Structural and compositional changes in very low density lipoprotein
triacylglycerols during basal lipolysis
Jyrki J. A
˚
gren
1,2
, Amir Ravandi
1
, Arnis Kuksis
1
and George Steiner
3
1
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada;
2
Department of Physiology,
University of Kuopio, Finland;
3
Department of Medicine and Physiology, The Toronto Hospital (General Division),
Toronto, Ontario, Canada
Triacylglycerols secreted by liver and carried by very low
density lipoprotein (VLDL) are hydrolysed in circulation by
lipoprotein and hepatic lipases. These enzymes have been
shown to have positional and fatty acid specificity in vitro.If
there were specificity in basal lipolysis in vivo, triacylglycerol
compositions ofcirculating and newly secreted VLDL would
be different. To study this we compared the composition of
normal fasting VLDL triacylglycerol of Wistar rats to that
obtained after blocking lipolysis by Triton WR1339, which
increased plasma VLDL triacylglycerol concentration about
4.7-fold in 2 h. Analyses of molecular species of sn-1,2- and

triacylglycerol composition of circulating VLDL resembles
that of the VLDL newly secreted by the liver, although very
few studies have examined the effects of basal lipolysis on
circulating VLDL.
VLDL triacylglycerols are hydrolysed by lipoprotein and
hepatic lipases [4]. These enzymes have been shown to have
positional and fatty acid specificity in vitro [5,6]. However,
most studies concerning substrate specificity have been
performed with human or bovine milk lipoprotein lipase
using chylomicrons or synthetic triacylglycerols, including
alkyl ethers, as substrates [5–8]. The properties of human
and bovine milk lipoprotein lipase may differ [9] as may the
properties of milk and endothelial lipoprotein lipase [10].
Because the biosynthesis of intestinal chylomicron and
hepatic triacylglycerols proceed along different routes [11],
structurally dissimilar triacylglycerols would have been
subject to endogenous lipolysis during clearance of post-
prandial chylomicron triacylglycerols and of VLDL triacyl-
glycerols further complicating the interpretation of earlier
results.
Previous studies [12–14] have shown that Triton WR1339
(a nonionic detergent) blocks lipolysis by inhibition of
lipoprotein and hepatic lipases, which leads to accumulation
of VLDL triacylglycerols. In one study [15], the fatty acid
composition of serum lipids was shown to differ between
control serum and serum collected 6 h after Triton injection.
However, only the major fatty acids were measured in
serum total triacylglycerols, while the fatty acids of liver
triacylglycerols were not determined. As Triton WR1339
has been shown to disturb lysosomal lipolysis in liver [16], it

Somnatol (50 mgÆkg
)1
) at 12.00 hours. A cannula was
inserted into femoral vein and Triton WR1339 (600 mgÆkg
)1
)
or saline was injected (12.30–13.00 hours). Two hours
after injection blood was drawn and rats were killed by
heart puncture. The blood samples were taken into 5%
EDTA and plasma was immediately separated by centrifu-
gation. Plasma was overlaid with a NaCl solution
(d ¼ 1.006 gÆmL
)1
) and VLDL was separated by ultracen-
trifugation at 37 000 r.p.m. in a 70.1 Ti rotor (Beckman) for
16 h at 16 °C. Livers were removed and stored at )70 °C. All
experiments were performed under protocols approved by
the Animal Care Committee and the University of Toronto.
Separation of triacylglycerols
Lipids from VLDL and liver samples were extracted with
chloroform/methanol (2:1, v/v) [20]. Triacylglycerols were
separated by TLC on silica gel H plates using heptane/
isopropyl ether/acetic acid (60 : 40 : 4, v/v/v) as the devel-
oping solvent. The triacylglycerol band was scraped off,
extracted in chloroform/methanol (2 : 1) and stored in
chloroform at )20 °C.
Analysis of fatty acid methyl esters
Triacylglycerols were subjected to acidic methanolysis using
6% H
2

