Electrospray ionisation mass spectrometric analysis of lipid restructuring in the carp (Cyprinus carpio L.) during cold acclimation
1 Department of Cell Sciences, School of Biological Sciences, University of
Southampton, Southampton, SO16 7PX, UK
2 Child Health, Division of Infection, Inflammation and Repair, School of
Medicine, Southampton General Hospital, Southampton, SO16 6YD, UK
3 Integrative Biology Research Division, School of Biological Sciences,
University of Liverpool, Liverpool, L69 7ZB, UK
* Author for correspondence (e-mail: cossins{at}liverpool.ac.uk)
Accepted 16 August 2002
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Summary |
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Key words: cold acclimation, carp, Cyprinus carpio, phospholipid, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, monounsaturated fatty acid, ESI-MS
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Introduction |
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Traditional methods of analysing the phospholipid components of biological
membranes have tended to concentrate upon their component fatty acids, rather
than examining the biologically relevant molecules in their entirety, thereby
oversimplifying the processes occurring. Changes in lipid composition during
temperature-induced membrane restructuring are extremely complex. The
structure of membrane phospholipids is itself complicated, the physical
properties of each molecular species being dependent upon headgroup, fatty
acid chain length, unsaturation of individual fatty acids and the positional
distribution of the two fatty acids within the molecule
(Stubbs and Smith, 1984;
Hazel and Williams, 1990
).
Inclusion of the first unsaturated bond causes the greatest change in the
physical properties of any fatty acid, with subsequent unsaturation having
progressively less effect (Coolbear et al.,
1983
; Stubbs and Smith,
1984
).
The results from Trueman et al.
(2000), which stimulated this
further analysis, showed that cooling-induced lipid restructuring in carp
liver microsomes was accompanied by co-ordinated expression of various lipid
biosynthetic enzymes. Fatty acyl
9-desaturase activity (EC No.
1.14.99.5) was an integral part of this response;
9-desaturase is
responsible for incorporating the first double bond into fatty acids at the
C9C10 position, causing maximum disordering. Previous studies have
primarily tended to examine this conversion of the saturated fatty acids
palmitate (16:0) and stearate (18:0) into their monounsaturated derivatives
palmitoleate (16:1) and oleate (18:1). Analysis solely of the desaturase
pathways, although important, ignores the complexity of the molecular species
structure of membrane phospholipids and the mechanisms by which membrane
fluidity is maintained. In addition to
9- and other desaturases, a
number of additional mechanisms contribute to the regulation of membrane
phospholipid composition, including fatty acid elongases, acyltransferases,
de novo synthesis, intracellular transport processes and
phospholipase activities.
Previous analyses of the effects of temperature on the molecular structures
of membrane phospholipids have been hampered by the laborious nature of the
analytical procedures involved. These have generally involved sequential steps
of thin layer chromatography, enzymatic hydrolysis by phospholipase C,
formation of dinitrobenzoyl (DNB)- diacylglycerol derivatives, and resolution
of DNB-diacylglycerol species by high-performance liquid chromatography
(HPLC). In addition to the errors inherent in any multi-step procedure,
identification of eluted species is often problematic, relying on relative
retention time. Moreover, co-elution of multiple species has meant that
complete identification of individual species has required repeat analyses
using mobile phases of different polarities
(Bell and Tocher, 1989).
Recently, the application of electrospray ionisation mass spectrometry
(ESI-MS) to the analysis of molecular species compositions of membrane
phospholipids has attracted great interest. ESI-MS provides a rapid and
sensitive quantitative technique for the detailed characterisation of
phospholipid molecular structures, and a variety of tandem MS/MS procedures
can provide unequivocal assignments of both composition and positional
assignment of fatty acids within each phospholipid species
(Brügger et al., 1997;
Han and Gross, 1995
). In the
present study, we have used ESI-MS to determine detailed changes to the
molecular species of carp liver microsomal phospholipids over five days after
a change in environmental temperature. The results demonstrate clearly both
the relatively restricted nature and the precise regulation of the adaptive
process involved.
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Materials and methods |
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Cooling regime
Animals were gradually cooled from 30°C to the specific temperatures of
23°C, 17°C and 10°C and were maintained at those temperatures for
five days (see Trueman et al.,
2000, for full details of the stepwise cooling programme). All
temperature changes were at a rate of 1°Ch-1 for a maximum rate
of change of 7°C day-1
(Schunke and Wodtke, 1983
).
One group of fish was maintained at 30°C throughout the experiment and
sampled on day 5. For each sampling temperature, replicate fish were killed by
pithing, their livers were removed and liver microsomal fractions were
isolated.
