Chemistry and Physics of Lipids 195 (2016) 47–57

Organization of lipids in avian stratum corneum: Changes withtemperature and hydration

Alex M. Champagnea,*, Heather C. Allenb,c, Robin C. Bautista-Jimenezd,Joseph B. Williamsd

aDepartment of Biology, University of Southern Indiana, Science Center 1255 8600 University Blvd., Evansville, IN 47712, USAbDepartment of Chemistry and Biochemistry, The Ohio State University, 1102 Newman and Wolfrom Laboratory 100 W 18th Ave., Columbus, OH 43210, USAcDepartment of Pathology, The Ohio State University, 129 Hamilton Hall 1645 Neil Ave., Columbus, OH 43210, USAdDepartment of Evolution, Ecology, and Organismal Biology, The Ohio State University, Aronoff Laboratory 318 W 12th Ave., Columbus, OH 43210, USA

A R T I C L E I N F O

Article history:Received 25 August 2015Received in revised form 11 November 2015Accepted 8 December 2015Available online 17 December 2015

Keywords:BirdsInfrared spectroscopyThin layer chromatographyWater lossSkinStratum corneum

A B S T R A C T

In response to increases in ambient temperature (Ta), many animals increase total evaporative water loss(TEWL) through their skin and respiratory passages to maintain a constant body temperature, a responsethat compromises water balance. In birds, cutaneous water loss (CWL) accounts for approximately 65% ofTEWL at thermoneutral temperatures. Although the proportion of TEWL accounted for by CWL decreasesto only 25% at high Ta, the magnitude of CWL still increases, suggesting changes in the barrier function ofthe skin. The stratum corneum (SC) is composed of flat, dead cells called corneocytes embedded in amatrix of lipids, many of which arrange in layers called lamellae. The classes of lipids that comprise theselamellae, and their attendant physical properties, determine the rate of CWL. We measured CWL at 25, 30,35, and 40 �C in House Sparrows (Passer domesticus) caught in the winter and summer, and in sparrowsacclimated to warm and cold lab environments. We then used Fourier transform infrared spectroscopy tomeasure lipid–lipid and lipid–water interactions in the SC under different conditions of temperature andhydration, and correlated these results with lipid classes in the SC. As CWL increased at highertemperatures, the amount of gauche defects in lipid alkyl chains increased, indicating that lipid disorderis partially responsible for higher CWL at high temperatures. However, variation in CWL between groupscould not be explained by the amount of gauche defects, and this remaining variation may be attributedto greater amounts of cerebrosides in birds with low CWL, as the sugar moieties of cerebrosides lieoutside lipid lamellae and form strong hydrogen bonds with water molecules.

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1. Introduction

Increases in ambient temperature (Ta) present physiologicalchallenges for organisms to maintain heat and water balance, andthese environmental demands may become more severe withclimate change (Williams et al., 2012; IPCC, 2013). To maintain aconstant body temperature, endotherms increase evaporativewater loss from their respiratory passages and their skin inresponse to increases in Ta (Marder and Benasher, 1983; Marder,1983; Tieleman and Williams, 2002), but in doing so, imposeconstraints on their water budget. In birds, the major route ofwater loss is evaporative water loss, the sum of cutaneous waterloss (CWL) and respiratory water loss (RWL) (Williams andTieleman, 2005). In many small bird species, evaporative water

* Corresponding author.E-mail address: achampagne@usi.edu (A.M. Champagne).

http://dx.doi.org/10.1016/j.chemphyslip.2015.12.0010009-3084/ã 2015 Elsevier Ireland Ltd. All rights reserved.

loss accounts for five times the water lost through urine and feces(Bartholomew, 1972; Dawson, 1982; Williams and Tieleman,2000), and at thermoneutral temperatures, CWL accounts forapproximately 65–75% of total evaporative water loss (Tielemanand Williams, 2002; Ro and Williams, 2010). As Ta increases, mostbirds increase RWL such that at Ta near the upper lethal limit, CWLaccounts for only about 25% of total evaporative water loss (Marderand Benasher, 1983; Marder, 1983; Tieleman and Williams, 2002).However, this increase in RWL is also accompanied by an increasein CWL, and this increase in CWL occurs at a higher Ta in birdsadapted to hot, dry desert environments than in birds adapted tomesic environments (Tieleman and Williams, 2002). These datasuggest that the increase in CWL observed as Ta increases mayreflect changes in water barrier properties of the skin, and thatbirds are able to modify the skin’s response to Ta through naturalselection or acclimation to their environment.