dissolved in methanol/water (95 : 5) and applied to Sep-Pak
C18 column (Waters), which had been solvated with the
same solvent. Further 15 mL of this solvent was passed
through the column and NEU derivatives were then eluted
with acetone (10 mL).
HPLC/ESI/MS of diacylglycerols
The sn-1,2 and sn-2,3-diacylglycerols were separated [24]
and analysed as diastereomeric NEU derivatives with a
Waters 550 HPLC connected through a Waters 990
photodiode array detector to a Hewlett-Packard 5989A
quadrupole mass spectrometer equipped with a nebulizer-
assisted electrospray interface. Two normal phase silica gel
columns (Supelcosil LC-Si, 5 lm, 25 cm · 4.6 mm i.d.,
Supelco Inc., Bellefonte, PA, USA) in series were used and
0.37% isopropanol in hexane was used as a mobile phase at
a flow rate of 0.7 mLÆmin
)1
. Positive chemical ionization
was obtained by postcolumn addition of chloroform/
methanol/30% ammonium hydroxide (75 : 24.5 : 0.5, v/v)
at 0.6 mLÆmin
)1
. The capillary exit voltage was 220 V and
the mass range scanned was m/z 500–720. The relative
proportions of diacylglycerol species were calculated from
the areas of the single ion plots obtained from the mass
spectra. NEU derivatives of VLDL diacylglycerols were
collected after HPLC separation. A sufficient amount of
sample for fatty acid analyses was obtained from three
Triton-treated and two nontreated rats.

The values have been expressed as mean ± SD. The
Mann–Whitney U-test was used for comparisons of groups.
6224 J. J. A
˚
gren et al. (Eur. J. Biochem. 269) Ó FEBS 2002
RESULTS
Effect of Triton WR1339 on plasma and VLDL triacyl-
glycerol levels
Plasma and VLDL triacylglycerol concentrations were
4.8 ± 0.8 and 4.4 ± 0.9 mmolÆL
)1
in Triton-treated and
1.4 ± 0.2 and 0.9 ± 0.2 mmolÆL
)1
in nontreated rats,
respectively. This shows that blocking lipolysis by Triton
WR1339 increased plasma and VLDL triacylglycerol levels
about 3.5 and 4.7 times in 2 h, respectively. On the
presumption that the VLDL triacylglycerol concentration
in Triton-treated rats was the same as in nontreated rats
before injection it could be estimated that VLDL contained
at least 80% unmodified triacylglycerol in the Triton-treated
group. This percentage could be also somewhat higher if
there has been any removal of VLDL particles during the
treatment. The very small amount of VLDL diacylglycerol
(0.2 ± 0.0% of neutral lipids) in Triton-treated rats indi-
cate also a minor contribution of modified VLDL. Prelimi-
nary studies showed linear increase of plasma triacylglycerol
concentration at least for 4 h after Triton injection.
However, 4-h treatment was found to affect the fatty

rats in the Triton-treated group with lower proportions of
polyunsaturated fatty acids caused some differences in the
mean values. These rats did not differ, however, from the
other Triton-treated rats in their VLDL fatty acid compo-
sition.
Molecular species of
sn
-1,2 and
sn
-2,3-diacylglycerols
There were statistically significant differences between the
groups in about half of the measured sn-1,2- and sn-2,3-
diacylglycerol species (Table 3). In nontreated rats there was
less 16:0–16:1 and 16:1–16:1 in both sn-1,2- and sn-2,3-
diacylglycerols and less 18:0–18:1 and 18:1–18:2 in the
sn-1,2-diacylglycerols and 16:0–18:2 and 16:1–18:2 in the
sn-2,3-diacylglycerols whereas the proportion of 16:0–18:1
was higher in the sn-1,2-diacylglycerols. In addition, the
proportions of 16:0–20:4 and 16:0–20:5 were lower and
those of diacylglycerol species with a combination of
18- and 22-acyl carbon fatty acids were higher in both
sn-1,2- and sn-2,3-diacylglycerols in nontreated rats. A small
amount of 20:5–22:6 was also found in the sn-2,3-diacyl-
glycerols and its level was higher in nontreated rats.
There were not significant differences in the sn-1,2 and
sn-2,3-diacylglycerol composition of liver triacylglycerols
between the groups. Compared with VLDL the proportions
of 16:0–16:0, 16:0–18:0 and 16:0–18:1 were higher and those
of 16:0–18:2, 16:0–20:4 and 16:0–20:5 were lower in the liver
sn-1,2-diacylglycerol (Fig. 1A). In the sn-2,3-diacylglycerols