Isolation of liver microsomes
Liver microsomes were prepared at Liverpool University using a modification
of the method of Wodtke and Cossins
(1991). All procedures were
carried out at 0-4°C. Liver tissue was weighed, minced and homogenised in
four volumes (w/v) buffer (250 mmol l-1 sucrose, 20 mmol
l-1 Hepes, pH 7.4) using eight passes in a glassTeflon
homogeniser. The homogenate was centrifuged for 30 min at 10 000
g, the supernatant removed and CsCl added to give a final
concentration of 15 mmol l-1 before centrifuging again at 120 000
g for 80 min. The resulting pellet was gently resuspended in a
saline solution (150 mmol l-1 NaCl, 0.1 mmol l-1 EDTA,
20 mmol l-1 Hepes, pH 7.4), taking care not to disturb the glycogen
portion of the pellet, and was centrifuged at 120 000 g for 60
min. After centrifuging, the pellet was resuspended in a small volume of
saline solution and frozen at -80°C.
Mass spectrometric analysis of phospholipids
Total lipids were extracted from 50 µl of liver microsomes using
chloroform and methanol, according to Bligh and Dyer
(1959). After drying under
nitrogen gas, lipid was dissolved either in methanol:chloroform:water (7:2:1
v:v:v) for single-stage MS analysis and tandem MS analysis of PC, PI and PS or
in the same solvent containing 0.5% (w/v) sodium iodide for tandem MS analysis
of PE. All electrospray ionisation mass spectrometric (ESI-MS) analysis was
performed using a triple-quadrupole tandem mass spectrometer (Quattro Ultima,
Micromass, Manchester, England) equipped with a Z-spray electrospray
ionization interface. Samples were infused with a syringe pump (Model 11,
Harvard Apparatus, South Natick, MA, USA) at a flow rate of 5 µl
min-1 directly into the mass spectrometer. Dry heated nitrogen was
used as both the cone gas (70 1 h-1) and the desolvation gas (600 1
h-1), while dry argon was used as the collision gas (0.35 Pa).
Single-stage ESI-MS
Samples were analysed in both positive and negative ionisation modes with
applied capillary voltages of +3kV and -3kV and cone voltages of +90V and
-100V, respectively. Positive ionisation spectra were dominated by the PC
species, while negative ionisation spectra contained peaks for all
phospholipids present. All data were collected with a scan time of 2.5 s over
a mass:charge (m/z) range of 600-950 and were recorded as a signal
average of 10 scans at atomic resolution.
Tandem mass spectrometry
Although all phospholipids could be detected by single-stage MS,
quantification can be problematic owing to signal overlap, differential
ionisation and adduct formation. Tandem MS of phospholipids after electrospray
ionisation can be used to confirm the identity of ions and can be diagnostic
for a class of phospholipid within a complex mixture.
Tandem MS is accomplished by passing the precursor ion of selected
m/z into the second quadrupole (collision cell). Collision-induced
dissociation (CID) of the precursor ion with argon gas generates product ions,
which are subsequently analysed in the third quadrupole. For unambiguous
identification of individual phospholipids, the mass-selected precursor ion in
negative ionisation is subjected to CID and the resulting product ion spectrum
is routinely dominated by peaks corresponding to the fatty acid chains.
Moreover, depending on the collision voltage used, preferential loss of fatty
acyl products from either the sn-1 or the sn-2 position of
the glycerol backbone can also provide evidence for the stereospecificity of
individual phospholipids (Han and Gross,
1995). Such analysis, based on detection of fatty acyl product
ions and of the acyl ketene moieties formed by loss of acyl groups
(Han and Gross, 1995
;
Hsu and Turk, 2000
), was
performed to characterise all the molecular species assignments presented in
this paper. Under more specific conditions, molecular ions of certain classes
of phospholipids will generate diagnostic product ions
(Brugger et al., 1997
).
Subsequent precursor scans that search for these specific product ions will
then create a quantitative mass spectrum containing only peaks pertaining to
that particular class. Such class-specific fragmentation and subsequent
scanning modes established in our laboratory are outlined in
Table 1.
|
Precursor scan data were recorded with a scan time of 12 s over m/z ranges of 675-860 (PC) and 750-925 (PI). Neutral loss spectral data were recorded with a scan time of 2.5 s over an m/z range of 625-900.