In birds, the stratum corneum (SC) spans the outermost10–20 mm of the integument, and serves as the primary barrier

48 A.M. Champagne et al. / Chemistry and Physics of Lipids 195 (2016) 47–57

to CWL (Elias, 2004). The SC is composed of flat, dead cells calledcorneocytes embedded within a matrix of lipids (Bouwstra, 1997),most of which occupy the intercellular space in layers calledlamellae (Wertz and Downing, 1982). These lamellar lipids of theSC are thought to serve as the primary barrier to CWL (Simonettiet al., 1995; Meuwissen et al., 1998; Clement et al., 2012), and arecomposed primarily of cholesterol esters, fatty acid methyl esters,triacylglycerol, free fatty acids, cholesterol, ceramides, andcerebrosides (Menon et al., 1986; Haugen et al., 2003; Muñoz-Garcia et al., 2006; Ro and Williams, 2010). At normal skintemperatures, lipids are thought to form a dynamic mosaic ofdifferent phases, depending on lipid make-up, to form a mixture ofthe highly ordered orthorhombic phase, a more disordered gel orhexagonal phase, and the most disordered liquid crystalline phase,with the majority of lipids packing in the orthorhombic phase(Forslind et al., 1997; Bouwstra and Ponec, 2006; Damien andBoncheva, 2010). Lipids in the orthorhombic phase present thegreatest barrier to water loss (Damien and Boncheva, 2010), as lipidmolecules are packed close together, increasing hydrogen bondingbetween head groups and Van der Waals interactions betweenalkyl chains. Lipids in the gel or liquid crystalline phase are spacedfurther apart, and as a result, the normal trans configuration of thelipid alkyl chains is disrupted, causing gauche defects in the chains,creating “holes” in the lamellae through which water moleculescan pass (Potts and Francoeur, 1990). Furthermore, as SCtemperature increases, lamellar lipids undergo phase changes toreduce the proportion of lipids in the orthorhombic phase,therefore increasing the permeability of the SC to water (Goldenet al., 1986, 1987; Gay et al., 1994; Krill et al., 1992).

Although temperature is the main environmental driver of lipidpacking, water molecules can also cause lipid disorder in the SC. Watermolecules are thought to disrupt lipid packing by inserting betweenhydrophilic lipidheadgroupsoratthebeginningof lipidalkylchainstoincrease spacing between molecules (Golden et al.,1986; Alonso et al.,1996; Milhaud, 2004; Pandey and Roy, 2011), thus creating gauchedefects in the adjacent alkyl chains (Potts and Francoeur, 1990). Inmammalian SC, the number of gauche defects increases as the SC ishydrated above 40% w/w, indicating fewer lipids in the orthorhombicphase, and this added water also lowers the phase transitiontemperature in SC lipids (Golden et al., 1986; Gay et al., 1994).

The composition of lipid classes also influences the predominantphase state of lipids in the SC. Furthermore, lipid compositionmoderates changes in these phases that accompany changes intemperature and hydration in the SC. In general, lipids with morepolar head groups and longer saturated alkyl chains uniform inlength with neighboring lipids can pack more closely together andare less prone to gauche defects than lipids with nonpolar headgroups and shorter unsaturated chains of variable length (Casal andMcElhaney, 1990; Adams and Allen, 2013). Desert larks, which havemore polarlipidsand longeralkyl chainsthanmesic larks, have lowerrates of CWL than mesic larks as ambient temperature increases,suggesting greater thermal resistance to gauche defects andconcurrent changes in lipid packing (Tieleman and Williams, 2002).

Lipid composition of the SC also affects the degree to whichlipid head groups form hydrogen bonds with water, and thearrangement of these head groups may predict the effects ofhydration on lipid packing. In birds, the lipids thought to interactmost strongly with water in the SC are the cerebrosides whichcontain a glucose moiety attached to the head group that mayinteract with four to nine water molecules (Bach et al., 1982).Interactions between cerebrosides and water molecules arethought to slow the passage of water through avian SC and thusreduce CWL (Muñoz-Garcia et al., 2008b; Champagne et al., 2012,2015). Despite the prominent role that cerebrosides may play inreducing CWL in birds, cerebrosides are thought to be absent inmammal skin except in individuals with a pathological condition

called Gaucher disease, a disease characterized by dry, scaly skinand rates of CWL approximately 40 times higher than observed inhealthy individuals. In patients with Gaucher disease, CWL isthought to be higher because the bulky glucose moieties ofcerebrosides and their attendant water molecules disrupt packingof the lamellar lipids (Holleran et al., 1994). Because evidencesuggests that cerebrosides reduce, rather than increase CWL inavian SC, a fundamental difference must exist in the arrangementof cerebrosides within lamellae in the SC of birds and mammalswith Gaucher disease, or our understanding of the causes of suchhigh rates of water loss in these patients is flawed.

