Sociedade Brasileira de Espectrometria de Massas – BrMASS
GC-MS & LC-MS
Lipid profiles of melanocytes and melanoma cell lines
characterized by GC-MS of Methyl and d3-methyl FA derivatives.
Arquimedes Paixão de Santana-Filho1, Daniel Suss Riter1, Andersson
Barison2, Marcello Iacomini1, Lauro Mera de Souza1, Philip Albert James
Gorin1, Sheila Maria Brochado Winnischofer1, Guilherme Lanzi Sassaki1*
*[email protected]
1
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do
Paraná, Curitiba, PR, Cx.P 19046, CEP 81531-990, Brazil.
2
Departamento de Química, Universidade Federal do Paraná, Curitiba, PR,
Cx.P. 19081, CEP 81531-990, Brazil
Few studies explore the relationship between lipid composition changes and the
tumorigenicity of melanoma cell lines in comparison to normal melanocytes. The lipid
profiling of cancer cells are difficult to perform with the intact lipid extract, when
limited quantities of biological matrix are available to lipid extraction. An alternative is
the use of derivatization strategies coupled to GC-MS analysis(1), which can show the
fatty acid (FA) composition of the major lipid classes, cholesterol and other lipidassociate volatile compounds, being reliable to be used when very low quantities of
lipidic extract are available. Methanolysis were carried out on the lipid extracts
obtained from melanocytes (MA) and melanoma (B16-F10) cell lines, and several
conditions of reactions were tested to allow the optimum methanol: acetyl chloride
(AcCl) ratio, giving complete derivatized fatty acid methyl esters. The mixtures were
vortexed for 2 minutes and heated at 100ºC for different time intervals. The resulted
FAMEs were extracted by partition using 1 mL of hexane plus 0.5 mL of distilled water
to facilitate phase separation. The organic phase was collected and evaporated under a
stream of N2. Additionally, we performed the esterification reaction with the
AcCl:CH3OH or CD3OD reagent, resulting in non-deuterated and deuterated methyl
ester derivatives of the fatty acids. The cell lines showed different fatty-acid
compositions, with the MA cells having a minor proportion of the C16 and C16:1
FAMEs, when compared with the B-16 cell line (Fig. 2A and Table 3). For the C18 and
C18:1 FAMES this effect was the opposite, especially for C18:1, the MA cell line having
a higher relative amount of these two FAMEs than the B-16 cell line. Unfortunately,
simultaneous quantification of the FAME and d3-FAME was not possible for unsaturated
fatty acids, in part because the small delay in the R t showed for the C18:1 methyl-d3
derivatives than for the FA with lower carbon chains, but mostly by the fact that nearly
all fragment ions from unsaturated FAME or d3-FAME are formed after elimination of
the headgroup, that remains uncharged, so the ions formed are found in the mass
spectra of FAME and d3-FAME(2) However, the most important result was the
reproducibility in the quantification when the CH 3OH derivatives are compared with the
CD3OD derivatives. This feature enables the simultaneous analysis of two different
samples in the same chromatogram, providing a more reliable comparison between
them.
[1] Sassaki, G. L.; Souza, L. M.; Serrato, R.V.; Cipriani, T. R.; Gorin, P. A. J.; Iacomini, M. J.
Chromatogr. A 2008, 1208, 215 - 222.
[2] Thurnofer, S.; Vetter, W. J. Agric. Food. Chem. 2006, 54, 3209-3214
4º Congresso BrMass – 10 a 13 de Dezembro de 2011