The spectra obtained were processed using Masslynx software (Micromass, Manchester, UK). Preliminary studies (results not shown) established that samples were sufficiently diluted for all responses to be linear with concentration for each phospholipid class. Each phospholipid generated either a protonated [M+H]+ or sodiated [M+22]+ ion in positive ionisation and a deprotonated [M-H]- molecular ion in negative ionisation, together with associated 13C-containing ions. Molecular species compositions of individual ion peaks were assigned on the basis of a combination of product, precursor and neutral loss scans as appropriate. Results are presented in terms of the predominant molecular species determined for each ion peak and are expressed as mol % of the total in each phospholipid class.
Statistical analysis
Data were analysed for significant differences between warm-acclimated
control and cooled fish using one-way analysis of variance (ANOVA), adjusting
for multiple comparisons with Bonferroni's test. P0.05 was used
to establish statistical significance.
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Results |
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The molecular species analysis presented in Table 2 shows very clearly that, despite the restricted number of molecular species present, the compositions of PC, PE and PI were maintained very precisely and independently of each other. For instance, polyunsaturated molecular species containing 16:0 at the sn-1 position were predominant for PC and PE, while PI contained a higher proportion of species with sn-1 18:0. All monounsaturated PC species contained 16:0 or 16:1 at their sn-1 position, while monounsaturated PE species contained sn-1 18:0.
Effects of cooling on microsome phospholipid composition
Phosphatidylcholine
The total proportion of all PC species possessing saturated fatty acids at
the sn-1 position was significantly higher in the control fish
maintained at 30°C than in any of the cooled fish
(Fig. 2A). By contrast, PC from
fish kept at the three lower temperatures exhibited a greater proportion of
monounsaturated fatty acids at the sn-1 position than did PC from
30°C control fish (Fig.
2A). There were no significant compositional differences observed
at the sn-2 position of any of the molecular species measured. The
proportion of PCs possessing saturated fatty acids, monounsaturated fatty
acids or polyunsaturated fatty acids at this position was not affected by
temperature (Fig. 2A).
|
Phosphatidylethanolamine
The composition of PE molecular species altered in response to temperature
with a very similar pattern to that of PC. Again, the fatty acid distribution
at the sn-2 position was not influenced by temperature
(Fig. 2B), while the proportion
of PE species with a saturated fatty acid at the sn-1 position was
significantly greater at 30°C than at the lower temperatures. Conversely,
the proportion of PE species with a monounsaturated fatty acid at the
sn-1 position was significantly lower in the control fish
(Fig. 2B) compared with fish
maintained at all three lower temperatures.
Phosphatidylinositol
In contrast to both PC and PE, no PI species was detected with either a
saturated fatty acid or a monounsaturated fatty acid at the sn-2
position; all of the PI species contained a polyunsaturated fatty acid at the
sn-2 position. As with PC and PE, the proportion of saturated or
monounsaturated fatty acids at the sn-1 position of PI changed
significantly with temperature. At 30°C, the PI species contained
significantly more saturated fatty acids than at the lower temperature
(Fig. 2C), while the reverse
was observed for PI species containing monounsaturated fatty acids.
Individual molecular species
Phosphatidylcholine
The modulation of PC molecular species present in liver microsomes in
response to temperature was relatively restricted and modest. The proportions
of the three monounsaturated PC species, PC16:0/16:1, PC16:0/18:1 and
PC16:1/18:1, did not change significantly with acclimation temperature
(Fig. 3). By contrast, the
already low proportion of PC16:0/16:0 at 30°C (1.80±0.57%) was
significantly lower at 23°C (0.77±0.01%) and 17°C
(0.67±0.22%). The proportions of the major polyunsaturated PC species
changed in response to temperature, but the temperature dependence of such
change varied for individual species. For instance, the proportion of
PC16:0/22:6 decreased between 30°C (61.19±2.50%) and 23°C
(47.78±2.82%) and then remained constant down to 10°C, while the
proportion of PC16:1/22:6 only increased significantly between 17°C
(5.80±0.40%) and 10°C (9.92±1.23%)
(Fig. 3).
|
Phosphatidylethanolamine
Significant temperature-dependent changes were observed for proportions of
three of the four major PE species, but for none of the five minor PE species
(PE18:0/18:0, PE18:0/18:1, PE16:0/20:4, PE16:1/20:4, PE16:1/22:6;
Fig. 4). As for PC, the
proportion of the predominant PE species (PE16:0/22:6) decreased significantly
between 30°C (62.09±3.54%) and 23°C (46.31±9.23%) and
then remained constant down to 10°C. By contrast, proportions of the two
PE species containing both 22:6 and a monounsaturated fatty acid both
increased incrementally between 30°C and 10°C
(Fig. 4); the proportion of
PE18:1/22:6 increased from 7.57±2.69% at 30°C to 21.49±5.63%
at 10°C, while corresponding values for PE20:1/22:6 were 2.81±0.57%
and 14.43±3.47% at 30°C and 10°C, respectively. It is important
to note that the proportion of PE species containing 18:1 only changed with
temperature when that fatty acid was at the sn-1 and not the
sn-2 position. This was shown clearly by the progressive increased
proportion of PE18:1/22:6 with decreasing temperature in contrast to the
unaltered concentration of PE18:0/18:1.
