MRC Elsie Widdowson Laboratory

Stable Isotope Facility

The MRC Elsie Widdowson Laboratory (MRC EWL) Stable Isotope Facility, led by Dr Michelle Venables, builds on over 25 years of pioneering research experience initiated by Drs Andy Coward and Les Bluck. The facility provides highly specialised technical and analytical support for a range of research studies, and the UK nutrition surveys, frequently in conjunction with national and international collaborators. We specialise in the analysis and modelling of the stable isotopes of hydrogen (2H), Carbon (13C), Nitrogen (15N) and Oxygen (18O). Working alongside our collaborators on a variety of projects, we provide input to study design, individual dosing and collection kit preparation, sample analysis, data modelling and interpretation.

For further information and enquiries please contact the stable isotope team at stable.isotopes@mrc-ewl.cam.ac.uk.

Instrumentation 

  • Dual inlet-IRMS (Isoprime)
  • Continuous flow IRMS (AP 2003)
  • Continuous flow IRMS (Sercon ABCA-Hydra 20-22)
  • Continuous flow IRMS (Sercon ABCA)
  • GC-MS (Agilent 7890B-5977B)
  • GC‑MS (Agilent 6890-5973)
  • GC-CP- IRMS (Sercon orchid-Hydra 20-22)

Stable Isotope Facility Specialisms

  • Energy Expenditure and Physical Activity
  • Body Composition
  • Pregnancy, Lactation and Complementary Feeding
  • Glucose Metabolism (including insulin sensitivity)
  • Lipid Metabolism
  • Nitric Oxide Production
  • 13C breath tests (including gastric emptying)

Energy Expenditure and Physical Activity

The gold standard method for the measurement of total energy expenditure (TEE) in free-living subjects is the doubly labelled water (DLW) method, pioneered by Dr Andy Coward and members of MRC EWL in the UK.

The principle of the method is that following the collection of a pre-dose (baseline) sample, subjects receive a drink of water labelled with the stable isotopes 2H and 18O and then provide either saliva or urine samples for up to two weeks afterward. These samples are measured using isotope ratio mass spectrometry (IRMS). The difference in the rates of disappearance of the 2H and 18O from body water provide a measurement of carbon dioxide (CO2) production, which in turn can be used to estimate the rate of energy expenditure in kJ/day.

Recent collaborations include:

  1. National Diet and Nutrition Survey (Public Health England): Validation of methods of self-recorded dietary intake
  2. UK Biobank (MRC Epidemiology): Validation of physical activity monitors for determination of energy expended
  3. Erasmus MC: Applications to determine energy requirements pre- and post- interventions in clinical populations
  4. Ministry of Defence (UK): Measurement of energy expenditure during basic recruit training
  5. English Institute of Sport: Measurement of energy expenditure during periods of training
  6. The EU Childhood Obesity Programme (CHOP): MRC EWL has collaborated with multiple EU members testing the hypotheses linking the mode of infant feeding to later obesity and energy expenditure

Body Composition

At MRC EWL, we use the deuterium dilution technique to estimate Total Body Water (TBW). From this we can estimate Lean Body Mass and Fat Mass. We supply doses of deuterium oxide solution (2H2O) for studies and measure the deuterium enrichment in body water. We are experienced in modelling both the plateau and back extrapolation technique. Depending on which technique is used, they require either saliva samples over 5 hours or urine samples over 5 days, respectively.

Recent collaborations include:

  1. Optimized complementary feeding study on body composition (Umeå University, Sweden)
  2. Metabolic Reference Measurements in Adults and children (University of Cambridge): Validation of DXA measurements using TBW calculations in the four compartment model
  3. Body composition and metabolic profile in children with end stage liver disease (King’s College Hospital NHS Foundation Trust R&D): Clinical applications to answer questions relating to TBW in renal failure
  4. Efficacy of the motivational approach for treating childhood obesity (Institut d’Investigació Sanitària Pere Virgili, Spain). Body composition following lifestyle interventions

Lactation and Complementary Feeding

The ‘deuterium oxide dose to mother’ technique is an accurate method for calculating breast milk intake. The mother drinks a dose of 2H labelled water, and samples of urine and/or saliva are collected from both mother and baby. Additionally, it can also provide body composition data for the mother.

Recent collaborations include:

  1. Cambridge Baby Growth Study II (University of Cambridge)
  2. The effect of prenatal and early childhood nutritional supplementation on infant and early childhood body composition, growth and development (ENID) (MRC International Nutrition Group, The Gambia)

Glucose Metabolism (including insulin sensitivity)

Long-term diet and exercise interventions are beneficial in reducing obesity and preventing type 2 diabetes. Investigative studies of the effectiveness of these interventions invariably incorporate insulin sensitivity and secretion measurements and important aspects of glucose metabolism can be quantified by using the minimal model of glucose and insulin kinetics to interpret these data.

Incorporating stable isotopes into the glucose dose improves these tests allowing the separation of glucose disposal from endogenous production and increasing the precision of the physiological parameters measured. We have in-house expertise in insulin sensitivity modelling and measurement.

We reduced the amount of tracer used, and hence the cost, of a well-established intravenous glucose challenge for the assessment of peripheral insulin sensitivity. We accomplished this by substituting 13C-glucose for 2H-glucose and using the more precise technique of GC/C/IRMS for the analysis (Clapperton et al 2002 and Bluck et al 2005).

