula. The project will provide the tools and know-how for other scientist or industry to do these assessments. All data will be made open access within Metabolights and other relevant international repositories and will provide companies with sufficient background knowledge to test the metabolic efficacy of any new formula.

The final result of the proposed work will be a fully validated and translatable methodology that can be used to by other groups (science or industry). The methodology will give the ability to annotate the lipidomic profile from a DBS sample of an infant as normal or associated with sub-optimal growth. It should also be able to give evidence if the changes in diet lead to changes in the lipid profile that are associated with a healthy outcome. Each year we will organise practical workshops at MRC HNR on the methodology, which will be open to researchers from academia and industry. The objective of the workshops will be to train researchers to independently use dried blood spots in their lipid profiling studies using the SOP and to analyse their data.

This technology would for the first time give science community and industry the opportunity to determine if change to the formula lead to positive changes in the metabolism without having to wait for several years before the effect becomes measurable in the gross phenotypic data. The lipid profile also has the potential to be more sensitive than gross phenotypic data, which would yield a reduction of the number of infants necessary in a trial and thus a significant reduction of time and costs to test new formula.

Publications

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Acharjee A (2017) The translation of lipid profiles to nutritional biomarkers in the study of infant metabolism. in Metabolomics : Official journal of the Metabolomic Society

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Bond NJ (2017) massPix: an R package for annotation and interpretation of mass spectrometry imaging data for lipidomics. in Metabolomics : Official journal of the Metabolomic Society

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Furse S (2021) A pipeline for making 31P NMR accessible for small- and large-scale lipidomics studies. in Analytical and bioanalytical chemistry

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Furse S (2022) Altered Lipid Metabolism in Obese Women With Gestational Diabetes and Associations With Offspring Adiposity. in The Journal of clinical endocrinology and metabolism

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/M027252/1 30/09/2015 29/09/2016 £557,009
BB/M027252/2 Transfer BB/M027252/1 30/09/2016 29/09/2019 £453,545
 
Description We have validated the biomarkers for infant nutrition and we are in the process of validating biomarkers of infant growth and development. This work has been published and has been taken up and used by academia and industry.
The Diet and Health Research Industry Club (DRINC) steering committee summarised the project outcome as "High quality project with major implications for public health."
Exploitation Route Industry is aiming to use the nutritonal biomarkers to assess if newly developed formula is giving a metabolic response that is more like breastmilk than conventional biomarkers.
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology

 
Description We participated in the Cambridge Science Festival and in the Einsteins Garden of the Greenman festival. Both events allowed us to speak and interact with several 100s of people. We explained the complexity of breast milk and how lipid metabolism can give information about people's health and diet. In 2017 the work was presented on the NIHR BRC Cambridge open evening.
First Year Of Impact 2019
Sector Agriculture, Food and Drink,Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Children's Liver Disease Foundation funding
Amount £5,101 (GBP)
Organisation Children's Liver Disease Foundation (CLDF) 
Sector Academic/University
Country United Kingdom
Start 11/2017 
End 10/2018
 
Description ERA-HDHL call "Biomarkers for Nutrition and Health".
Amount € 1,034,440 (EUR)
Funding ID BB/P028195/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 04/2020
 
Description MRC Confidence in Global Nutrition and Health Research Initiative
Amount £53,900 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2018 
End 01/2019
 
Description Michael J Fox Foundation Biomarkers Fall 2018
Amount $500,000 (USD)
Funding ID not yet given 
Organisation Michael J Fox Foundation 
Sector Charity/Non Profit
Country United States
Start 03/2019 
End 02/2021
 
Description public-engagement funding
Amount £900 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 07/2016 
End 09/2016
 
Title Validated biomarkers for infant nutrition 
Description We have developed a lipid profiling method using dried blood spots from heel pricks to determine the diet of infants. In brief, the blood/analytes from a 3.2 mm DBS was extracted with 100 µl of MilliQ H2O in a well of a glass-coated 2.4 ml deep well plate (Plate+TM, Esslab, Hadleigh, UK), then 250 µl of MeOH was added. Lipids were partitioned into 500 µl of Methyl-tertiary-butyl ether. After centrifugation, the organic layer was concentrated and used for lipid analysis. Samples were infused into a Thermo Exactive benchtop orbitrap (Hemel Hampstead UK), using an Advion Triversa Nanomate (Ithaca US) and data acquired in both positive (+1.2 kV) and negative (-1.5 kV) mode voltages. All experiments were run with blank controls and two different quality control samples. In total, 218 lipid signals could be detected robustly using this method. The lipids have been identified as described previously (Koulman et al. 2014) and the identification is at level 2 of the Metabolomics Standards Initiative . 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact Research interest from different companies such as Danone and Mead Johnson 
 