|
Phosphatidylinositol
The composition of liver microsomal PI altered in response to temperature
to a greater extent than that of either PC or PE. As with PC and PE though,
there was a consistent pattern to these changes, with increased proportions of
PI species containing sn-1 monounsaturated fatty acids and decreased
proportions of PI species containing sn-1 saturated fatty acids
(Fig 5) at lower temperatures.
This was most apparent for the considerable and progressive decreased
proportion of PI18:0/20:4 from 52.2±9.1% at 30°C to
29.99±5.72% at 10°C. The parallel decreased proportion of
PI16:0/20:4 was of smaller magnitude and was only significant at 23°C and
17°C. By contrast, cooling from 30°C to 10°C significantly and
progressively increased the proportions of PI18:1/20:4 (from 16.9±2.7%
to 22.85±2.34%), PI18:1/20:5 (from 6.3±2% to 11.30±1.80%)
and PI20:1/20:4 (from 2.45±0.7% to 10.16±2.13%). Proportions of
PI species containing 22:6 (PI18:0/22:6 and PI18:1/22:6) did not alter
systematically with variation in temperature, although that of PI18:1/22:6 was
significantly higher at 17°C. The only PI species containing a
monounsaturated fatty acid not to respond to cooling at any temperature was
PI16:1/20:4.
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Discussion |
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ESI-MS analysis facilitated the analysis of the effects of decreasing temperature on PI as well as on PC and PE molecular species, and the different responses of these three phospholipid classes to temperature change are potentially important for maintenance of membrane function. The fundamental principles defining these changed compositions were essentially similar for all three phospholipid classes, with increased proportions of species containing monounsaturated fatty acids at the expense of species containing saturated fatty acids. The extent of these changes, however, both in magnitude and in the number of species affected, was greatest for PI, then for PE and finally for PC. Unfortunately, the poor availability of synthetic individual molecular species of PI has precluded comparison of the liquid crystal to gel transition temperatures of PI, PE and PC species. Consequently, it is not clear whether modulation of PI species composition in response to temperature will exert a disproportionate effect on membrane structure and function.
It is not surprising that cooling was not associated with any change to the
proportions of PC, PE or PI species containing polyunsaturated fatty acids
esterified at the sn-2 position of the glycerol backbone of the
molecule. As all three phospholipid classes already contained high proportions
of these fatty acids at 30°C, increasing their content still further at
lower temperatures would have exerted little effect on membrane structure or
fluidity. The greatest effect on the physical properties of a fatty acid is
apparent on introduction of the first unsaturated double bond into a saturated
molecule (Coolbear et al.,
1983; Stubbs and Smith,
1984
). This is reflected in the strategy adopted by carp liver
microsomes to adapt to lower temperatures, with saturated fatty acids being
replaced by monounsaturated fatty acids in all phospholipid classes. It is
important to recognise, however, that this response is highly regulated and
co-ordinated and cannot be simply a consequence of increased
9-desaturase activity providing more monounsaturated fatty acids for
esterification into phospholipids at lower temperature. While induction of the
9-desaturase contributes to the overall temperature response
(Trueman et al., 2000
), it
cannot explain why the response is restricted to selected individual molecular
species of phospholipids containing monounsaturated fatty acids or why such
changes were only apparent for fatty acids esterified at the sn-1 and
not at the sn-2 position.
This selectivity of the response to temperature can be seen most clearly for the composition of microsomal PC (Fig. 3), which was enriched specifically with PC16:1/22:6 and PC18:1/22:6 at lower temperatures at the expense of PC16:0/22:6. The lack of change to other PC species containing either 16:1, 18:1 or 22:6 emphasises the precision of the response. For instance, over the same temperature range that modulated the proportions of PC16:1/22:6 and PC16:0/22:6, no response was apparent for PC18:1/22:6, PC18:0/22:6, PC16:1/20:4 or PC16:1/18:1 proportions. Contrasting selectivity was also apparent for the response of PE composition to lower temperatures (Fig 4), as increased proportions were apparent only for PE18:1/22:6 and PE20:1/22:6 and not for PE16:1/22:6 or any other PE species. The observation that PE18:0/18:1 concentration did not alter with temperature also demonstrates the positional selectivity of the response, clearly showing that the lower temperatures only stimulated incorporation of the 18:1 monounsaturated fatty acid into the sn-1 and not into the sn-2 position of PE.
