Measurement of leucine and alpha-ketoisocaproic acid fluxes in the fetal/placental unit.

Many investigators are now using stable isotopes in place of radioactive isotopes because of ethical considerations in human research. Our laboratory has had a long history in the development of an ovine model for the study of the physiological and biochemical changes during pregnancy. We wanted to extend some of the hypotheses and experimental protocols developed while using this model system to the human. As a first step in this process, we carried out infusions using a mixture of both 14C and 13C isotopes of the essential amino acid L-leucine. Results from this study showed that turn-over rates calculated using the two isotopes were equal within experimental error (8.99 +/- 0.45 and 8.97 +/- 0.52 mumol/kg.min, respectively). A key step in the development of the techniques for this study involved the use of tert.-butyldimethylsilyl derivatives for the amino acids. Because of the strong M-57 peak seen in the mass spectra of these compounds, we were able to use a relatively inexpensive Hewlett-Packard mass-selective detector for these determinations. Enrichments in triplicate measurements of the same sample had a precision of +/- 0.03%. Similar precision data were obtained in enrichment measurements of the keto acids derived from the amino acids. The combination of the speed of the analysis and the excellent precision have provided us with the opportunity to study uptake and loss across an organ system (i.e. placenta, liver, etc). This tool is now being used to study the detailed flow of amino acids across the placenta during both normal and abnormal pregnancies.