Abstract
Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.
Highlights
Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma
We have examined the use of an LC-MS/MS-based method for quantifying the turnover of plasma apolipoproteins in mice given either 2H2O or Disclosure statement Manuscript received 6 October 2011 and in revised form 1 March 2012
Attention was focused on quantifying the isotope labeling and measuring the abundance of multiple proteolytic peptides, which, in total, yields an estimate of absolute rates of apoprotein flux in vivo
Summary
Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. Investigators determine the flux rates by collecting multiple samples from a given subject and fitting the labeling curves; clearly this is not practical in studies conducted in rodent models in which sample volumes are often a limiting factor (e.g., mice have ف1 ml of blood). As the t1/2 of water is relatively slow in rodents (e.g., ف2 days for 2H in a mouse), it is possible to maintain a constant labeling of the precursor pool for several hours by administering a single bolus of either water tracer [6], and this can be extended by allowing the animals access to enriched drinking water [4, 7]. Attention was focused on quantifying the isotope labeling and measuring the abundance of multiple proteolytic peptides, which, in total, yields an estimate of absolute rates of apoprotein flux in vivo
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