The effect of temperature acclimation in fish on the molecular species compositions of liver PI has not previously been examined. While temperature selectivity of the PI response was initially less obvious, this may reflect the very different composition of PI from PC and PE molecular species in carp liver microsomes (Table 2). PC and PE compositions were dominated by species containing 22:6n-3, but PI was predominantly composed at all temperatures of species containing 20:4n-6 at the sn-2 position. The details of the PI response, however, are intriguing and differ considerably from those of either PC or PE. The proportion of PI18:0/20:4, which remained the predominant PI species at all temperatures, decreased by over 40% between 30°C and 10°C (Fig. 5). The large magnitude of this decrease was not accompanied by any correspondingly large increased proportion of an individual PI species containing monounsaturated fatty acids. Instead, proportions of three such PI species, PI18:1/20:4, PI18:1/20:5 and PI20:1/20:4, increased in parallel with decreasing temperature. PI was the only phospholipid class where significant modification to the proportions of fatty acids esterified at the sn-2 position was observed, shown by the increased proportions of PI18:1/22:6 at 17°C and PI18:1/20:5 at 10°C.
These results confirm and extend those in the earlier study by Trueman et
al. (2000), which examined the
effects of differential cooling on the fatty acid composition of the
phospholipid classes PC and PE. We demonstrate the enhanced analysis possible
by analysis of individual molecular species instead of total fatty acid
compositions. For instance, inspection of the PC fraction shows clearly that
the significant increased proportion of total 16:1n-9 in PC after
cooling for a period of five days (Trueman
et al., 2000
) reflected the increased contribution of a single PC
species (PC16:1/22:6) with no alteration to other PC species containing 16:1
(Fig. 3). Similarly, Trueman et
al. (2000
) found transient
changes in the total 16:1, 18:1 and 20:1 contents of the PE fraction depending
upon acclimation temperature and length of acclimation. Molecular species
analysis shows that the increased total 20:1 reflected solely the greater
quantities of PE20:1/22:6 at lower temperatures
(Fig. 4) and also explained the
transient nature of the temperature response of total 18:1n-9 in PE.
Lower temperatures (Fig. 4)
were associated with a range of non-significant changes to the contents of a
range of PE species containing 18:1n-9. The variation in these
changes was sufficient to mask the significant increased proportion of
PE18:1/22:6 when combined for total fatty acid analysis.
While considerable attention has been given to the effects of temperature
acclimation on membrane phospholipid compositions
(Stubbs and Smith, 1984;
Cossins, 1994
;
Hazel, 1995
;
Hazel and Williams, 1990
), the
basic mechanisms that regulate membrane phospholipid compositions have not
been clearly identified. By comparison, extensive studies in rat liver have
demonstrated a complex interaction of phospholipase and acyltransferase
activities that regulates dynamic processes of acyl remodelling. For instance,
analysis of phospholipid synthetic pathways using radiochemical HPLC has
consistently demonstrated an initial synthesis of PC16:0/18:2 by rat liver
in vivo (Burdge et al.,
1994
) and in isolated rat hepatocytes
(Tijburg et al., 1991
), with
subsequent conversion to other PC species by processes of acyl exchange. Such
acyl remodelling is responsible for the incorporation of 20:4n-6 into
rat liver PC, while 22:6n-3 is introduced into rat liver PC by
N-methylation of PE. One study that followed the incorporation
pattern of exogenously added radiolabelled phospholipids has indicated that a
complex interaction of deacylation/reacylation, elongation and desaturation
reactions is similarly involved in the early restructuring of PC molecular
species from plasma membrane fractions of isolated trout hepatocytes
(Williams and Hazel, 1995
).
The precise details of such acyl remodelling of phospholipids in fish liver,
however, is still unknown. Such information will be essential to determine the
mechanisms regulating membrane adaptations to temperature change. In this
context, the great sensitivity and selectivity of phospholipid molecular
species analysis by ESI-MS should prove especially valuable. ESI-MS cannot
only provide detailed compositional information but is ideally designed for
metabolic studies of phospholipid synthesis and turnover using deuteriated
substrates (Hunt et al.,
2001
).
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Acknowledgments |
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References |
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