Development of a LC-MS assay to quantify multiple metabolic intermediates to assess thermogenesis

Ben McNeill, University of Edinburgh

 

Aims

Obesity and associated metabolic diseases such as type 2 diabetes are increasing in prevalence globally, highlighting the need for novel treatments. Brown adipose tissue (BAT) activation increases energy expenditure and improves glucose and lipid levels by utilising multiple substrates to generate heat during cold-induced thermogenesis (CIT). BAT activation is reduced in obesity and diabetes but the mechanisms driving this are unclear. In my BHF-funded PhD (just commencing year 3), I am investigating the key role of the succinate receptor in BAT thermogenesis. Quantifying succinate concentrations in my samples is essential and crucially colorimetric kits are inaccurate and substantially overestimate concentrations. My aims are:

1) - develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay that can quantify not only succinate but also other important tricarboxylic acid (TCA) cycle intermediates (e.g. succinate, fumarate, malate, oxaloacetate, citrate, α-ketoglutarate) and metabolic substrates (e.g. glucose, lactate, pyruvate, glutamate) simultaneously in order to determine if these key intermediates are dysregulated in obesity.

2) – to quantify circulating and BAT concentrations of these intermediates in lean and diet-induced obese mice during both warm and cold exposure.

3) – to quantify circulating concentrations of these intermediates in lean and obese humans during both warm and cold exposure.

 

Evaluation

Using this award, we collaborated with the mass spectrometry core in QMRI to develop a LC-MS/MS assay (using a QTRAP 6500) to quantify multiple metabolic intermediates. Development was slower than we had anticipated, initial testing required optimization of different columns, samples preparation and mobile phases as the limit of detection was not sufficient to detect many of the compounds at the expected in vivo concentrations. However, following optimization we were able to measure multiple compounds in plasma simultaneously including glucose, lactate, pyruvate, and intermediates of the tricarboxylic acid (TCA) cycle including citrate, alpha-ketoglutarate, succinate, fumarate and malate. We were not able to quantify oxaloacetate or isocitrate with this assay, however we discussed this with collaborators in the field who confirmed these compounds are commonly not detectable in vivo. Therefore, aim 1 of this grant was completed successfully. 

Following development of the assay, we then analysed these compounds in plasma from both male and female mice that had either been fed a high fat (58% of energy as fat) or control diet (7.5% fat) for 12 weeks (n=8 mice per group so 32 samples). There were dramatic sex-specific effects, as all of the concentration of all the above compounds apart from alpha-ketoglutarate were significantly lower in the female mice, for the TCA cycle intermediates these were generally reduced by 40%. The high fat diet increased glucose concentrations only in male mice, but surprisingly all the other intermediates were unaltered by high fat diet in both male and female mice. Therefore, aim 2 was also completed successfully.

 

Grant awarded: £4,991.00