Triacylglycerols 93.1 ± 0.5 91.1 ± 0.7
c
C48 (+ C49) 3.2 ± 0.8 2.9 ± 0.5
C50 (+ C51) 13.2 ± 1.3 11.0 ± 0.6
b
C52 33.8 ± 1.3 33.5 ± 1.2
C54 20.6 ± 0.7 18.4 ± 0.7
c
C56 17.2 ± 0.9 19.2 ± 0.9
b
C58 5.1 ± 0.5 6.1 ± 0.5
c
Statistical comparison between the groups:
a
P < 0.05;
b
P < 0.02;
c
P < 0.01.
Ó FEBS 2002 Modification of VLDL triacylglycerols by lipolysis (Eur. J. Biochem. 269) 6225
The proportions of some major VLDL triacylglycerol
species and their reverse isomers, calculated on the basis of
stereospecific positional distribution of fatty acids, are
presented in Table 5. In nontreated rats, the proportions of
most triacylglycerol species with 50 or fewer acyl carbons or
containing 18:0 were lower. Otherwise the fatty acid in the
sn-1 position seemed to have greatest influence on hydro-
lysis. The proportions of triacylglycerol species with 18:2
were mostly lower, and those with 16:0 were higher in
nontreated rats whereas there were not much difference in

treatment and were able to obtain VLDL fraction with 4.7
times greater triacylglycerols concentration than in non-
treated rats. Although the possibility of unknown side-
effects of Triton treatment cannot be ruled out, none of the
known effects of Triton WR1339 gives reason to suppose
that it had affected the composition of accumulated VLDL
triacylglycerols in circulation. Furthermore, similar fatty
acid compositions of liver triacylglycerols and phospholi-
pids (data not shown) in both groups indicate that the
composition of secreted VLDL triacylglycerols was not
affected by Triton WR1339 during the treatment.
The effect of lipolysis on plasma VLDL neutral lipids in
nontreated rats was evident from a reduced triacylglycerol
and increased diacylglycerol proportion. The higher pro-
portion of cholesterol esters in nontreated rats could also
result from the removal of triacylglycerols. However, the
proportion of free cholesterol was not higher in nontreated
rats indicating that the ratio cholesterol ester : cholesterol in
VLDL is modified in the circulation and that Triton
treatment affects these events also. It was shown earlier that
lecithin cholestrol acyltransferase activity is decreased as
Triton WR1339 displaces apolipoprotein A-I in the high
density lipoprotein particles [28] but its other possible effects
on cholesterol metabolism or transfer are unknown. The
greater proportion of 54 and 56 acyl carbon triacylglycerols,
Table 2. Fatty acid composition of VLDL and liver triacylglycerols. Fatty acid methyl esters were prepared from VLDL and liver triacylglycerols by
acidic methanolysis and analysed by GLC. Results are expressed as mole percentages and are mean ± SD for five rats per group.
Triton-treated
VLDL Liver
Triton-treated Nontreated Triton-treated Nontreated

