Developments in bile acid kinetic measurements using 13C and 2H: 105 times improved sensitivity during the last 40 years†

Abstract

Bile acid kinetics involve the measurement of pool sizes and turnover rates of individual bile acids. The technique is based on isotope dilution and was first described in the 1950s using radioactive 14C-labelled cholic acid (CA). It took until the 1970s before stable isotopes were introduced for this purpose (13C, 2H) and isotope analysis methods were developed for CA and chenodeoxycholic acid (CDCA) applying gas chromatography/electron impact mass spectrometry. Until the 1980s, the isotope enrichment measurements were performed in bile samples aspirated from the duodenum. Thereafter, methodology became available allowing measurements to be performed in blood requiring at least 2 ml serum samples. Simultaneous measurement of kinetics of metabolically dependent CA and deoxycholic acid using 13C and 2H labels was introduced. Until the 1990s, this technique was only possible in adult humans due to the large sample sizes. Introduction of pentafluorobenzyl bromide derivatisation and electron capture negative ion mass spectrometry (GC/ECN-MS) reduced the sample volume to 50 μ l serum. This allowed isotope abundance measurement of CA in rats and in mice. However, repetitive collection of 100 μl blood samples in mice is too invasive (collection via the orbita) and exhaustive. Therefore, the method development is now focussing on enhanced sensitivity and reduction of blank effects originating from the sample preparation. The final goal is to determine CA isotope enrichments in 20 μl mouse blood obtained from the tail vein. This paper shows the feasibility of reaching this goal.