Description BioFN project 
Organisation Erasmus University Rotterdam
Department Department of Public Health
Country Netherlands 
Sector Academic/University 
PI Contribution taking the lead on the BioFN project to obtain funding to study infant lipid metabolism in relation to body fat distribution of childhood obesity risk
Collaborator Contribution Providing clinical expertise and access to samples of the sophia pluto cohort from Rotterdam and providing bioinformatics capability from DTU.
Impact This collaboration led to the success funding application
Start Year 2016
 
Description BioFN project 
Organisation Technical University of Denmark
Department Department of Systems Biology
Country Denmark 
Sector Academic/University 
PI Contribution taking the lead on the BioFN project to obtain funding to study infant lipid metabolism in relation to body fat distribution of childhood obesity risk
Collaborator Contribution Providing clinical expertise and access to samples of the sophia pluto cohort from Rotterdam and providing bioinformatics capability from DTU.
Impact This collaboration led to the success funding application
Start Year 2016
 
Description Dutch Famine Cohort 
Organisation University of Amsterdam
Department Swammerdam Institute for Life Sciences
Country Netherlands 
Sector Academic/University 
PI Contribution Collaboration on the effect of famine during pregnancy on lipid metabolism
Collaborator Contribution Amsterdam provide samples of the cohort
Impact The collaboration aims to understand the effect of prenatal famine on lifelong lipid metabolism
Start Year 2017
 
Description High-throughput single cell unbiased lipidomics assay in human iPSC-derived dopamine neurons. 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution The development of single cell lipidomics approaches to study the lipid metabolism of dopageneric neurons in relation to parkinson's disease
Collaborator Contribution Changes in lipid metabolism have been strongly associated with Parkinson's disease (PD). Lipids have the potential to be important biomarkers for PD, but the lack of appropriate tools to measure total lipids (lipidome) in human dopamine neurons has limited advances in this field. Here, we propose to develop a novel unbiased and high-throughput single cell assay to determine total lipids. This will allow us to identify and validate lipid metabolites in dopamine neurons, which may serve as biomarkers for PD.
Impact project is ongoing
Start Year 2019
 
Description Understanding Society Health Innovation Panel: Biomeasure And Health Data Collection From The Innovation Panel Of The Uk Household Longitudinal Study 
Organisation University of Essex
Country United Kingdom 
Sector Academic/University 
PI Contribution The measurement of biomarkers in dried blood spots
Collaborator Contribution Organise the dried blood spot collection and other data collection
Impact still in progress
Start Year 2019
 
Description bibins 
Organisation Queen Mary University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution To collaborate on the BiBins study
Collaborator Contribution providing samples of the Bibins cohort
Impact samples have been analysed, data analysis is in progress
Start Year 2017
 
Description infant gut microbiome metabolism 
Organisation Quadram Institute Bioscience
Country United Kingdom 
Sector Academic/University 
PI Contribution In this collaboration we try to understand how nutrition impacts on the gut microbiome. We determine how the lipid profile of changes when different types of milk are incubated with different gut microbiomes.
Collaborator Contribution The IFR helps with the determining the composition of the gut microbiome.
Impact na
Start Year 2015
 
Description infant lipid metabolism 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Providing lipid profiling of infant samples (dried blood spots) and breast milk samples
Collaborator Contribution Providing samples
Impact this work let to multiple publications and the successful BBSRC-DRINC project.
Start Year 2013
 
Description Participated in LifeLab Peterborough 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Using the Fortune telling molecules to explain nutritional biomarkers.
Year(s) Of Engagement Activity 2018
URL https://www.camlifelab.co.uk/peterborough
 
Description The Fortune Telling Molecules, explaining nuritional biomarkers to the audience of the Einstein's Garden at the Green man Festivel 
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 Over 4 days we have spoken to hunderts of people about nutritional biomarkers and got them involved in a hands-on activity.
Year(s) Of Engagement Activity 2018
URL https://www.greenman.net/news/discover-whats-landing-in-einsteins-garden/
 
Description engage with public at Cambridge Science Fesitival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact We, the Lipid Profiling group at MRC Human Nutrition Research study fats in the blood of large numbers of people. Everyone has thousands of different fat molecules in their blood. With mass spectrometry we try to measure as many of these fats as possible. This has allowed us to complete a number of puzzles about which fats are related to future health and disease - we call these fats 'markers'. We will tell you about how we found markers for predicting the future risks of heart attacks and diabetes.
Year(s) Of Engagement Activity 2016
URL http://www.sciencefestival.cam.ac.uk/events/blood-fat-and-future
 
Description participated as book in the human library 
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 I participated in the human library as the book "From the Breast to the Brain, our First Fatty Food" and discussed with members of the public who were interested the research into breastmilk.
Year(s) Of Engagement Activity 2016
URL http://humanlibraryuk.org/