Two models currently attempt to explain the position ofcerebrosides within lamellar lipids in birds. In the first model,cerebrosides, free fatty acids, cholesterol, and short chain ceramidescoexist in a gel phase in the center of two outer layers of ceramides inthe orthorhombic phase to form a trilayer of lipids (Muñoz-Garciaet al., 2008b). This model is a modification of the sandwich modelproposed for mammalian SC (Bouwstra et al., 2000), with cerebro-sides functionally replacing cholesterol in the middle of the lipidtrilayers. In the second model, cerebrosides are part of lipid bilayers,with different lipid phases existing across the plane of each bilayerdepending on lipid domain formation (Champagne et al., 2012,2015).The key differencebetweenthesetwomodels is the locationofglucose moieties on cerebrosides. In the modified sandwich model,glucose moieties interdigitate with alkyl chains of adjacent lipids.Therefore, as the glucose binds with water molecules, these watermolecules will interact with alkyl chains and potentially lead togreater numbers of gauche defects, thus disrupting lipid packing(Golden et al., 1986). In the bilayer model, glucose moieties arelocatedinthehydrophilic spacebetweenlamellae,andastheglucosebinds with water, lipid alkyl chains should be unaffected.

In this study, we measured temperature-dependent CWL andlipid composition of the SC in House Sparrows (Passer domesticus)caught in the winter or summer in Ohio, or acclimated to low or highTa in the lab. Using Fourier transform infrared spectroscopy (FTIR),we then related CWL and lipid composition to the prevalence ofgauche defects and the strength of hydrogen bonds as SCtemperature and hydration increased. Our study provides compel-ling evidence that an increase in gauche defects, and thus atransitionwithin a subset of lipids from more to less ordered phasesis partially responsible for elevated CWL at high temperatures inbirds. An additional factor governing the response of CWL totemperatureappears tobe the polarityof lipid head groups and theirability to bind with water molecules. Our data also provide supportfor the bilayer model of cerebroside arrangement in the SC of birds,an arrangement that may differ from that seen in mammals. Takentogether our findings provide a potential comparative tool forpractitioners studying Gaucher disease and also offer insights intothe ability of birds to physiologically adapt to a changing climate.

2. Material and methods

2.1. Capture and temperature acclimation of birds

We used mist nets to capture adult house sparrows in Columbus,OH, USA (4000000N, 8301000W) during August 2010 and January 2011,and measured CWL in all the birds caught in January within 4 days ofcapture (winter birds; n = 12). The August birds were divided intothree groups, one group that we measured within 4 days of capture(summer birds; n = 10), and two groups that we acclimated for21 days to either high (warm acclimated; n = 13) or low (coldacclimated; n = 9) temperature. All sparrows were fed on a diet ofsunflower seed, millet, and boiled eggs during captivity.

We placed the high and low temperature acclimation groups inwire cages in environmental chambers (Percival [Boone, IA], modelsE-30B and I-30BLL), housing five or six sparrows per cage. All

A.M. Champagne et al. / Chemistry and Physics of Lipids 195 (2016) 47–57 49

sparrows were exposed to a photoperiod of 14L:10D to approximatephotoperiodic conditions at the time of their capture. We exposedthe warm acclimated group to a constant Ta of 37 �C, and the coldacclimated group to 8 �C. To minimize changes in absolute humidityassociated with these temperature differences, we used a vacuumpump to pull atmospheric air through two columns of Drieritebefore it entered the chambers housing the warm acclimated group,and placed pans filled with water beneath the cold acclimated birds’cages. We measured humidity and temperature continuously withHOBO (Bourne, MA) ProSeries data loggers, and found that warmand cold acclimated groups experienced an absolute humiditybetween8 and 12 g/m3. Forcomparison betweencontrol groups andacclimation groups, weused Weather Underground(www.wunder-ground.com) to find the average temperature and humidity in the21 days prior to the capture of each control group. Summer birdswere exposed to an average high temperature of 27 �C and anaverage low of 16 �C. Absolute humidity ranged from 5.86 to 19.91 g/m3. Winter birds experienced an average high temperature of �3 �Cand an average low of �13 �C, with humidity ranging between0.99 and 6.36 g/m3. Experiments were approved by IACUC at TheOhio State University (2009A0074-R1).

2.2. Cutaneous and respiratory water loss

We measured CWL for all birds using standard flow-throughrespirometry (Tieleman and Williams, 2002; Ro and Williams, 2010).Our systemseparated CWL from RWL by placing a mask over the bird’sbill to capture respiratory water, and also used a layer of mineral oil totrap feces, thus excluding them as a source of water in measurements.Before each measurement, we fasted birds for 2–3 h to ensurepostabsorptive conditions, then placed the bird in a stainless steelmetabolic chamber replete with a water jacket (29.5 cm � 21.5 cm� 28 cm) and a plexiglass lid sealed by a rubber gasket and screws. Acirculatingwaterbath(Neslab RTE7)allowedustocontrol theTa of thechamber, which we measured with a thermocouple and a BailyBatt-12 thermocouple reader. We measured CWL and RWL at 25, 30,35 and 40 �C. To minimize the continuous amount of time each birdspent in the chamber, we divided measurements for each individualinto two sessions, one in which we took measurements at 25 and35 �C, and the other inwhich we took measurements at 30 and 40 �C.Birds were allowed to recover for approximately 20 h betweensessions. For each session, we started at the lower temperature,which is within the birds’ thermoneutral zone (Hudson and Kimzey,1966) and allowed CWL and RWL readings to stabilize. We thenaveraged CWL and RWL over 10 min, increased Ta, and recorded asecond 10 min average before ending the session.

