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Format:
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Author:
O'Rourke, Bruce D.
Dept./Program:
Chemistry
Year:
2007
Degree:
MS
Abstract:
The use of stable isotope dilution for the study of substrate metabolism requires the accurate measurement of the tracer to tracee ratio (TTR). Gas chromatography mass spectrometry (GCMS) has frequently been used for this assessment. The study of free fatty acid (FFA) flux, intracellular and extracellular FFA recycling and whole body lipolysis relies on the precise measure of the rate of appearance (R[subscript a]) of FFA and glycerol. The R[subscript a] of palmitate has been shown to be representative of the total FFA R[subscript a]. The determination of the TTR of palmitate and glycerol by GCMS has been problematic. Previously, it has been reported that the isotopic ratio of labeled to unlabeled palmitate is dependent on the amount of palmitate entering into the GCMS. Solutions, which involve maintaining control of the amount of derivatized palmitate injected or the use of mathematical algorithms to adjust the MS derived isotopic ratio, have been proposed as means to correct for this concentration dependence. Here we compare two commonly used derivatives of palmitate and examine the extent of the changes in isotopic ratio with increasing palmitate content.
We also present a novel and reliable approach for the determination of the TTR of palmitate in human plasma, using positive chemical ionization (PCI) selected ion monitoring (SIM) GCMS, which is free of the concentration dependence seen in other methods. The most commonly used isotope to track the R[subscript a] of glycerol has been [1,1,2,3,3-²H₅] glycerol (d₅-glycerol). The methods for measuring the isotopic ratio of plasma glycerol using the tristrimethylsilyl derivative and the tris-triacetate derivative and electron impact (EI) SIM GCMS retain only three of the five carbons of glycerol. Other methods have been reported using the tris-t-butyldimethylsilyl derivative which can suffer from poor derivatization efficiency.
Negative chemical ionization (NCI) SIM GCMS has been used in combination with the tris-heptafluorobutryl ester of glycerol. We demonstrate that, with this analytical method, the isotopic ratio, as (M+5)/M, is dependent on the amount of total derivative of glycerol. WE show that this method does not exhibit this concentration dependence and can be used reliably for the determination of the TTR of glycerol in human plasma. Here we present PCI SIM GCMAS methods for the measurement of the isotopic ratio of plasma palmitate and glycerol that require little preparative time, produce accurate, reliable results and are suitable for the analysis of large batches of samples from stable isotope infusion studies.