The synthesis of 13c-bilirubin and its use in the validation of bilirubin kinetic studies in rats

Sturrock, Edward David

The synthesis of 13c-bilirubin and its use in the validation of bilirubin kinetic studies in rats

Thesis / Dissertation

1994

Publisher

University of Cape Town

Department

Department of Medicine

Faculty

Faculty of Health Sciences

Abstract

The total synthesis of [ 10-13C] bilirubin IXcx., the principal waste product of haem degradation, is described. Site specific labelling was accomplished by the Vilsmeier formylation of one of the dipyrrolic fragments using [1-13C] dimethylformamide. The penultimate dehydrohalogenation reaction was complicated by a competing elimination reaction which yielded a bridged biliverdin derivative. The base catalysed reaction affords a novel [ 10-13C]-8, 12-bis(2-methoxycarbonylethyl)-7, 13, 17-trimethyl-2, l 8-propano-3vinylbilin-1, 19(21H,24H)-dione in which the 2 and 18 positions of the macrocycle are bridged with a propano tether; the structure has been established using single crystal Xray and 1 H nuclear Overhauser effect studies. The labelled biliverdin was reduced to [ 1013C]bilirubin IXcx. and the desired product was thus prepared in an overall yield of 6% from the step where 13C was introduced with quantitative isotope incorporation. [ 14C]bilirubin was prepared, biosynthetically, using [14C]aminolaevulinic acid. Bilirubin kinetics in 4 rats were measured by the analysis of the plasma disappearance of [ 14C]bilirubin in a two-compartment model. The plasma half-life of the first and second exponentials were 1.97 and 32.8 min respectively. The data were used to determine model independant parameters k12, k21 and k20. In the proposed model, plasma unconjugated bilirubin exchanges with a hepatic unconjugated bilirubin pool. Bilirubin is eliminated from the system via the proposed hepatic pool. These studies provide an analysis of the kinetics of unconjugated bilirubin in rats and are intended to serve as a reference point for studies using a stable isotope of bilirubin. The plasma disappearance of [ 10-13C]bilirubin IXa in three rats was studied using mass spectrometry to measure the bilirubin 813C. Validation of the experimental procedure in terms of range and reproducibility of the detection method was carried out. The half lives of the initial and terminal exponentials were 2.27±2.5 and 22.8±12.9 minutes. Despite the large 95% confidence limits calculated for these clearance curves they serve as an important foundation for future bilirubin kinetic studies using nonradioactive isotopes of bile pigments.

Keywords

13c-bilirubin

sythesis

Reference:

Collections

PhD / Doctoral

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