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Patel S., Nelson D.R. and Gibbs A.G. 2001. Chemical and Physical Analyses of Wax Ester Properties. 7 pp.
Journal of Insect Science , 1.4. Available online: insectscience.org
Insect
Science
insectscience.org
Received: 23 March 2001, Accepted 4 April 2001, Published April 24, 2001
Abstract
Wax esters are major constituents of the surface lipids in many terrestrial arthropods, but their study is complicated by their diversity. We
developed a procedure for quantifying isomers in mixtures of straight-chain saturated and unsaturated wax esters having the same molecular
weights, using single-ion monitoring of the total ion current data from gas chromatography-mass spectrometry. We examined the biological
consequences of structural differences by measuring the melting temperatures, Tm, of >60 synthetic wax esters, containing 26-48 carbon
atoms. Compounds containing saturated alcohol and acid moieties melted at 38-73oC. The main factor affecting Tm was the total chain
length of the wax ester, but the placement of the ester bond also affected Tm. Insertion of a double bond into either the alcohol or acid
moiety decreased Tm by ~30oC. Simple mixtures of wax esters with n-alkanes melted several oC lower than predicted from the melting
points of the component lipids. Our results indicate that the wax esters of primary alcohols that are most typically found on the cuticle of
terrestrial arthropods occur in a solid state under physiological conditions, thereby conferring greater waterproofing. Wax esters of
secondary alcohols, which occur on melanopline grasshoppers, melted >60oC below primary esters of the same molecular weight and
reduced Tm of the total surface lipids to environmental values.
Abbreviation:
FTIR Fourier-transform infrared spectroscopy
SIM Single ion monitoring
TIC Total ion current
levels of unsaturated fatty acids and alcohols (Kattner et al., 1996; grasshopper, Melanoplus sanguinipes, as follows. Cuticular lipids
Phleger, 1998; Saito and Murata, 1998). were extracted from frozen grasshoppers using a 10-minute hexane
A major technical problem in the analysis of wax esters is wash, followed by a 1-minute wash. Waxes were separated from
that isomers having the same molecular weight (and retention times other lipid constituents using silica gel chromatography. Total surface
on gas chromatograms) are often present. Researchers typically lipids from individual grasshoppers were applied to a column in a
digest the waxes and analyze the resulting fatty acids, thereby losing pasteur pipet. Alkanes were eluted with hexane, and wax esters were
information about the specific molecular species of wax esters eluted with a hexane:chloroform (98:2 v/v) mixture. The purity was
present. Despite these problems, the chemical composition of wax checked by thin-layer chromatography and GC, and was >98% wax
esters has been described in numerous species, but the biological ester. Base-catalyzed hydrolysis of the wax esters, followed by GC
significance of wax ester differences is unclear. In both terrestrial analysis of the products (Nevenzel et al., 1985), indicated that the
and marine organisms, the physical properties of wax esters may be major alcohol and acid components contained 16-23 carbons, as
functionally important. For example, surface lipids of insects provide reported previously (Blomquist et al. 1972).
a better waterproofing barrier when they are in a solid rather than
fluid state (Gibbs, 1998; Rourke and Gibbs, 1999; Rourke, 2000), Gas Chromatography-Mass spectrometry
so one would expect insects to synthesize waxes with high melting Gas chromatographymass spectrometry (GCMS) was
temperatures. On the other hand, melted lipids are less dense than performed on a Hewlett-Packard HP 5890A gas chromatograph
solid lipids, so marine organisms that use wax esters for buoyancy equipped with a pressure programmable cool oncolumn injection
would gain greater lift if their waxes were in a fluid state. port. The column consisted of a 1 m retention gap connected to a
Most biophysical analyses of wax esters have used complex 12.5 m X 0.2 mm capillary column of crosslinked dimethyl silicone
mixtures containing waxes and other lipid components (e.g. Basson Ultra 1 (Hewlett-Packard) and was coupled to a HP 5970B
and Reynhardt, 1988a,b; Sessa et al., 1996). In this paper, we report quadrupole mass selective detector. The carrier gas was helium, and
a method for determining the relative amounts of isomers of straight- the initial column temperature was between 150 and 200 oC and
chain saturated wax esters, based on single-ion monitoring values was programmed to increase to 320oC at 3 or 4o/min and held at
from gas chromatography-mass spectrometry (GC-MS) analyses. 320oC. The system was operated and data collected with a Hewlett-
We also describe the physical properties (melting temperatures, Tm) Packard 5970C computer. The system was calibrated, and the quality
of over 60 pure synthetic wax esters, most of which occur naturally. of the chromatogram monitored, with a standard mixture consisting
We examine how structural variations (acid and alcohol chain length, of: methyl heptadecanoate, methyl icosanoate, 3-methyltricosane,
ester position, unsaturation) affect Tm values. If waxes melt at tricosanyl acetate, octacosane, tetracontane, and tricosanyl
ecologically relevant temperatures, then lipid phase transitions may heptadecanoate, run at six concentrations. The mass spectral data
have significant effects on insect water balance. for the wax esters were analyzed by single-ion extraction of the
data (Nelson et al., 2000). The integrated areas for the total ion
Methods and Materials current (TIC) and the single ions obtained by single-ion monitoring
(SIM) of the data were then compared to obtain the factor needed to
Wax Esters convert SIM area values to TIC areas, so that wax esters could be
Synthetic wax esters of primary alcohols were purchased quantified based solely on their SIM values if present in a mixture.
