302s Biochemical Society Transactions (1995) 23
Desatumtion and estenfication of palmitic and steak acids in
cultured hepatocytes
JENNIFER S BRUCE, SlON RICHARDS, GUILLAUME
LEGRAND AND ANDREW M SALTER
Department of Applied Biochemistry and Food Science,
University of Nottingham, Sutton Bonington Campus,
Loughborough, LE I2 5RD
Considerable evidence exists to suggest that not all long chain
saturated fatty acids are metabolized in an identical manner, for
example, dietary stearic acid does not increase plasma
cholesterol to the same extent as palmitic acid [I] Several
reasons have been suggested for this difference, including
effects on intestinal absorption [2], rate of desaturation [3] and
rate of incorporation into cellular lipids [4] We have previously
reported reduced plasma VLDL cholesterol concentrations in
hamsters fed tristearin compared to tripalmitin [5] In the
present study this was investigated further by considering the
desaturation and esterification of these fatty acids, in
comparison to oleic acid, by monolayer cultures of hamster
hepatocytes
Hamster hepatocytes were prepared and maintained in
monolayer culture essentially as previously described for rat
hepatocytes [6]
After overnight incubation, cells were
transferred to serum free medium containing 2gA bovine serum
albumin (BSA) and 300pM fatty acid bound to BSA (61 Cells
were incubated for up to 8h, after which lipids were extracted
from both cells and medium and individual lipid classes were
separated by thin layer chromatography
Table I: Removal of fatty acid fmm the medium and
estenfication into differrent lipid fractions in cultured
hepatocytes
lower for stearate. The relative proportion of the total
triacylglycerol which was secreted at the 4h time point was
greater for oleate (21. I I”/) than for palmitate and stearate
( 1 5.28 and 17.75%, respectively). incorporation of each of the
radiolabelled fatty acids into secreted phospholipid appeared to
saturate at 30min. without any further increase with time.
In a further experiment the extent of desaturation of
palmitate and stearate was examined.
Hepatocytes were
incubated with fatty acids as indicated above. After 4h cellular
lipids were extracted and separated, and the phospholipid and
triacylglycerol fractions were then saponified. The fatty acids
released were separated by silver phase thin layer
chromatography and radioactivity associated with saturated and
monounsaturated bands determined. Table 2 shows that more
stearate than palmitate was desaturated and that a greater
proportion of unsaturated fatty acid was found in the
phospholipid fraction compared to the triacylglycerol.
However, the proportion of fatty acid desaturated was relatively
low for both fatty acids suggesting that in neither case is this a
major route of metabolism
Table 2: Relative proportion of
major cellular lipids
fatty acid
3)
4)
5)
’
shows that palmitate was removed from the medium at
rate than oleate or stearate The rate of incorporation
acids into cellular triacylglycerol and phospholipid was
for up to 4h and decreased in the order,
palmitate>oleate>stearate The proportion of stearate which was
incorporated into phospholipid relative to triacylglycerol was
similar for oleate (0 52) and palmitate (0 53), but considerably
higher for stearate (0 69) Incorporation of fatty acid into
secreted triacylglycerol was linear for up to 8h The rate of
secretion was similar for oleate and palmitate but considerably
triacylglycerol
C-stearate
esu ts are expresse as t e percentage o t e tota atty aci
recovered in each fraction found to be unsaturated
These results suggest that differences in the rate of fatty acid
uptake and incorporation into triacylglycerol and phospholipid
may, at least i n part, explain some of the different effects
dietary palmitic and stearic acid have on lipoprotein
metabolism By contrast, hepatic desaturation of the fatty acids
appears to account for a relatively small proportion of the total
metabolized and is a less likely explanation for these observed
differences
2)
Table I
a faster
of fatty
linear
phospholipid
H-palmitate
1)
medium and secreted triacylglycerol and nMoles fatty a c i d h h g
cell protein for incorporation into cellular triacylglycerol and
phospholipid Removal of fatty acids from the medium and
incorporation into cellular lipids was measured over 4h while
secretion was measured over 8h
fatty acids desaturated in
6)
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