Abstract
In this research project two topics were studied, initiated with the effect of post-column
mixed organic solvents on the ionization of several LC-(ESI)MS/MS methods. A well
known effect in LC-MS/MS methods is ion suppression, which leads to a decreased
sensitivity. This is for instance caused by co-eluting less volatile compounds with the
analyte. However, post-column mixed organic solvents prior to the electrospray
ionization- (ESI) interface, could possibly increase the ionization efficiency.
In order to examine effects for the positive and negative ionization mode, the VUmc
metabolic laboratory LC-MS/MS methods for S-adenosylmethionine (SAM) and Sadenosylhomocysteine (SAH), D- and L-2-hydroxyglutarate (D- and L-2-HG), sialic acid
(SA) and homocysteine - methionine were selected for the post-column experiments. For
every method a selection was made from the following organic solvents: 1- and 2propanol, methanol, acetone, acetonitrile, tetrahydrofurane, ethanol and 2 mM acetyl
acetone in 50% methanol. After post-column mixing at different flow rates of these
solvents during measurements, peak areas were obtained, and for enhanced signals the
signal to noise (S/N) ratios were calculated. Maximal enhancement of the signals was
obtained during post-column mixing of 2-propanol for SAH. However, the S/N ratios did
not show a similar elevation progress, indicating an increased noise level by post-column
mixed 2-propanol. No general effects on ionization were observed for the examined LCMS/MS methods during post-column mixing organic solvents. Furthermore, no clear
differences were obtained between positive and negative ionization.
The second part of this research project focused on the LC-MS/MS measurement of
mitochondrial beta-oxidation acyl-coenzyme A (acyl-CoA) intermediates, extracted from
cultured fibroblasts. Fatty acids are stepwise degraded into acetyl-coA units by the
mitochondrial beta-oxidation, in which process energy is obtained. The mitochondrial
beta-oxidation pathway consists of various enzymatic conversions. An impaired enzyme
of this pathway causes a beta-oxidation disorder, leading to accumulated intracellular
acyl-CoAs of a certain carbon chain length, and elevated extracellular acylcarnitine
analogue levels. Former studies of beta-oxidation in cultured fibroblasts have often
determined acylcarnitine levels in the culturing medium. By the measurement of intra
cellular acyl-CoAs and acylcarnitines in culturing medium of the same fibroblasts,
differences could be shown between these methods.
During this project, fibroblasts were cultured in the presence of palmitate or Lpalmitoylcarnitine. Furthermore, a LC-MS/MS method was developed which separates
short (C2) from long chain (maximum C16) acyl-CoAs. Acyl-CoAs were measured in
lysates from harvested cultured fibroblasts. Additionally, acylcarnitines were determined
in culturing medium with the LC-MS/MS method of the VUmc metabolic laboratory. In
experiments with L-palmitoylcarnitine clear signals originating from beta-oxidation acylCoA intermediates were measured. Additionally, a cultured medium-chain acyl-CoA
dehydrogenase (MCAD) deficient fibroblast cell line was tested showing a difference in
the pattern compared with control samples. In measurements of acyl-CoAs in cell lysates
however, not all intermediates were observed and signals were often low. Obtained acylCoAs in the comparison of control- and MCAD deficient fibroblasts showed very low
signals and no difference between the cell lines. This is possible caused by residual
enzyme activity in obtained cell lysates. Therefore, the measurement of acylcarnitines in
culturing medium, compared with acyl-CoAs in cultured fibroblast lysates, is at this
moment a better method to determine beta-oxidation flux.
This is possible caused by degradation of acyl-CoAs through residual enzyme activity in
obtained cell lysates. The measurement of acylcarnitines in culturing medium, compared
to acyl-CoAs in cultured fibroblast lysates, is therefore currently a better method to
determine beta-oxidation flux.