GC/CIRMS Isotope ratio mass spectrometer with on-line chromatographic separation and conversion facilities for 13C, 15N, 18O and 2H for metabolic research.

To study intestinal absorption, post-absorptive metabolism and fermentation of nutrients and endogenous components in humans, stable isotopes (13C, 15N, 2H, 18O ) are extremely useful because of their non-radioactive properties. The most powerful analytical technique to analyse stable isotopes in body fluids is isotope ratio (IR) mass spectrometry (MS) with on-line facilities for gaschromatographic (GC) separation and conversion (C): GC/CIIRMS. This grant application concerns the third generation GC/C/IRMS with on-line facilities for separation and conversion of 13C, 15N, 18O and 2H. This instrument can measure different labels in one sample, which allows measurement of at least two different metabolic processes in one experimental set-up. This technique and other features of the proposed apparatus will allow us to gain significant progress in various research lines, among these: Bioavailabillty of nutrients; (mal)absorption and fermentation, with the topics: 1. digestion/absorption of lipids; 2. digestion of lactose (in vivo measurement of lactase activity); 3. fermentation of lactose (acetate production); 4. colonic bacterial growth rate (15N-incorporating activity). ad 1. Oral administration of a mixture of the triglyceride 13C-trioleate and 2H-oleate as free fatty acid, followed by serum analysis of the ratio 13C-oleate/ 2H-oleate. This gives an indication of the two major steps in lipid digestion: luminal lipase activity and (micellar) solubility capacity. These data lead to a better differential diagnosis and treatment of lipid malabsorption syndromes. ad 2. Oral administration of a mixture of 13C-lactose and 2H-glucose, followed by serum analysis of the ratio 13C-glucose / 2H-glucose, indicates the in vivo physiological lactase activity. These data lead to a better diagnosis and treatment of lactose intolerance, ad 3. 13C-carbohydrates (starch, lactose) that are not absorbed in the small intestine spill over into the colon, where they can be fermented, leading to desired (protection against colon cancer) or undesired (diarrhea) effects. Measurement of 13C-acetate in serum is one of the indicators of fermentation which can be monitored to study the complex process of fermentation. ad 4. Another important process in the colon is the relative bacterial growth rate compared to substrate influx in the colon. For the search of rate-limiting steps in this process, the N-retention can be estimated by measuring 15N-ureate in urine after oral consumption of 13C-lactose-15N-ureide, a substrate that is not digested in the small intestine. In contrast to the first generation, this third generation GCICIIRMS can also measure metabolites in smaller volumes, which is important for studies in prematures and transgenic mice. Also isotope studies with metabolites at lower concentrations may become possible by improved electronic performance and improved chromatographic separation, due to a better combustion interface design. The purchase of this third generation GC/C/IRMS will create new experimental possibilities and open up new research areas in (human) metabolic research.