To measure CWL as a function of Ta solely through the lipids ofthe SC without confounding factors such as differences betweenskin temperature and Ta, cutaneous perfusion of blood vessels, orcloacal evaporation (Ophir et al., 2002; Hoffman et al., 2007), wealso measured CWL of dead birds in the same system (Ro andWilliams, 2010). After measuring CWL in the live bird, we sacrificedit by cervical dislocation, re-attached the mask system to eliminateevaporative water loss from the mouth and respiratory passages,sealed the cloaca with wax to eliminate cloacal evaporation, andrepeated CWL measurements at 30, 35, and 40 �C in order. Wewaited one hour after the chamber was sealed to ensure that theambient air had washed out of the system before takingmeasurements (Ro, 2009), and then averaged CWL over a fiveminute period at each temperature.

To correct for any differences in body mass between individuals,we used Meeh’s equation (Meeh, 1879) with Rubner’s constant of10 (Rubner, 1883) to calculate CWL as a function of surface area foreach individual, and we also divided RWL by body mass to calculatemass specific RWL (Ro and Williams, 2010; Tieleman and Williams,2002).

2.3. Isolation of SC

To isolate the SC and characterize the lipids within, we sacrificedthe bird, plucked feathers, peeled the skin away from the body, andpinned the skin on a Teflon sheet covered with filter paper. Wesaturated the filter paper with a 0.5% trypsin solution in phosphatebuffered saline (PBS), and incubated the skin at 4 �C for 24–48 h toseparate the epidermis from the dermis. After this period, theepidermis was immersed in a fresh 0.5% trypsin solution in PBS andincubated for 3 h at 37 �C to isolate the SC from the rest of theepidermis. The trypsin solution was rinsed with distilled water, andthe SC was placed in a 10 mL vial and freeze-dried overnight toextract water. From each SC, we isolated a random sampleapproximately 5 mm in diameter for infrared spectroscopy. To testfor differences in the spectral properties of SC from different bodyregions, we also isolated samples of SC from the breast and wing,respectively, of winter control birds, as birds acclimated to a winterenvironment have shown differences in skin temperature of over14 �C between body regions (Hill et al.,1980). The remainder of eachsample was used for lipid composition analysis.

2.4. Transmission FTIR spectroscopy

Fourier Transform infrared spectroscopy (FTIR) passes infraredlight (4000–400 cm�1) through a sample to study the characteris-tic vibrations and associated vibrational structure of molecules.Chemical bonds within molecules vibrate at unique frequencies,and absorb infrared light at that same frequency. These character-istic absorptions are detected by FTIR spectroscopy as transmit-tance ratios and converted to absorbance peaks. The intensity ofthese peaks can provide information on the relative abundances ofcertain molecules or bond types, and the frequency at which thesepeaks are recorded provide information on relative bond strength,local environment, and molecular ordering.

To obtain infrared spectra for SC, we placed freeze-driedpieces of SC approximately 5 mm in diameter between twoinfrared transparent CaF2 windows (Pike Technologies) 25 mmin diameter and 4 mm thick. We secured these windows in abrass sample holder connected to a water bath (Neslab RTE 7)to regulate temperature. We monitored sample temperature bydrilling a small hole into one of the CaF2 windows, and securinga 36 gauge thermocouple inside with epoxy. The sample holderwas placed in a PerkinElmer infrared spectrometer (Spectrum100) with a nitrogen purge to eliminate ambient CO2 and watervapor. The infrared beam passed through the CaF2 windows andthe sample and to a DTGS detector. All spectra were taken at aresolution of 0.25 wavenumbers (cm�1) and based on anaverage of 20 scans.

To measure the effect of skin temperature on the molecularinteractions in the SC, we obtained transmission spectra for thesame piece of freeze-dried SC from 25 to 50 �C at 5 �C intervals. Tomeasure the effects of water molecules on molecular interactionsin the SC, we first obtained transmission spectra for freeze-driedpieces of SC at 37 �C, the typical skin temperature for birds atthermoneutral temperatures (Hill et al., 1980). We then exposedthe same samples to 55% relative humidity (RH) by suspendingthem over a saturated solution of Mg(NO3)2 in a sealed container at37 �C for 24 h (Winston and Bates, 1960) and then obtaininedspectra at 37 �C. After freeze-drying the samples a second time, weexposed them to 100% RH by suspending them over nanopurewater at 37 �C for 24 h, and then took spectra at 37 �C. We verifiedall RH values with HOBO (Bourne, MA) ProSeries data loggers. Forboth humidity levels, we also determined water content of SC,relative to freeze-dried SC, gravimetrically using a microbalance(Mettler Toledo).

