Biomedical applications of stable isotopes

 

Summary

An overview is presented about biomedical applications of stable isotopes. The aims of metabolic studies in the areas of glucose, fat, cholesterol and protein metabolism are explained as well as the principle of breath testing and the techniques to study body composition and energy expenditure. Much attention is paid to the analytical considerations based upon metabolite concentrations, sample size restrictions and the availability of stable isotope labeled substrates. The instrumental conditions for Gas Chromatography / Reaction / Isotope Ratio Mass Spectrometry and Gas chromatography / Mass Spectrometry are discussed as well as the present use and future perspective of Infrared Spectrometry for clinical and biomedical stable isotope applications.

Introduction

The application of stable isotopes in clinical diagnostics and biomedical research is mainly focused on metabolic in vivo studies. Metabolic studies are carried out in a large range of disciplines related to cardiovascular disease, diabetes, kidney disease and a number of inborn errors of metabolism. The majority of the work is dealing with the interaction between the exogenous influx of nutrients via the diet and the endogenous metabolism of the same or related metabolites. The key question is how endogenous metabolism is regulated and affected by an exogenous flux into the pool. On a chemical level we are dealing with a diversity of compounds: carbohydrates, proteins, fats, cholesterol. On a metabolic level the major issues are synthesis and catabolism, where catabolism can mean conversion into metabolic products, oxidation or excretion. In the steady state situation increased exogenous influx is counteracted by decreased synthesis and increased catabolism. The opposite effects are observed in the case of decreased exogenous influx. However, in pathological situations the regulatory system may work insufficiently leading to accumulation or depletion of stores. Recent observations have indicated that regulation systems may cross borders between various metabolic pathways since regulating elements are involved in different pathways. This is exemplified by the fact that diabetes, originally defined as a disturbance in glucose metabolism, also exert disturbances in lipid metabolism. Metabolic research can be performed at different levels. Much effort is put into the measurement of nuclear receptors and gene expressions involved in the production and regulation of proteins participating in the metabolic pathway. However, this can only be done in experimental animals collecting tissue samples of organs of interest. Furthermore these measurements are static and organ bound. To study the effect of disease or manipulation by drugs or surgery, metabolic fluxes need to be measured in order to create a real time reflection of the situation. The only reliable technique to obtain a dynamic picture of the process is the use of stable isotopes. Techniques used to measure isotope enrichments are mainly based on mass spectrometry. The demands on the analytical procedure and instrumentation are diverse since different metabolites are involved and studies are to be performed in human adults or infants and in experimental animals such as rats and mice. The different types of applications are shortly mentioned and the analytical requirements are defined. The present use of infrared techniques and the future perspective in the area of metabolic research will be discussed.

Stable isotope applications

Glucose metabolism

Carbohydrates are the major nutrient in humans. Starch and sugars are present in almost every meal such as bread, potato, rice, fruits, vegetables, snacks, sweets, cookies, etc. Excess glucose derived from the diet is stored as glycogen in the liver and used for conversion of glucose to fat. When fasting, glycogen is broken down to glucose and secreted into blood. Long time fasting requires additional production of glucose initiated by conversion of other compounds such as lactate, glycerol. and amino acids to glucose (gluconeogenesis). The liver is the central organ in regulation of glucose metabolism. Depending on the feeding status, the breakdown of glycogen, gluconeogenesis and fatty acid synthesis from glucose must be up or down regulated. In this process insulin plays an important role. Measurement of endogenous production of glucose is generally determined applying a continuous infusion of deuterated glucose (6,6-²H₂-glucose). In the fasting state the deuterium enrichment is determined by the rate of isotope infusion and the rate of production. In the fed situation the enrichment is determined by the total rate of production, consisting of the rate of infusion and the sum of endogenous production and the influx from the intestine. When the bioavailability of glucose due to digestion of a carbohydrate bolus is to be measured, this carbohydrate is labeled with ¹³C. Measurement of ¹³C enrichment combined with measurement of deuterium enrichment enables to differentiate between glucose molecules coming from the intestine and those originating from endogenous synthesis. Since endogenous synthesis is dose dependently suppressed by the flux of exogenous glucose, which is time dependent, a non steady state situation exists, requiring non steady state calculations. This way, the bioavailability of glucose can be calculated applying a correction for metabolism. When carbohydrate digestion is very fast, as is the case with disaccharides, correction of metabolism is possible by a simultaneous oral administration of 6,6- ²H₂ glucose. A number of monosaccharides are commercially available in a synthetic highly enriched labeled form, labeled in selected positions or uniformally. Highly enriched disaccharides are less frequent available and very expensive. Highly enriched polysaccharides as appearing in natural foods, are not commercially available. Corn derived naturally enriched starches and also lactose isolated from milk from corn fed cows are often used in Europe. These products are less than 0.02% (APE) enriched compared to the body glucose in European subjects. Hepatic fluxes of glucose to and from glycogen are measured using simultaneous constant infusion of three stable isotope labels: U-¹³C₆ glucose, 1-²H-galactose and 2-¹³C glycerol. These substrates monitor fluxes through the glucose-6-phosphate pool. Glucose oxidation is measured as breath ¹³CO₂ excretion after administration of ¹³C-glucose. Measurement of ¹³CO₂ as a measure of carbohydrate digestion in the small intestine appears not accurate when the digestion rate is low. This is due to ¹³CO₂ production in the colon, leading to an erroneously high ¹³CO₂ response in breath.