from Nu-Check Prep or Sigma Chemical Co., or were synthesized
by modification of the method previously described (Nelson et al., Lipid Physical Properties
1990), based on that of Iyengar and Schlenk (1969). Equal molar Melting temperatures (Tm) of lipid samples were determined
amounts (about 0.1 mmole) of the alcohol and the acid chloride using Fourier-transform infrared (FTIR) spectroscopy (Gibbs and
were separately dissolved in 4 ml of dry diethyl ether or Crowe, 1991). Approximately 50 µg was dissolved in hexane and
tetrahydrofuran. The solutions were slowly mixed together in a flask deposited as a thin film on a CaF2 window, which was then placed
of a reflux apparatus and refluxed for 1 hr. Five 100 µl portions of in a temperature-controlled cell holder in a Perkin-Elmer Systems
dry pyridine (distilled and stored over a molecular sieve) were then 2000 FTIR spectrometer. The sample temperature was increased in
added at 10 min intervals. Sufficient solvent was then added to increments of ~1oC, with an infrared spectrum being collected at
allow refluxing overnight. The reaction mixture was transferred to each temperature. As alkyl chains melted, the frequency of -CH2-
a separatory funnel and washed sequentially with 6N sulfuric acid, symmetric stretching vibrations increased from ~2849 cm-1 to ~2854
6N sodium hydroxide, and water. The organic phase was dried over cm-1, and was used as an index of lipid melting. The midpoint of the
magnesium sulfate, filtered through glass wool, the solvent removed phase transition (Tm) was calculated by fitting plots of frequency vs.
and the wax ester weighed. The wax was dissolved in a minimum temperature to a logistic equation.
volume of chloroform and spotted on a 20x20 cm plate of 250 µm
thick silica gel G. The plate was developed in hexane:diethyl Results
ether:formic acid (80:20:1; v/v/v). The area containing the wax ester
was removed and the esters eluted with 4 bed volumes of chloroform. Gas chromatography-mass spectrometry
The eluate was dried and the ester redissolved in 95% ethanol with We used GC-MS to confirm that all wax esters studied were
heating. The solution was then cooled, and the wax ester crystals greater than 98% pure and were of the predicted structures. Wax
collected. esters can be identified by the chain lengths and unsaturation of
Wax esters of secondary alcohols were isolated from the their alcohol and acid chains, respectively. For example, arachidyl
Patel S., Nelson D.R. and Gibbs A.G. 2001. Chemical and Physical Analyses of Wax Ester Properties. 7 pp. Journal of Insect Science , 1.4. Available 3
online: insectscience.org
Figure 3. Effects of carbon number on melting temperatures of saturated wax Figure 5. Effects of unsaturation on melting temperatures of wax esters.
esters of primary alcohols. For a given number of carbon atoms, each point
represents an isomer whose ester bond is in a different position.
related to the position of the ester linkage were observed. Lipid Tm these, the fatty acid moiety, containing 14-22 carbons, forms an
values were higher for compounds having alcohol and acid chains ester bond with 11-tricosanol or other secondary alcohols. These
of the same length on either side of the ester moiety. When the ester wax esters therefore have a fundamentally different geometry from
link was moved to less central locations along the molecule, the Tm the synthetic wax esters studied above, being T-shaped rather than
decreased by 1-5oC (Figure 4). linear. In spite of the fact that wax esters from M. sanguinipes are
Unsaturation significantly affected the properties of wax saturated and contain ~40 carbon atoms, they melted at 5-10oC
esters. For waxes containing 18 carbon atoms in both alcohol and (Figure 6). We note that tricosanyl 1-stearate, an isomer of the major
acid moieties, insertion of a single cis-9 double bond reduced the wax ester of M. sanguinipes, melts at ~68oC (Figure 3).