Fig. 1. Absorbance spectrum from house sparrow stratum corneum between3800 and 2500 wavenumbers (cm�1). OH stretching and CH2 S stretchingabsorbance regions are indicated by arrows. The OH stretching region is shownwith two component peaks separated by peak-fitting software into a peak at�3500 cm�1 (dashed line) and a peak at �3300 (dotted line).

50 A.M. Champagne et al. / Chemistry and Physics of Lipids 195 (2016) 47–57

2.5. Spectral analysis

We used the peak analyzer function of OriginPro 8.6 (OriginLabCorporation, Northampton, MA) software to identify the frequen-cies and areas of absorbance peaks between 4000 and 1800 cm�1.For each spectrum, we subtracted the baseline and specified fivepeaks to be fitted: OH stretching peaks at �3500 and �3300 cm�1,a CH stretching peak at �3080 cm�1, a CH2 asymmetric (AS)stretching peak at �2950 cm�1, and a CH2 symmetric (S) stretchingpeak at �2850 cm�1 (Fig. 1). We fit Gaussian peaks to both OHstretching peaks, and fit Lorentzian peaks to the CH and CH2 peaksto achieve the best fit. All peaks were fit to an R2 value of 0.98 orgreater. For our analysis, we focused on two variables: (1) Thefrequency and relative areas of the OH stretching peaks at�3500 and �3300 cm�1. These peaks provide information onthe strength of intermolecular hydrogen bonding, and thereforecan indicate the strength of bonds between lipid head groups aswell as the relative amount of water influenced by elements suchas the sugar moiety of cerebrosides in the SC. The peak at�3500 cm�1 generally indicates hydrogen bond strength typical offree water or water perturbed by the presence of ions and solutes(Liu et al., 2004), whereas the peak at �3300 cm�1 indicates waterin a more strongly hydrogen bonded environment with adjacentwater molecules, sometimes in a tetrahedral bonding structuresimilar to that seen in ice (Du et al., 1993). Thus, a greater area ofthe peak at �3300 cm�1 relative to the peak at �3500 cm�1

indicates stronger hydrogen bonding character between watermolecules or hydroxyl groups (Schneider et al., 1979). As thesepeaks change in size, the relative positions of the peaks maychange, and these peak positions may also provide information onthe strength of hydrogen bonding, as low frequency peaks indicatestronger hydrogen bonding than high frequency peaks (Gallinaet al., 2006). Because we performed repeated measurements onthe same samples of SC, we assumed that the OH stretchingcontribution from lipid head groups and sugar moieties wasconstant across measurements. (2) The frequency of the CH2 Sstretching peak at �2850 cm�1 is an indicator of the relativeamount of gauche defects within lipid alkyl chains. More gauchedefects cause this peak to shift to a higher frequency. We chose to

Concentrati

evaluate this peak rather than the other two CH peaks because itoverlaps less with the absorbance frequencies of the OH stretchingband, thus increasing reliability (Lewis and McElhaney, 2007).

2.6. Lipid extraction and thin layer chromatography

The portion of each bird’s SC not used for infrared spectroscopyunderwent lipid extraction. After we determined the dry mass ofeach SC, we extracted the intercellular lipids by placing the SC insuccessive baths of chloroform:methanol 2:1, 1:1, and 1:2 v/v for 2 heach. Each bath also contained 50 mg/L of the antioxidant butylatedhydroxytoluene (BHT). We combined the extracts and evaporatedthe solvent with a stream of nitrogen in an evaporimeter (N-EVAP,model 11155-O, Organomation Associates, Inc., Berlin, MA, USA), andthen re-constituted the lipids in enough 2:1 chloroform:methanolwith 50 mg/L BHT to fully dissolve the sample (�100–300 mL).