As administration of the reduced amount of tracer does not affect extant glucose kinetics, 13C-glucose can be used in combination with the more physiologically relevant standard oral glucose tolerance tests in the Oral Dose Intravenous Label Experiment (ODILE) (Bluck et al 2006 and Bluck et al 2013).

A novel method was also developed to measure different aspects of

glucose metabolism using a 2H labelled meal test (Pennant et al 2006 and Pennant et al 2006). The test involves the addition of the stable isotope into a drink or meal alongside a 13C intravenous dose. This sophisticated but physiological test has being optimised and validated in subjects with type 1 diabetes (Pennant et al 2008).

Recent collaborations include:

  1. The effects of green tea catechins (GTC) and chlorogenic acid (CGA) on insulin sensitivity and cardiovascular function in women with Polycystic Ovary Syndrome (PCOS), obesity and insulin resistance (University of Cambridge)
  2. Coffee and glucose metabolism: clinical, crossover and randomized glucose disposal assay using stable isotopes (University of Brazil)
  3. Tolerance of fasting in small for gestational age vs. appropriate for gestational age adults (Steno Diabetes Center, Denmark)

Lipid Metabolism

Metabolic dyslipidaemia is characterized by high circulating triglyceride and low HDL levels and is frequently accompanied by hepatic steatosis. Increased hepatic lipogenesis contributes to both of these problems. The fact that insulin fails to suppress gluconeogenesis but continues to stimulate lipogenesis in obese and lipodystrophic insulin resistant mice has led investigators to propose the existence of a selective post-receptor defect in hepatic insulin action.

As part of a large collaborative study in which severely insulin resistant patients with insulin receptor mutations were characterized, we measured the degree of lipogenesis these patients exhibited. This involved measurements of body water enriched by a large oral dose of heavy water (2H2O) and deuterium incorporation into plasma triglyceride. Combined, these can be used to determine the fractional synthetic rate of fatty acids, and hence assess the degree of de-novo lipogenesis.

Prior studies have implicated MHCII (major histocompatibility complex Class II molecule) instability or low CLIP (class II-associated II peptide) affinity in autoimmune pathogenesis. The regulation of MHCII turnover in vivo is largely unexplored, however, due to a lack of methodology. To address this, heavy water (2H2O) is being used as a non-interfering label for studying in vivo the dynamics of proteins (by 2H labelling of nonessential amino acids) and, simultaneously, of the cells that harbour them (by 2H labelling of DNA).

Recent projects and collaborations include:

  1. Genetics in severe insulin resistance (Wellcome Trust Clinical research Facility, University of Cambridge)
  2. Investigating Serum Palmitate and Myristate-containing Triglycerides as Markers of Hepatic De-Novo Lipogenesis using Lipidomic Mass Spectrometry and Stable Isotope Methodology (University of Cambridge)
  3. Comparison of strategies to deliver dietary lipid to human volunteers (MRC EWL)

Nitric Oxide Production

We have developed an inexpensive and non-invasive method for measuring whole body NO in man (Siervo et al 2011), which we have used to show reduced NO production in the metabolic syndrome.  Furthermore we have shown an association between NO production and insulin sensitivity in a group of normal weight and obese subjects

Nitric oxide (NO) is an important signalling molecule which regulates a number of physiological processes. It promotes blood vessel relaxation and maintains vascular tone, and plays a prominent role in cardiovascular health. Impairments in nitric oxide production and insulin sensitivity are expected to go hand-in-hand in a number of disease states.

Nitric oxide production is difficult to assess directly as the molecule itself has a very short half-life in blood and tissue. Rates of whole-body production are assessed from rates of generation of its metabolites, nitrite (NO2) and nitrate (NO3).  Both these are found widely in a normal western diet, which confounds the assessment of NO production by a simple measure of urinary excretion.  However stable isotope methods obtain estimates of NO production with sufficient precision to be useful.

Recent collaborations include:

  1. The effects of green tea catechins (GTC) and chlorogenic acid (CGA) on insulin sensitivity and cardiovascular function in women with Polycystic Ovary Syndrome (PCOS), obesity and insulin resistance (University of Cambridge)
  2. Nitric Oxide Production and Insulin Sensitivity in Obese Subjects with Metabolic Syndrome (University of Cambridge)
  3. Systemic nitric oxide production in Tanzanian children with sickle cell disease (Muhimbili University of Health & Allied Sciences and London School of Hygiene & Tropical Medicine)

13C breath tests (including gastric emptying)

The 13C-breath test provides a safe and non-invasive diagnostic tool for a number of clinical conditions, including gastrointestinal function and nutritional status.

The principle of the breath test is based on the administration of a substrate with a functional group labelled with naturally occurring 13C. During digestion, the substrate is broken down and metabolised before being released in breath. Isotope ratio mass spectrometry is used to measure the 13C content of the breath before and after ingestion of the labelled substrate.

The rate of appearance or level of enrichment of the 13C in the breath is determined by the rate-limiting step located at the site of the organ or enzyme function under investigation. The breath test is a non-invasive probe of how well the function is performing, and can be used to detect impairment of physiological processes and the clinical status of the subject.

Breath tests can investigate processes such as:

  • organ function, such as gastric emptying, liver dysfunction, pancreatic dysfunction and intestinal integrity
  • bacterial colonisation such as helicobacter infection in the stomach and bacteria affecting the small intestine

Recent collaboration:

  1. The effects of changing the liquid/solid content of an isoenergetic test meal on gastric emptying (University of Cambridge)