a
0.4 ± 0.1 0.5 ± 0.1
20:4n-6 1.7 ± 0.1 1.7 ± 0.2 1.6 ± 0.2 2.0 ± 0.4
20:5n-3 1.5 ± 0.1 1.7 ± 0.1
a
0.8 ± 0.2 1.2 ± 0.4
22:4n-6 0.4 ± 0.1 0.5 ± 0.2 0.4 ± 0.1 0.5 ± 0.1
22:5n-3 1.8 ± 0.2 2.2 ± 0.3
a
1.6 ± 0.5 2.2 ± 0.6
22:6n-3 5.3 ± 0.4 6.7 ± 1.1
b
4.3 ± 1.5 5.6 ± 0.8
Statistical comparison between the groups:
a
P < 0.05;
b
P < 0.02;
c
P < 0.01.
6226 J. J. A
˚
gren et al. (Eur. J. Biochem. 269) Ó FEBS 2002
containing most of the 20- and 22-carbon fatty acids in
plasma VLDL [30] of nontreated rats corresponds with the
differences observed in the fatty acid and enantiomeric
diacylglycerol composition.
Specificity of endogenous lipases
Lipoprotein and hepatic lipases hydrolyse VLDL triacyl-
glycerols and they have been shown to have positional

30:1 14:0–16:1 0.6 ± 0.2 0.4 ± 0.1 0.3 ± 0.1 0.2 ± 0.1
32:0 16:0–16:0 4.8 ± 0.9 4.6 ± 0.3 2.5 ± 0.5 2.1 ± 0.4
32:1 16:0–16:1 4.9 ± 0.5 3.6 ± 0.2
c
2.5 ± 0.3 2.0 ± 0.3
a
32:2 16:1–16:1 2.3 ± 0.3 1.3 ± 0.1
c
1.1 ± 0.2 0.7 ± 0.1
b
33:0 16:0–17:0 + 15:0–18:0 0.6 ± 0.2 0.6 ± 0.1 0.3 ± 0.1 0.3 ± 0.1
33:1 16:1–17:0 + 15:0–18:1 1.4 ± 0.2 1.1 ± 0.1
a
0.6 ± 0.1 0.5 ± 0.1
34:0 16:0–18:0 2.2 ± 0.2 2.2 ± 0.7 1.6 ± 0.1 1.3 ± 0.2
34:1 16:0–18:1 + 16:1–18:0 19.3 ± 0.9 22.3 ± 0.8
c
7.0 ± 0.4 6.5 ± 0.5
34:2 16:0–18:2 + 16:1–18:1 21.7 ± 1.1 22.4 ± 0.5 9.4 ± 0.5 7.9 ± 0.5
c
34:3 16:1–18:2 + 16:0–18:3 6.5 ± 0.8 7.0 ± 0.9 4.5 ± 0.4 3.6 ± 0.2
c
34:4 16:1–18:3 + 14:0–20:4 0.8 ± 0.1 0.4 ± 0.1
c
0.6 ± 0.1 0.3 ± 0.0
c
35:1 17:0–18:1 + 15:0–20:1 0.9 ± 0.0 1.1 ± 0.1
b
0.4 ± 0.1 0.6 ± 0.2
35:2 17:0–18:2 + 15:0–20:2 1.2 ± 0.1 1.2 ± 0.1 0.9 ± 0.1 0.9 ± 0.2

38:5 16:0–22:5 + 16:1–22:4 0.6 ± 0.2 0.8 ± 0.1 1.8 ± 0.5 2.2 ± 0.2
38:5 18:0–20:5 + 18:1–20:4 +18:2–20:3 0.6 ± 0.1 0.9 ± 0.1
c
0.9 ± 0.1 1.2 ± 0.1
b
38:6 16:0–22:6 + 16:1–22:5 0.9 ± 0.3 0.9 ± 0.1 2.1 ± 0.3 2.0 ± 0.3
38:6 18:1–20:5 + 18:2–20:4 0.3 ± 0.1 0.4 ± 0.1
b
0.4 ± 0.1 0.6 ± 0.2
38:7 16:1–22:6 0.4 ± 0.1 0.6 ± 0.1 0.9 ± 0.2 0.7 ± 0.1
40:4 18:0–22:4 0.0 ± 0.0 0.1 ± 0.1
c
0.2 ± 0.1 0.3 ± 0.1
40:5 18:0–22:5 + 18:1–22:4 0.1 ± 0.0 0.3 ± 0.1
c
1.1 ± 0.1 1.3 ± 0.3
40:6 18:0–22:6 + 18:1–22:5 0.3 ± 0.1 0.8 ± 0.2
c
3.1 ± 0.3 3.7 ± 0.2
c
40:7 18:1–22:6 + 18:2–22:5 0.7 ± 0.2 1.4 ± 0.2
c
5.3 ± 0.8 6.2 ± 0.4
a
40:8 18:2–22:6 0.4 ± 0.1 1.0 ± 0.1
c
3.3 ± 0.4 3.9 ± 0.4
a
40:9 18:3–22:6 0.1 ± 0.1 0.3 ± 0.1
c