To analyze the amounts of lipid classes in the SC by thin layerchromatography (TLC), we used 20 cm � 20 cm glass plates coatedwith silicic acid 0.25 mm thick (Adsorbosil-Plus 1, Altech, Deer-field, IL, USA). A 2:1 chloroform:methanol solution run to the top ofthe plates removed contaminants prior to sample loading. We thenactivated plates by heating them in an oven for 30 min at 110 �C,and scored the silicic acid to create 13 lanes on each plate. For eachsample, we ran one plate to detect relatively polar lipids, such ascerebrosides, ceramides, and cholesterol, and a separate plate todetect non-polar lipids, such as free fatty acids, triacylglycerol,methyl esters, and cholesterol esters (Supplemental Figs. 1 and 2).On the polar plate, we classified ceramides as ceramide I, the leastpolar, through ceramide III, the most polar, because polarity ofceramides has been shown to be important in CWL (Muñoz-Garciaet al., 2008b). We used a Hamilton syringe with a Teflon coated tipto load a set of five standards comprised of each lipid classdissolved in 2:1 chloroform:methanol at known concentrations oneach plate. Our standards were diluted by 1/2, 1/4, 1/8, and 1/16th,respectively, relative to the most concentrated standard to producea range of lipid concentrations with which we compared thesamples. After loading the standards, we loaded each sample intotwo lanes, and then developed the plates. To separate polar lipids,we developed the plate with chloroform:methanol:water40:10:1 run 10 cm from the bottom, followed by chloroform:methanol:acetic acid 190:9:1 run 15 cm from the bottom, andfinally hexane:ethyl ether:acetic acid 70:30:1 run to the top. Wedeveloped nonpolar plates with hexane:ethyl ether:acetic acid80:20:2 run to the top (Ro and Williams, 2010). After developingthe plates, we allowed them to dry, sprayed the entire plate with asolution of 3% cupric acetate in 8% phosphoric acid, and heatedthem in an oven for 30 min at 180 �C. This procedure chars thelipids to allow visualization. We scanned the plates on a Hewlett-Packard scanner and quantified the optical density of each lipidclass with TN image (Nelson, 2003). We then plotted the opticaldensities of our standards against their known concentrations toconstruct standard curves for each lipid class, to which wecompared the optical densities of our samples. Where our samplesdid not exactly align with our standards, we used known Rf valuesfrom previous studies that used an identical solvent system toassign each sample to the correct lipid class (Ro and Williams,2010). After calculating concentrations of our dissolved sampleswith our standard curve, we calculated the quantity of each lipidclass per gram of SC as:

on of dissolved lipidðmg=mLÞ � Total amount of solutionðmLÞWeight of SCðgÞ

A.M. Champagne et al. / Chemistry and Physics of Lipids 195 (2016) 47–57 51

Supplementary material related to this article found, in the onlineversion, at http://dx.doi.org/10.1016/j.chemphyslip.2015.12.001.

2.7. Statistics

We performed all statistical tests with SPSS 23.0 (IBM, Armonk,NY, USA), with statistical significance set at p � 0.05. All values arepresented as means � SE. To test for differences in CWL, RWL, OHstretching peaks, and the CH2 S stretching peak as a function oftemperature or humidity, treatment group, and the interaction, weused a repeated measures general linear model with Tukey posthoc tests to discern differences between main effects. We reportdifferences between groups and interaction terms only whensignificant. When the interaction term was significant, we used aone-way ANOVA with each temperature or humidity factored bytreatment group, with Tukey post hoc tests to identify specificdifferences between groups at each temperature or humidity.

To test for differences in FTIR spectra between SC from thebreast and wing in winter controls as temperature or hydrationincreased, we used a repeated measures general linear model totest for differences in the ratio of the OH stretching peak areas andthe CH2 S stretching peak position as a function of temperature orhydration, body region, and the interaction. To test for differencesin CWL as a function of lipid disorder and treatment group, we usedstepwise regression to correlate CWL at each temperature with thefrequency of the CH2 S stretching peak at the correspondingtemperature. We used the CWL from dead birds for this analysisbecause their skin temperature matched Ta. To test for differencesin SC lipid amounts and proportions between groups, we used aone-way ANOVA with each lipid class factored by treatment groupwith Tukey post hoc tests to specify differences between groups.We also used stepwise regression to correlate amounts andpercentages of each lipid class with CWL at 25, 30, 35, and 40 �C. Alllipid proportions were logit transformed [ln Y/(1 � Y))] to normal-ize the data prior to analyses.

To explore the interactions between lipid classes and their effectson CWL, we used principal component analysis (PCA) on the amountof each lipid class (Shaw, 2003). This analysis yielded uncorrelatedcomposite variables, the principal components. We used theprogram ‘Factor analysis’ in SPSS without rotation to extractcomponents with eigenvalues greater than one as our selectioncriterion. We then used stepwise linear regression to determineassociations between each principal component and CWL for eachindividual bird at 25, 30, 35, and 40 �C. We also attempted to use PCAon the proportions of each lipid class, but we did not find enoughcorrelation between variables to justify the use of PCA (Kaiser–Meyer–Olkin measure of sampling adequacy = 0.50).

Fig. 2. Cutaneous water loss in live (A) and dead (B) birds as a function of temperature focold acclimated groups combined (filled triangles, solid line). Standard error bars are b

3. Results

3.1. Cutaneous and respiratory water loss

CWL changed significantly as a function of temperature(F(1.04,51.84) = 55.33, p < 0.001), group (F(3,37) = 14.82, p < 0.001),and the interaction between temperature and group(F(4.20,51.84) = 2.58, p = 0.05). Warm and cold acclimated groups didnot differ at any temperature, and therefore were treated as a singlegroup (p > 0.78). Summer birds had higher rates of CWL than allother groups at all temperatures (p < 0.03). Winter birds had lowerCWL than summer birds at all temperatures (p < 0.001), and lowerCWL than the acclimated groups at 35 and 40 �C (p < 0.02; Fig. 2A) .