Tm from 61oC to 27 or 37oC, depending on whether the double bond
occurred in the alcohol or acid chain, respectively (Figure 5). Oleyl Mixtures of wax esters with alkanes
oleic ester, which is unsaturated in the 9-position on both chains, Although wax esters are abundant on many arthropod
melted slightly below 0oC. species, the major component of the surface lipids is usually long-
chain hydrocarbons (de Renobales et al., 1991). Interactions between
Wax esters of secondary alcohols lipid classes can significantly affect bulk lipid properties (Gibbs,
The lesser migratory grasshopper, M. sanguinipes, contains 1995). We used FTIR to examine the properties of simple mixtures
~30% wax esters of secondary alcohols (Blomquist et al., 1972). In between pairs of wax esters, containing 21-30 carbons, and n-
Figure 4. Effects of ester bond position on Tm. Numbers indicate the total Figure 6. Melting curves for cuticular lipids isolated from Melanoplus
number of carbon atoms for each series of points. Ordinal data are expressed sanguinipes. Wax ester and hydrocarbon fractions were separated using silica
as the difference in chain length between the alcohol and acid moieties. Each gel chromatography. These melting curves were obtained using lipids isolated
point indicates a separate determination of Tm. from a single adult grasshopper.
Patel S., Nelson D.R. and Gibbs A.G. 2001. Chemical and Physical Analyses of Wax Ester Properties. 7 pp. Journal of Insect Science , 1.4. Available 5
online: insectscience.org
Wax ester properties and insect water balance maintain the surface lipids in a solid, impermeable state, despite
Wax esters are important components of the surface lipids the presence of some unsaturated components.
of many terrestrial arthropods (de Renobales et al., 1991; Buckner, An exception to the apparent waterproofing benefits of wax
1993). Most cuticular wax esters are saturated and contain at least accumulation is provided by the secondary wax esters of
30 carbon atoms, so they will melt above 50oC (Figure 3). This is grasshoppers from the genus Melanoplus. These melt at 5-10oC,
above the range of body temperatures typically experienced in nature and their presence reduces the Tm of the total surface lipids by ~10oC
(Heinrich, 1993), so these compounds will tend to remain in a solid (Figure 7). Cuticular lipids from M. sanguinipes melt at 35-50oC
state under physiological conditions. (Gibbs et al., 1991; Gibbs and Mousseau, 1994; Rourke and Gibbs,
Dragonflies, honeybees, and a few other species contain 1999; Rourke, 2000), within the ecologically relevant temperature
significant quantities of monounsaturated cuticular wax esters (Jacob range for this species (Chappell, 1983; Rourke, 2000). Thus,
and Hanssen, 1979; Blomquist et al., 1980), having a total of 36-50 deposition of wax esters of M. sanguinipes may increase rates of
carbon atoms. Saturated wax esters of this size melt at 60-80oC evaporative water loss. These compounds do not have any known
(Figure 3), so we can calculate that the unsaturated waxes will melt function in communication or predator deterrence, so their presence
above 30oC (Figure 5). We note, however, that most of the waxes on is problematic. It may be that some lipid melting is desirable, for
these species are saturated, so the overall Tm of the wax esters will example to aid in dispersal of lipids over the surface of the cuticle.
probably be at least 45oC. Thus, deposition of wax esters on these Alternatively, the additional thickness of the lipid layer provided by
insects will tend to raise Tm above environmental temperatures and wax esters may offset the effects of lower Tm.
Arthropod cuticles contain numerous other lipids in addition
to wax esters, particularly hydrocarbons. These may interact with
wax esters to affect the properties of the overall lipid mixture
(Riederer and Schneider, 1990; Gibbs, 1995; Dodd and Afzal-Rafii,
2000). Our data suggest that interactions between wax esters and
hydrocarbons reduce lipid Tm by no more than 5oC (Figure 7). Thus,
the effects of cuticular wax esters on insect water balance are clear.
Even in species having unsaturated waxes, these compounds will
reduce cuticular permeability by maintaining lipid Tm values above
environmental temperatures. The novel wax esters of melanopline
grasshoppers are an exception to this rule.
Acknowledgements
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