Similar examination of 18:1–18:2 indicates that its smaller
proportion in nontreated rats was due to differences in 18:2–
18:1 whereas the values for 18:1–18:2 were about the same
(Fig. 2B). These findings demonstrate that the modification
of circulating VLDL triacylglycerols by basal lipolysis is
only partly revealed by the analysis of fatty acid composi-
tion because of the uncertainty of the origin of each fatty
acid.
Previous work has reported that the polyunsaturated 20-
and 22-acyl carbon fatty acids of human chylomicrons are
released by bovine milk lipoprotein lipase more slowly than
the shorter chain fatty acids [7], and that 22:6n-3 is released
more readily than 20:4n-6 or 20:5n-3 [7,33]. On the other
hand, human chylomicrons enriched with polyunsaturated
fatty acids were hydrolysed faster by human milk lipopro-
tein lipase than chylomicrons containing more saturated
fatty acids [34]. In the present study, triacylglycerol species
containing highly unsaturated fatty acids were also differ-
entially affected by lipolysis. In the sn-1,2 diacylglycerol the
Fig. 1. Proportions of selected sn-1,2-diacylglycerol (A) and sn-2,3-
diacylglycerol (B) species derived from VLDL and liver triacylglycerols
of Triton-treated rats. NEU derivatives of sn-1,2- and sn-2,3-diacyl-
glycerols derived from VLDL and liver triacylglycerols were analysed
by HPLC/MS as described in Materials and methods. Results are
expressed as mean ± SD. Statistical comparison between VLDL and
liver: *P <0.05.
Table 4. Positional distribution of fatty acids in plasma VLDL triacylglycerols. Fatty acid compositions of sn-1,2- and sn-2,3-diacylglycerols
recovered from the HPLC separation were determined from three Triton-treated and two nontreated rats. Stereospecific positional distribution of
fatty acids was calculated from diacylglycerol and total triacylglycerol fatty acid compositions. Results are expressed as mean ± SD.
Triton-treated Nontreated

22-acyl carbon polyunsaturated fatty acids in the sn-1, and
possibly also in the sn-3, but not in the sn-2 position of
VLDL triacylglycerol retards the hydrolysis. This would
explain the divergent changes in the diacylglycerol moieties
containing these fatty acids, and possibly also the differences
found in the studies with VLDL and chylomicrons as the
positional distribution of fatty acids is not similar in these
lipoproteins. VLDL triacylglycerol is derived from liver
cytosolic triacylglycerol through hydrolysis and reesterifica-
tion, and possibly by lipolysis from cellular phospholipids
[29,30,35] The major source of chylomicron triacylglycerol
is the 2-monoacylglycerol pathway in which original dietary
fatty acids are retained in the sn-2 position, e.g. 22:6n-3 in
fish oils [26]. This means that, in addition to difference
between VLDL and chylomicrons, there could be also
substantial differences in the distribution of polyunsaturated
fatty acids within chylomicrons depending on the relative
contribution of endogenous and dietary fatty acids and on
the nature of dietary lipids.
Contrary to retarding hydrolysis when located in the sn-1
position of VLDL triacylglycerol, 20- and 22-acyl carbon
fatty acids in the sn-2 position seemed to advance lipolysis.
It could be speculated that these fatty acids affect the
structure of triacylglycerol molecule in a way that facilitates
the action of lipolytic enzymes. It is also possible that
hepatic lipase has a specific role in hydrolysis of triacylgly-
cerol species with these fatty acids. It has been shown
that the hydrolysis of 20:4 containing triacylglycerol and
diacylglycerol was slower in rat postheparin plasma when
hepatic lipase was inhibited [36]. Hepatic lipase is capable of

