592 M. Adiels et al.
partially deuterated products are formed during tracer
recycling,2 it is a potential drawback of the method to have to
measure the mþ2 peak. However, this does not seem to
impact on the results of the modelling since the differences in
estimated parameters were small and non-systematic
(Table 1). We also tested the impact on the amplitude itself
of the enrichment curves by scaling the amplitude (between
0.5- and 2-fold) and found a difference less than 1% in all
estimated parameters. Thus, we suspect that even if the two
methods give different amplitudes, the small observed
difference in results is not due to the difference in amplitude
(which was 9–34% in the analyzed time series). Amplitude
variation (within the given range) does not influence the
results of the model. This is an inherent effect of the design
and implementation of the model and reflects the fact that the
production rate of VLDL is invariant of the contribution of
different glycerol sources. On the other hand, the relative
contribution from different glycerol sources cannot be
determined using this experimental setup.
current circumstances, and this allows for comparison
between results derived using these methods. However,
the possibility that there are circumstances where it is
important to measure the intact molecule cannot be ruled
out, and here we present such a method suitable for use with
EI-MS. In practice, the saponification protocol with deriva-
tization with MTBSTFA is the recommended protocol
whereas the transesterification protocol is applicable when
the free fatty acids are also to be measured.
Acknowledgements
The authors thank Mike Stolinski for helpful discussions and
Hannele Hilden, Helina Perttunen-Nio, Anne Salo, Elin
Stenfeldt and Maria Arnell for excellent technical assistance.
This work was supported by the the Swedish Foundation for
Strategic Research, European Atherosclerosis Society, the
Novo Nordisk Foundation, Emelle fond, Sigrid Juselius
Foundation Finland, the project ‘‘Hepatic and adipose tissue
and functions in the metabolic syndrome’’ (HEPADIP,
European Commission as an Integrated Project under the
6th Framework Programme (Contract LSHM-CT-2005-
018734), and by ETHERPATHS (Contract FP7-KBBE-222639).
Even if there was no significant difference in the outcome
of the kinetic model, applications may still exist where it is
important to be able to measure the intact glycerol molecule.
Furthermore, the findings in this study also strengthen the
conclusions in earlier studies using the old protocol for the
measurement of glycerol isotopic enrichment since it was not
known how much these results were influenced by the
fragmentation of the glycerol molecule after ionization.7
Previously, EI-MS has been used with HFBA derivatiza-
tion, but the analytical response is of low intensity.11 Since
fluorine, and other halogens, are highly electronegative,
compounds containing these halogens preferentially form
negative ions and they are often analyzed by negative
chemical ionization. The MTBSTFA method presented here
gives a high signal, suggesting that lower initial amounts of
material could be used and thus reduce the amount of blood
drawn from the subject. HFBA, on the other hand, has the
advantage that it seems to be able to derivatize glycerol in the
presence of salts, such as directly after transesterification of
VLDL-triglycerides or directly in the glycerol-containing
aqueous phase of plasma,2 resulting in less time-consuming
procedures.
REFERENCES
1. Parhofer KG, Barrett PH. J. Lipid Res. 2006; 47: 1620.
2. Patterson BW, Mittendorfer B, Elias N, Satyanarayana R,
Klein S. J. Lipid Res. 2002; 43: 223.
3. Harris WS, Connor WE, Illingworth DR, Rothrock DW,
Foster DM. J. Lipid Res. 1990; 31: 1549.
4. Zech LA, Grundy SM, Steinberg D, Berman M. J. Clin Invest.
1979; 63: 1262.
5. Malmstrom R, Packard CJ, Caslake M, Bedford D, Stewart P,
Yki-Jarvinen H, Shepherd J, Taskinen MR. Diabetologia 1997;
40: 454.
6. Adiels M, Taskinen MR, Packard C, Caslake MJ, Soro-Paa-
vonen A, Westerbacka J, Vehkavaara S, Hakkinen A, Olofs-
son SO, Yki-Jarvinen H, Boren J. Diabetologia 2006; 49: 755.
7. Adiels M, Packard C, Caslake MJ, Stewart P, Soro A, Wester-
backa J, Wennberg B, Olofsson SO, Taskinen MR, Boren J.
J. Lipid Res. 2005; 46: 58.
8. Beylot M, Martin C, Beaufrere B, Riou JP, Mornex R. J. Lipid
Res. 1987; 28: 414.
9. Wolfe RR. Radioactive and Stable Isotope Tracers in Biomedicine.
Wiley-Liss: New York, 1992; 424.
10. Ackermans MT, Ruiter AF, Endert E. Anal. Biochem. 1998;
258: 80.
11. Gilker CD, Pesola GR, Matthews DE. Anal. Biochem. 1992;
205: 172.
CONCLUSIONS
12. Flakoll PJ, Zheng M, Vaughan S, Borel MJ. J. Chromatogr. B
Biomed. Sci. Appl. 2000; 744: 47.
13. Schwenk WF, Berg PJ, Beaufrere B, Miles JM, Haymond
MW. Anal. Biochem. 1984; 141: 101.
14. Lindgren FT, Jensen LC, Hatch FT. In Blood Lipids and
Lipoproteins: Quantitation, Composition, and Metabolism, Nel-
son GJ (ed). Wiley-Interscience: New York, 1972; 181–274.
15. Egusa G, Brady DW, Grundy SM, Howard BV. J. Lipid Res.
1983; 24: 1261.
16. Chong MF, Fielding BA, Frayn KN. Am. J. Clin. Nutr. 2007;
85: 1511.
17. Wolfe RR, Chinkes DL. Isotope Tracers in Metabolic Research:
Principles and Practice of Kinetic Analysis. Wiley-Liss: New
Jersey, 2005.
We have compared three different protocols for the
derivatization of glycerol from triglycerides derived from
lipoproteins. We optimized the derivatization reaction with
MTBSTFA with respect to time, temperature and the
inclusion of TBDMCS. We then compared the results from
kinetic modelling using data derived from our original
protocol using acetic anhydride, the optimized protocol
(optimized from Flakoll et al.12), and a new simpler protocol
for preparing VLDL-triglyceride glycerol, also using
MTBSTFA. We conclude that the results from kinetic
modelling using glycerol enrichment curves derived with
the different methods did not differ significantly, under the
18. McMurry J. Organic Chemistry. Brooks/Cole Publishing
Company: Pacific Grove, CA, 1996.
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 586–592
DOI: 10.1002/rcm