Fat metabolism

Besides fat uptake from the intestine, fatty acids palmitic acid, stearic acid and oleic acid are synthesized in the liver and triglyceride particles are assembled and secreted into blood. Dietary fats are hydrolyzed by pancreatic lipase enzyme and the free fatty acids and monoglycerides are reassembled into chylomicrons and taken up by the lymph. Fat digestion, fatty acid absorption and endogenous fatty acid synthesis are subject of metabolic tracer studies with stable isotopes. To study fat digestion and absorption, ¹³C labeled natural or synthetic triglycerides (labeled in fatty acid) and fatty acids are applied and the ¹³C enrichment measured in breath CO₂ or plasma triglyceride fatty acids. Fatty acid synthesis, palmitic acid as a model compound, is measured by Mass Isotopomer Distribution Analysis (MIDA). This principle is based on the knowledge that palmitate is a polymeric product of acetate. Therefore 1-¹³C, 2-¹³C or 1,2-¹³C₂ acetate is infused and incorporation of ¹³C into palmitate is measured in the VLDL lipid fraction in plasma. The VLDL fraction contains the fatty acids synthesized in the liver.

Protein and amino acid metabolism

Protein balance must be neutral in the adult situation. This means that the synthesis and breakdown of protein are about equal. Children in the growing phase must have a positive balance, synthesizing more protein than is broken down. Catabolic disease states may lead to increased protein breakdown and a negative protein balance. Measurement of synthesis and turnover of proteins is measured using labeled (¹³C, ²H, ¹⁵N) amino acids in a continuous infusion mode. Whole body protein metabolism is measured solely by isotope dilution in the amino acid pool. Dilution of label occurs by input of unlabeled amino acid by protein breakdown. Valine and leucine are the most applied labeled essential amino acids. Synthesis and breakdown of specific proteins is measured by collection of a protein sample. This may involve collection of a blood sample or a biopsy of specific tissue such as muscle. Specific amino acid pathways are also subject of stable isotope related research. Examples are arginine in relation to NOx formation and glutamine as a conditionally essential amino acid.

Cholesterol metabolism

Cholesterol metabolism is an important health issue since accumulation cholesterol and in particular LDL-cholesterol is associated with an increased risk for cardiovascular disease. The advice to reduce cholesterol intake is too simple. The cholesterol metabolism is very complex and it is often forgotten that the human body synthesizes about twice as much cholesterol as is normally taken up by the food. A number of factors are involved to control cholesterol levels: absorption of dietary cholesterol from the intestine, endogenous synthesis, in particular in the liver, secretion of cholesterol into the intestine via the bile and conversion in the liver of cholesterol to bile salts. All these processes must be coordinated in order to balance the so called cholesterol homeostasis. Dynamic measurements of these processes are necessary and can be performed applying stable isotopes. To determine cholesterol absorption from the intestine, two labeled cholesterols are used. One (for instance ²H) is given orally with a test meal, the other (for instance ¹³C) is administered intravenously at the same time. The absorbed portion of the oral label will slowly be mixed with the intravenous label. After about 2 days the ratio of the two enrichments becomes constant in time. This ratio reflects the absorption coefficient of the orally administered labeled cholesterol when the same amounts of both labels has been administered. In line with fatty acid synthesis, also cholesterol synthesis involves a polymerization of acetate units. Thus, using a ¹³C-acetate infusion, the plasma cholesterol pool becomes enriched in ¹³C. The MIDA method enables the calculation of the fractional synthesis rate and absolute synthesis rate. Catabolism of cholesterol to bile acid is measured by the isotope dilution technique involving ¹³C or ²H labeled primary bile acids cholic acid and chenodeoxycholic acid. From the isotope dilution kinetics, the pool sizes and synthesis rates of both bile acids can be determined simultaneously.

Energy metabolism and body composition

Energy utilization is measured using the doubly labeled water (²H₂ ¹⁸O) technique. From the knowledge that after administration ¹⁸O is lost in body water and CO₂, whereas ²H is lost only in water, the decays of ²H and ¹⁸O enrichment in body water leads to the calculation of energy expenditure. After administration of singly labeled ²H₂O the ²H enrichment in body water allows calculation of body composition. Body water volume is calculated as the distribution volume and lean body mass by the known relationship between body water mass and lean body mass. Fat mass is determined by subtraction of body water mass plus lean body mass from the total body mass.

Breath tests

Age number of breath tests have been developed in order to determine specific organ functions in vivo in patients. ¹³C-labeled substrates have designed to be cleaved by selective enzymes in specific organs. Examples: ¹³C-methacetin is solely demethylated by hepatic microsomal enzymes, ¹³C-ketoisocaproic acid solely by hepatic mitochondrial enzymes. In any application one must realize that the site of sampling (breath) and the organ function tested may be separated by interfering physiological processes. Generally it may be stated that ¹³CO₂ breath tests will discriminate well when organ function is heavily impaired.