Mass-specific RWL changed as a function of temperature for allgroups (F(1.47,54.56) = 79.14, p < 0.001). Mass-specific RWL remainedthe same between 25 and 30 �C (p > 0.99), but increased from56.65 � 3.59 to 192.10 � 21.99 mgH2O g�1 between 30 and 35 �C(p < 0.001), and increased further to 479.53 � 42.41 mgH2O g�1

between 35 and 40 �C (p < 0.001). In parallel with these changes inCWL and RWL, the percentage of CWL relative to total evaporativewater loss changed as a function of temperature (F = 105.25,p < 0.001). CWL comprised 61.4 �1.8% of total evaporative waterloss at 25 �C, but made up only 27.8 � 1.5% of total evaporativewater loss at 40 �C.

3.2. Cutaneous water loss in dead birds

As temperature increased, CWL in dead birds increased(F(1.35,37.67) = 116.33, p < 0.001). Dead birds also exhibited signifi-cant differences between groups (F(3,28) = 16.27, p < 0.001) and inthe interaction between temperature and group (F(4.04,37.67) = 6.09,p = 0.001). CWL in dead birds did not differ between warm andcold-acclimated birds (p > 0.98), we again considered them to be asingle group. Summer birds had higher rates of CWL than all othergroups at 30, 35, and 40 �C (p < 0.003). Additionally, wintersparrows had lower CWL than all other groups at 30, 35, and 40 �C(Fig. 2B; p < 0.006). CWL was higher in live birds than dead birds atall temperatures (p < 0.001). Furthermore, the rate of increase inCWL between 30 and 35 �C and between 35 and 40 �C wassignificantly higher in live birds than in dead birds (p = 0.001 andp = 0.02, respectively).

3.3. FTIR differences between SC from different body regions

We found no differences in the frequency of the CH2 S stretchingpeak between SC taken from the wing and SC taken from the breastin winter birds as a function of temperature (F(1,20)< 0.001,p > 0.99) or hydration (F(1,18)< 0.001, p > 0.99). This pattern

r summer (filled circle, dashed line), winter (open circle, dotted line), and warm andased on the mean for each group at each temperature.

52 A.M. Champagne et al. / Chemistry and Physics of Lipids 195 (2016) 47–57

continued in the ratio of the �3300 to the �3500 cm�1 OHstretching peaks, as body region again did not differ as a function oftemperature or hydration (F(1,20) = 0.89, p = 0.36 and F(1,18) = 0.05,p = 0.83, respectively). For subsequent analysis of spectra, we chosespectra from the wing or breast in each individual based on whichspectrum had the greatest amount of signal relative to noise.

3.4. FTIR responses to changes in temperature

As skin temperature increased, the frequency of the CH2 Sstretching peak increased (F(2.92,104.95) = 77.17, p < 0.001). Thechange in frequency of this peak did not differ between groups(F(3,36) = 0.83, p > 0.49), but the interaction between group andtemperature indicated that different groups exhibited changes infrequency at different rates (F(8.75,104.95) = 2.05, p = 0.04). However,we could not detect differences between groups at any singletemperature (F(3,37)< 2.65, p > 0.06), and thus considered changesin the CH2 S stretching peak with temperature to be the same for allgroups (Fig. 3a). The average frequency of the CH2 S stretching peakincreased from 2850.02 � 0.03 cm�1 at 25 �C to 2850.56 � 0.05cm�1 at 50 �C.

We identified two OH stretching peaks, the first of whichabsorbed infrared light at 3294 �1 cm�1 (�3300 cm�1) at 25 �C,and increased in frequency as temperature increased to afrequency of 3299 � 1 cm�1 at 50 �C (F(2.95,106.36) = 41.44, p < 0.001).The area of this peak did not change as a function of temperature(Fig. 4a; F(1.90,68.35) = 2.66, p = 0.08)

The second OH stretching peak absorbed infrared light at3474 �15 cm�1 (�3500 cm�1) and did not change in frequency as afunction of temperature (F(1.00,36.04) = 1.58, p = 0.22). The area of thispeak decreased as a function of temperature for all groups (Fig. 4b;F(1.81,65.09) = 43.95, p < 0.001), indicating a loss of weakly hydrogenbound water as temperature increased, or a decrease in thetransition moment strength of hydrogen bonds.

3.5. Gravimetric uptake of water in SC samples

When we exposed freeze-dried SC to 55 and 100% RH, thepercent water by weight (w/w) of the SC increased as a function ofhumidity (F(1.13,33.74) = 104.61, p < 0.001), treatment group(F(3,30) = 3.49, p = 0.03), and the interaction (F(3.37,33.74) = 2.83,p = 0.05). This difference was driven by greater water uptake afterexposure to 100% RH in the cold acclimated group compared withthe winter control group (p = 0.04), but no other differences weredetected between humidity or treatment groups. Overall, assum-ing that freeze-dried samples contained 0% w/w, SC samplesincreased water content to 3.79 � 0.95% w/w and 35.39 � 3.12% w/w after being exposed to 55% and 100% RH, respectively.

