Assessment of In Vivo Metabolism Using Stable Isotope Techniques

Assessment of in vivo metabolism using stable isotope techniques 

Stable isotopes are non-radioactive atoms of the same chemical element, which differ only in their number of neutrons [19]. Many elements also have radioactive (non-stable) isotopes. The most commonly used stable isotopes in studies of macronutrient metabolism are (D or deuterium), C, and 0, while Mg, Mg, a, Ca, Ca, Fe, Fe, Zn, and °Zn are the most commonly used stable isotopes for studies of mineral metabolism. The most commonly used radioactive isotopes are and (tritium) [19]. More than 6(X)0 stable isotope-labeled compounds (tracers) are commercially available for use in metabolic studies. Examples for some of these tracers are [1- C] leucine, [1- C, N] leucine, [ring- Hj] phenylalanine, and [6,6]-D2 glucose. It is currently accepted that these compounds have neghgible biological side-effects, which renders them ethically acceptable for use in children [20]. 

Following intravascular or oral application, the tracer is metabolically indistinguishable from the equivalent unlabeled compound of interest (tracee). The metabolic fate of the compound can be assessed qualitatively and quantitatively by measuring the relative abundance of tracer and tracee and/or their respective breakdown products with time. The detectable mass difference of tracer and tracee allows the analysis of compounds extracted from plasma by either GC-MS or LC-MS [21]. Both require nanogram or picogram sample size (analytical range is 0.1-100 mol%, precision 0.2%). The detection limit is considerably less than 

1 mol%, when tracers with multiple stable isotope labels (for example, ring-Dj phenylalanine) are used [22], Stable isotopes in breath (i.e., 2 and C02) are analyzed using an isotope-ratio mass spectrometer (IRMS, microgram sample size, analytical range 0.001-10 atom% excess, precision 0.00005 atom% (5 ppm)) [19]. Combustion-IRMS essentially has the same analytical capabilities as IRMS but allows the combustion of tissue samples with subsequent analysis of gaseous isotope enrichment [19]. Stable isotopes of minerals are typically analyzed by thermal ionization mass spectrometry (TIMS) or inductively coupled plasma mass spectrometry (ICP-MS) with high precision and sensitivity [19]. 

The advantages of stable isotope-labeled compounds compared with their radioactive counterparts are manifold. Most importantly, several different stable isotope tracers can be safely administered simultaneously to the same subject without limiting future studies. The plasma volume which is needed for one study to analyze isotope enrichment is small, allowing even pre-term infants to be studied. On average 0.5 ml of plasma is needed for one study. The intramolecular location of one or more label(s) is determined easily, which allows the assessment of metabolic pathways [19]. 

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