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S. Salentinig et al. / Chemistry and Physics of Lipids 190 (2015) 43–50
than longer chain fatty acids (Laszlo et al., 2008; Compton et al.,
triglyceride using mass spectrometry. A combination of (i) fully-
protonated tricaprin; (ii) tricaprin with fully-deuterated fatty acids
on the sn-1/3 positions, a deuterium atom on the C2 position of the
glycerol backbone, and non-deuterated fatty acid on position sn-2;
and (iii) tricaprin with the fully deuterated fatty acid on the sn-2
position and non-deuterated fatty acid on the sn-1/3 were used to
investigate the stereoselectivity of the lipase action, fatty acid
chain migration and enable product determination with mass
spectrometry. In mass spectrometry, deuterated adducts occur at
higher m/z values compared to their hydrogenated counterparts as
a result of the mass difference of deuterium and hydrogen. As a
consequence, mass spectra from the hydrolysis of the deuterated
components provides detailed qualitative information on the
stereochemistry of the hydrolysis reaction. In the absence of
deuteration, mass spectrometry would not be able to determine
the identity of the structural isomers of the digested products due
to the similarity in the mass values of the different isomers of
monoglycerides or diglycerides. For NMR, deuteration provides
2007; Boswinkel et al., 1996).
The formation of sn-2 monoglyceride in equilibrium conditions
is enhanced up to 80% yield by the presence of self-assembled
structures (Kodali et al., 1990; Holmberg and Osterberg, 1988;
Holmberg and Österberg, 1990; Mazur et al., 1993). This is
biologically-relevant, as these structures such as mixed micelles
and more complex liquid crystalline structures are observed in
monoglyceride/free fatty acid/bile salt mixtures (Hernell et al.,
1990; Hofmann and Borgström, 1964; Mazer et al., 1980;
Schurtenberger et al., 1985; Salentinig et al., 2014) and during
the in-vitro digestion of triglyceride emulsions (Salentinig et al.,
2011) and milk fat (Salentinig et al., 2013; Salentinig et al., 2015).
These structures also form the basis for the transport and
absorption of lipids and support the solubilisation and absorption
of oil-soluble food components (e.g., carotenes, vitamin A, D, E, and
K) in the aqueous system of the gastrointestinal tract (Salentinig
et al., 2014; Salentinig et al., 2011; Salentinig et al., 2013; Salentinig
et al., 2010). Compared to molecular transport, micelles and
vesicles enhance the number of fatty acid and monoglyceride
molecules available for uptake by the enterocytes. From a
monomeric point of view, fatty acid is transferred 3-fold faster
than the corresponding monoglyceride under pH conditions of the
small intestine (Narayanan and Storch, 1996). The size of the fatty
acid is extremely important as the physiological effects of
medium-chain fatty acids are distinctly different from the long-
chain fatty acids more commonly found in our diet. Medium-chain
triglycerides (MCTs) are generally considered a good biologically
inert source of energy that humans find relatively easy to
metabolize and are a common excipient in pharmaceutical
lipid-based formulations (Bach and Babayan, 1982). Until the
early 1980s, MCTs were predominantly available only as a
constituent of butter, coconut oil, and other natural sources.
However, since that date, processes have been developed to
produce them in large quantities to be used primarily for
therapeutic application in a number of conditions and for the
treatment of disorders of lipid absorption. Thus a complete
understanding of the MCT digestion process on the molecular level
is imperative. Knowledge and understanding of how these food
and supplement components are treated and utilized during the
digestion process opens opportunities for functional food products
that may help to avoid or cure health issues including obesity,
vitamin malabsorption, the risk of coronary heart disease and
cancer (Austin, 1991; Unger and Zhou, 2001; Katan, 2000).
However, there is a lack of information on the stereoselectivity
and fatty acid chain dynamics in digestion processes catalysed by
pancreatic lipase. This information is also important in industrial
processes that utilize lipase technology for the modification of oils
and fats to produce high-value added products, such as cocoa
butter equivalents, human milk fat substitutes, and other specific-
structured lipids. Enzymatic interesterification is a catalytic
reaction that occurs when an enzyme is introduced into oil and
rearranges the fatty acids on the glycerol backbone of a triglyceride
2
another nucleus (i.e., H) and frequency dimension, which allows
cleaner spectral windows to be probed for changes when a mixture
2
of hydrogen containing reagents is used: the H labeled fatty acid
chains of interest in the triglycerides can be solely probed without
1
any interference from the H signals of the other components. This
approach allows direct observation of the signals during the
digestion process and allows monitoring of the appearance and
disappearance of signals and assists in confirming the identity of
the final digestion products that are determined by mass
spectrometry.
2. Materials and methods
2.1. Materials
Selectively-deuterated tricaprin with two fully-deuterated
capric acid chains on the sn-1 and sn-3 positions and one
deuterium atom on the C2 position of the glycerol backbone, as
well as deuterated tricaprin with fully-deuterated fatty acid chain
on the sn-2 position were synthesized as described below.
62
Tricaprin (C33H O6, >98%) was purchased from Tokyo Chemical
Industry Co., Ltd., Tokyo, Japan. Bile salt (sodium taurodeoxycho-
late >95%), NaOH, NaOD, HCl and DCl (p.a. grade) were purchased
from Sigma Aldrich (St. Louis, MO, USA). Pancreatin extract (USP
grade pancreatin activity) was from Southern Biologicals (Nuna-
2
wading, Victoria, Australia). D O (99.8%) was supplied by AECL
(Ontario, Canada). Ultra-pure water (resistivity >18 M
Vcm) was
used for the preparation of all samples.
The deuteration of the capric acid was performed by
hydrothermal reactions using a Mini Benchtop 4560 Parr Reactor
ꢀ
(600 mL vessel capacity, 3000 psi maximum pressure, 350 C
maximum temperature). Thin layer chromatography was used
(referenced with the protonated compound) to estimate the purity
1
2
and to develop separation protocols. H (400 MHz) and H NMR
(61.4 MHz) spectra were recorded on a Bruker 400 MHz spectrom-
eter at 298 K. Chemical shifts, in ppm, were referenced to the
residual signal of the corresponding NMR solvent. Deuterium NMR
was performed using the probe’s lock channel for direct observa-
tion.
(
Xu, 2000). For example, tripalmitin treated with oleic acid in the
presence of 1,3-specific pancreatic lipase gives products where the
palmitate is retained at the sn-2 position, whereas oleate is
introduced at sn-1 and sn-3, producing a human milk fat substitute
such as Betapol. In practice, pure starting materials are not used
Electrospray ionization mass spectra (ESI–MS) were recorded
(
Akoh and Xu, 2002).
for the deuterated fatty acid on a 4000 QTrap AB Sciex
Here we report the synthesis of selectively deuterated tricaprin,
spectrometer. The overall percentage deuteration of the molecules
was calculated by MS using the isotope distribution analysis of the
a medium chain length triglyceride, to study the intermediate and
1
final products of the in situ digestion by pancreatic lipase using H
different isotopologues. This was calculated taking into consider-
2
13
and H NMR and mass spectrometry (MS) under biologically
ation the
C natural abundance, whose contribution was
relevant conditions (pH, T, bile salt and lipase concentration). The
use of selective deuteration provides a direct method to identify
unequivocally the final products of digestion of this symmetrical
subtracted from the peak area of each M + 1 signal to allow for
accurate estimation of the percentage deuteration of each
isotopologue.