Stable Isotope Methodology

Stable isotope tracers provide a safe and powerful tool to investigate carbohydrate, fat, and protein metabolism, especially to asses substrate fluxes in vivo in humans. Isotopes are elements which share the same place in the periodic table. As such, they have the same number of protons, but differ in their atomic mass, which enables us to distinguish between them. Common stable isotopes such as 1H, 12C, 14N and 16O make up most of our biological environment. In contrast, 2H, 13C, 15N and 18O do also occur in nature though they are by definition rare (0.02-1.11%). These rare stable elements, can be incorporated in organic molecules like glucose, fatty acids and amino acids and can subsequently be applied as tracers to study carbohydrate, fat and protein metabolism. In various nutritional studies these labelled substrates have been administered orally to determine their subsequent oxidation rates. For example, the oxidation rate of ingested, 13C-labelled carbohydrate can be quantified by determining the amount of 13CO2 that is excreted in the expired breath.



Besides only determining the oxidation rate of orally administered labelled substrates, stable isotope tracers can be applied to provide information on the dynamics of (endogenous) substrate turnover. Substrate utilisation rates from different endogenous fuel stores can be determined by combining indirect calorimetry with dynamic tracer dilution techniques. The basics of dynamic tracer dilution can be explained when tracer is added to a particular body pool at a constant rate (for example, into the circulation by way of continuous infusion) and samples (for example blood, breath, muscle and/or urine samples) are collected at defined intervals in time. As the applied tracer will equilibrate throughout the entire pool, the rate of flux of the tracer and, therefore its tracee, can be calculated. The movement of the tracee (for example, the substrate flux that is investigated) can simply be calculated as the rate of entry of the applied tracer into the pool (infusion rate) divided by the extent of labelling of the pool (the steady-state enrichment). Of course, this is a very simplified example, as there are various ways to apply all kinds of different tracers and to collect a wide variety of samples from different sampling sites. In addition, the calculation of substrate kinetics depends largely on the assumption of the (most appropriate) physiological model (for example static- or dynamic, single- or multiple-pool models) and mathematical approach (for example non-steady state or steady-state calculations).


Carbohydrate metabolism
In a wide variety of metabolic studies, intravenous infusions and/or the ingestion of labeled glucose tracers are applied to determine the oxidation rate of plasma or gut derived glucose, respectively. By quantifying total fat and total carbohydrate oxidation rates using indirect calorimetry, the respective use of muscle glycogen can be (indirectly) estimated. As such, changes in the use of the different endogenous carbohydrate sources can be followed for example during exercise conditions, allowing us to study the impact of training status, exercise intensity and/or sports nutrition on skeletal muscle metabolism. Other applications are the use of continuous glucose tracer infusion to study hepatic glucose output during pharmacological treatment of type 2 diabetes. Furthermore, many of our studies focus on modulating post-prandial blood glucose homeostasis. With the combined use of intravenous infusions with labeled glucose and the ingestion of food products or ingredients with another glucose tracer, we are able to define effective nutritional interventions that improve postprandial blood glucose homeostasis.



Fat metabolism
In a wide variety of metabolic studies, continuous intravenous infusions of labelled fatty acids (predominantly palmitate) are applied to determine the appearance, disappearance and/or oxidation rate of plasma derived free fatty acids. By quantifying total fat and total carbohydrate oxidation rates, using indirect calorimetry, the respective use of other fat sources (the sum of intramuscular and lipoprotein derived triglycerides) can be (indirectly) estimated. The latter allows the quantification of the use of the different endogenous and/or exogenous fat sources. As the appearance and disappearance rate of plasma fatty acids and/or glycerol can be determined using the appropriate tracers, we are also able to measure whole-body, adipose tissue, and/or muscle tissue lipolytic rate. Of course, conversion of carbohydrate to fat, and or storage of plasma derived fatty acids in the muscle triacylglycerol pool and/or adipose tissue are all processes that can be studied in such human in vivo settings.


Protein metabolism
Protein metabolism at the whole-body, muscle and organ levels in various physiological conditions is based on direct and indirect measurements of protein synthesis and breakdown, and exchange rates of amino acids and amino acid tracers between tissues. In our lab we have the expertise, equipment and methodology to use these amino acid tracers and study the effect of several exercise and nutritional interventions on whole-body and muscle protein turnover.



General model of protein metabolism used in the whole-body methods. Q, whole-body amino acid turnover or flux; Ra, rate of appearance in the plasma free amino acid pool; Rd, rate of disappearance from the plasma free amino acid pool; phe, phenylalanine; tyr, tyrosine.


Stable isotope tracers are widely used in biomedicine to study metabolic pathways in vivo, because they are functionally identical to the compound of interest (tracee), but distinct in some physical characteristics that enable their precise detection. Calculation of substrate kinetics traditionally measures the rate of appearance (Ra) which in steady state conditions equals the rate of disappearance (Rd) of the substrate (=flux). In a single pool model it is assumed that the infusion of tracer, sampling, and Ra of substrate occurs from a single, homogenous, instantly mixing pool. In metabolic studies, the blood compartment is usually viewed as a single pool, which implicates that blood sampling is allowed for the calculation of whole-body flux. In this model the rate of disappearance of amino acids from the blood compartment equals the rate of oxidation + protein synthesis, whereas the rate of appearance of amino acids equals the rate of protein breakdown + the rate of appearance of meal protein from the gut.
The use of the whole-body tracer balance methodology enables us to accurately measure the effect exercise and nutritional interventions on protein synthesis, breakdown, and oxidation at a whole-body level. A limitation of this technique, however, is that it does not provide information on the contribution of individual tissues to protein metabolism. As such, it does not allow the direct measurement of muscle protein synthesis rates.



