I am going to determine acetate and propionate kinetics in sheep. So, my professor advised me to use stable isotope, although I know some method of radio-isotope for VFA metabolism and the acetate kinetics of stable isotope by Moore. Can anybody help me with other established techniques of VFA metabolisms using stable isotopes? It will be highly appreciated if anyone can help me regarding this matter.
Dear Rahman, I can't give you provide you with an established technique for studying VFA metabolism in ruminants using stable isotope but I might be able to make some suggestions. Depending on your study goal the approaches will differ. If you are interested in VFA synthesis rate, you could start thinking about 13C tracer studies using 13C labelled sugar to introduce 13C into the precursor pool. You would than have to collect blood samples and analyse them for 13C abundance in VFAs by GC/MS or GC-IRMS. Which analytical technique you use will depend on the amount of tracer administered and the amount of tracer incorporated into the VFAs.
Conversely, if you are interested in metabolic breakdown (oxidation) of VFAs you could give your animals injections of 13C labelled acetate, propanoate or butyrate, either as bolus or as primed-constant infusion. These techniques have been used in many different metabolic studies including humans not just animals, so you should find publication describing these in detail. Rate of oxidation can be monitored quite easily by collection breath samples from your animals (well, how easy depends on how co-operative they are) and by analysing the rate of appearance of 13C in the exhaled CO2. In essence, the underlying principle is that of a 13CO2 breath test. Rate of appearance and dose recovery will tell you something about the rate of VFA metabolism. Hope this provides you with a good starting point.
Dear Prof Augenstein, Thanks for your time to response.
Although you give a brief discussion on use of stable isotope in metabolic study, but it is really help me a lot to enrich my way of thinking. Actually in our lab, earlier we used 1-13C acetate infusion technique by a priming dose injection through jogular vein follwed by 4 hour continous infusion. And blood was collected for every 30 mins. of last 2 hours of infusion period. And we usually see the isotopic enrichment for the last 2 hour and calculate the turnover rate of acetate using GC/MS technique followed by the method of Moreau et al. (2002). But this days, some how we can not find the isotopic enrichment by the same procedure that we did earlier. So we are looking for any new method for procedure to solve this problem.
Dear Sir, it will be highly appreciated if you can help me in this regard.
Thanks a lot in advance...
Not knowing which level of 13C enrichment you are using I can't really comment. Also, is the acetate enriched in C1 (carboxyl) or C2 (methyl) or both?
If the level of 13C enrichment is not high enough you might not be able to detect 13C with GC/MS. Conventional MS can only reliably detect 13C enrichment at a level exceeding 0.5 atom% excess (> 1.6 atom%). The dilution space for acetate in mammals is pretty large since acetate is the ultimate precursor for fatty acid biosynthesis and steroid biosynthesis.
Isotope Ratio MS instrument are much more sensitive since they are built and optimized to detect 13C abundance changes at natural abundance level and are thus ideal for compounds with a 13C abundance of < 1.6 atom%.
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