and reverse isomer content of triacylglycerols were calculated from the compositions of fatty acids in the sn-1, sn-2 and sn-3 positions of
triacylglycerols.
Written isomer Reverse isomer
Triton-treated Nontreated Triton-treated Nontreated
16:0–16:0–18:1 3.53 3.51 0.34 0.23
16:0–18:1–16:0 1.77 1.74
16:0–18:1–18:1 5.98 7.17 0.57 0.47
16:0–18:2–16:0 1.83 1.76
18:0–18:1–18:1 0.52 0.24 0.15 0.02
16:0–18:1–18:2 2.94 3.67 0.54 0.32
16:0–18:2–18:1 6.19 7.28 0.59 0.48
18:1–18:1–18:1 1.93 1.95
16:0–18:2–18:2 3.04 3.72 0.56 0.32
18:1–18:2–18:1 2.00 1.98
18:2–18:1–18:1 1.84 1.33 0.95 1.00
16:0–16:0–22:6 1.26 1.43 0.03 0.07
16:0–18:1–22:6 2.13 2.92 0.04 0.14
18:1–16:0–22:6 0.41 0.39 0.09 0.27
16:0–18:2–22:6 2.21 2.97 0.05 0.14
18:2–16:0–22:6 0.39 0.27 0.04 0.14
18:1–18:1–22:6 0.69 0.80 0.15 0.56
18:1–18:2–22:6 0.71 0.81 0.16 0.57
18:2–18:1–22:6 0.66 0.54 0.07 0.29
18:2–18:2–22:6 0.68 0.55 0.08 0.29
Ó FEBS 2002 Modification of VLDL triacylglycerols by lipolysis (Eur. J. Biochem. 269) 6229
synthesis. It has been found earlier that 10% of liver-free
diacylglycerol is sn-2,3-diacylglycerol [30]. Another possible
source for these differences is the use of phospholipids for
VLDL triacylglycerol synthesis [29]. As liver phospholipids
contain more polyunsaturated fatty acids, and especially

in the sn-2 position observed in our study could be also a
consequence of hydrolysis only in the primary position
leaving them to diacylglycerols and monoacylglycerols; the
direction of these glycerols may contribute to the divergent
distribution of polyunsaturated fatty acids.
In view of the marked difference in the composition of the
triacylglycerols of nascent and circulating VLDL, some
speculation would seem to be justified about the physiolo-
gical significance of their nonrandom lipolysis. The general
preferential hydrolysis of the unsaturated triacylglycerols
may be related to their higher solubility. However, a retrieval
of the essential fatty acids, which may have facilitated the
VLDL secretion, could also be involved. The preferential
attack on the sn-1-position may serve to avoid flooding of the
lipoprotein and cell membrane surfaces with sn-1,2-diacyl-
glycerols, which may promote glycerolipid resynthesis as well
as compromise the sn-1,2-diacylglycerol signalling pathway.
Furthermore, formation of sn-2,3-diacylglycerols may pre-
vent their accumulation on the lipoprotein surfaces because
of stereochemical incompatibility. Other hypotheses could be
advanced about a preferential release of the saturated and
monounsaturated fatty acids for the purposes of oxidation as
well as about special metabolic roles of specific molecular
species of diacylglycerols or triacylglycerols.
In conclusion, the results of this study show that the basal
lipolysis causes significant modifications in the fatty acid
Fig. 2. Measured and calculated proportions of (A) 16:0–18:2 and (B)
18:1–18:2 in the sn-1,2-diacylglycerol moieties of VLDL triacylglycerols
in Triton-treated and nontreated rats. Measured proportions were
obtained from HPLC/MS analyses of sn-1,2-diacylglycerols and cal-

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