To circumvent the limitation of whole-body tracer methodology, we use a different method to measure tissue or protein-specific synthesis rates; the amino acid tracer incorporation method. In most of these studies the amino acid tracer is provided by a continuous intravenous infusion until a steady state is obtained in the precursor pool for protein synthesis. Repeated muscle samples are then taken at steady-state. The protein is precipitated from the biopsy samples, hydrolyzed and the amino acids, after derivatization are analyzed for tracer enrichment using gas chromatography-mass spectrometry (GC-MS) or gas chromatography-isotope ratio mass spectrometry (GC-IRMS) methodology. The rise in tracer enrichment in the protein-bound amino acid fraction, over a given sampling time, is divided by the steady-state tracer enrichment in the precursor pool to give the fractional synthetic rate (FSR) of the protein, which is the percentage of the existing pool that has been synthesized over that time period.


An important feature in the above mentioned method is to maintain a steady state of the amino acid tracer in the precursor pool. When investigating a nutritional intervention involving protein and/or amino acids this implies that the nutrition must be fed periodically during the infusion period to prevent a drop in amino acid tracer concentration; which would create a non steady-state. These multiple bolus’ designs, however, do not represent the normal physiological process of protein intake. Only with use of an intrinsically labeled protein source (amino acid tracer incorporated into protein) can the normal physiological (single bolus) protein intake be mimicked, and provide us with a true indication for protein digestion rate and the subsequent muscle protein synthetic response. Recently, we have produced such an intrinsically labeled protein source with a high enrichment of amino acid tracer, and have successfully used it in human intervention studies. Together with the above mentioned expertise and methodology we have been able to take our muscle protein metabolism research to a higher level and we aim to provide a significant contribution to the current knowledge on this topic.


Stable isotope analyses
Though stable isotope methodology provides a excellent and safe means to investigate whole body substrate kinetics in vivo in humans, a large capital investment in technological instrumentation and substantial support facilities are needed for sample preparation, purification, and analysis. This support is provided by the Stable Isotope Research Center (SIRC) in Maastricht that is located in the Academic Hospital in Maastricht.



Due to the differences in molecular mass between the stable isotope tracer and its tracee, the relative abundance of tracer (tracer enrichment) can be measured by mass spectrometry (MS). Isotope-ratio mass spectrometry (IRMS) provides an extremely precise and accurate means to measure isotopic abundance, which is a necessity due to the minute amounts of tracer that are applied, the subsequent dilution in the body pool(s) followed by the appearance of label in the collected samples at an even lower abundance. The isotope-ratio mass-spectrometer is a dual inlet double-collector fixed-magnetic-field instrument designed for precise determination of, for example the 13CO2/12CO2 ratio. As such, using IRMS an isotopic abundance of 0.001 atom% excess (corresponding with a tracer/tracee ratio of less than 0.00001!) can be detected, with a precision of 0.0005 atom% excess. Depending on the collected samples, the compound that contains the labelled atom of interest, first needs to be extracted, separated, derivatised, isolated and/or oxidised to provide a gaseous product suitable for isotope-ratio mass analysis (like for example 13CO2). So, after sample preparation, gas chromatography (GC), and/or combustion (C), CO2 is injected in an ion source under vacuum. The gas molecules are bombarded by a stream of electrons, whereby they acquire a positive charge and are accelerated into a magnetic field. Here, the ionised gas molecules become segregated as a consequence of differences in molecular mass and strike individual collector plates. In doing so, the ions generate currents that are proportional to their number and enable their quantitfication, which provides us with the isotope ratio, for example the 13CO2/12CO2 ratio.


Gas chromatography combustion isotope ratio mass spectrometry



As has been mentioned above, several different analytical procedures need to precede IRMS, depending on the collected samples. In many studies the appearance of 13CO2 in the collected breath samples is determined by GC-IRMS; after isolating CO2 by gas chromatography (GC), isotopic abundance was quantified by IRMS. In order to determine plasma 13C-glucose and plasma 13C-palmitate enrichment, glucose and palmitate first need to be extracted from the plasma samples. After extraction, separation and derivatisation the acquired volatile derivates are determined by GC-IRMS with the inclusion of an extra combustion phase (C) as the derivate first needs to be converted to CO2 before entering the isotope-ratio mass spectrometer (GC-C-IRMS). In addition, plasma 2H2 glucose enrichment is determined by electron-ionisation gas chromatography mass spectrometry as 2H can not be used in conventional combustion systems as it produces 2H2O and CO2 as products.


The continuing advances in the technology and methodology used to determine isotope enrichment, will further increase the applicability of stable isotope methodology in metabolic research. Increasing the precision and accuracy of mass spectrometry technology would decrease the present minimal requirements for tissue sample size and improve the potential to quantitate very low isotopic abundance in tissue samples collected from large body pools with a relatively low turnover rate. This would facilitate, for example, the quantification of lipolysis rates in abdominal adipose tissue, amino acid turnover in skeletal muscle tissue and could in the future even enable us to measure nucleic acid turnover, thereby initiating a whole new spectrum of applications for stable isotope tracer methodology. In fact, the vision of Rudolf Schoenheimer on the use of stable isotope tracers in biomedical research is, even after more than 50 years, still up-to-date, as he wrote: “The chemical constituents of the living body represent links in a chain of continuous reactions in which apparently all organic substances, even those of the storage material, are involved. It is with this aspect of the dynamic processes of life that the biochemist is especially concerned. The isotopes of those elements which are present in natural organic compounds, presented to the biochemist by the physical chemist will certainly furnish a better insight into the details of this intricate mechanism.”