Fig. 3. Changes in the frequency of the CH2 S stretching peak in response to changes in (Abased on the mean for each group at each temperature or hydration level. Standard er

3.6. FTIR response to changes in hydration

The frequency of the CH2 S stretching peak changed as afunction of hydration in all groups (F(2,76) = 4.94, p = 0.01). Thefrequency of the peak was 0.14 � 0.04 cm�1 higher at 55% relativeto freeze-dried SC (p = 0.004), but the frequency did not differbetween freeze-dried SC and that exposed to 100% RH at 37 �C(p = 0.60) or between 55% and 100% RH (p = 0.26; Fig. 3b).

The frequency of the OH stretching peak at �3300 cm�1

increased with hydration for all groups (F(2,76) = 23.91, p < 0.001),ranging from 3296 � 1 cm�1 in freeze-dried skin to 3306 � 2 cm�1

after being exposed to 100% RH (p < 0.001). Peak area also variedwith hydration (F(1.48,56.32) = 10.71, p < 0.001), increasing in areaafter being exposed to 100% RH (Fig. 4c; p = 0.01), indicating thatsome of the added water exhibited strong hydrogen bonding. The�3500 cm�1 peak varied in frequency as a function of humidity inall groups, ranging from 3505 � 2 cm�1 in freeze-dried SC to3548 � 4 cm�1 after being exposed to 100% RH (F(1.63,62.11) = 47.27,p < 0.001). The area of this peak also varied with humidity in allgroups (F(1.39,52.80) = 22.13, p < 0.001). Area of this peak increasedwhen freeze-dried SC was exposed to 55% RH (p = 0.006), and againwhen exposed to 100% RH (Fig. 4d; p = 0.001), indicating that someadded water exhibited weaker hydrogen bonding. As a result of thechanges in area of the �3300 and �3500 cm�1 peaks, the ratio ofthe �3300 to the �3500 cm�1 peak varied with humidity(F(2,76) = 15.46, p < 0.001). The ratio decreased as freeze-dried SCwas exposed to 55% RH (p = 0.003), indicating that added waterdecreased the overall hydrogen bonding strength in the SC initially,but the ratio did not change again after being exposed to 100% RH(Fig. 5; p = 0.21).

3.7. Correlations between the CH2 S stretching peak and CWL in deadbirds

As the frequency of the CH2 S stretching peak increased withincreasing temperature in the SC, CWL in dead birds atcorresponding temperatures also increased (p = 0.005, R2 = 0.40).Winter birds had lower rates of CWL at any given frequency(p < 0.001), and summer birds increased water loss at a faster raterelative to the frequency of the CH2 S stretching peak comparedwith other groups (p < 0.001).

3.8. Lipid composition and CWL

We identified cholesterol esters, fatty acid methyl esters,triacylglycerol, free fatty acids, cholesterol, three classes ofceramides, and cerebrosides in the SC of all groups. We found asignificant difference between groups in the total quantity of lipids

) temperature and (B) hydration expressed as RH exposure. Standard error bars areror bars are based on the mean at each temperature or hydration level.

Fig. 4. Changes in the area of the (A) �3300 cm�1 OH stretching peak and (B) �3500 cm�1 OH stretching peak in response to changes in temperature, and changes in the areaof the same two peaks, C and D, respectively, in response to changes in hydration expressed as humidity exposure. Standard error bars are based on the mean at eachtemperature or hydration level.

A.M. Champagne et al. / Chemistry and Physics of Lipids 195 (2016) 47–57 53

(F(3,38) = 4.27, p = 0.01), as summer birds had more total lipids thanwarm-acclimated (p = 0.01) and winter birds (p = 0.04). Whencomparing amounts of lipid classes in the SC, triacylglycerol(F(2,38) = 11.59, p < 0.001), cholesterol (F(2,38) = 4.58, p = 0.02), cer-amide III (F(2,38) = 6.40, p = 0.004), and cerebrosides (F(2,38) = 6.06,p = 0.005) all differed significantly between groups. Because we didnot find any significant differences between warm and cold-acclimated groups for any lipid class (p > 0.34), we considered thema single group for analysis. Summer sparrows had moretriacylglycerol than all other groups (p < 0.001), more cholesterolthan winter birds (p = 0.01), and more ceramide III than all groups(p < 0.04). Winter sparrows had more cerebrosides than theacclimated groups (p < 0.005; Fig. 6).

Because total lipid quantity differed among groups, we alsocompared proportions of lipid classes between groups. Triacyl-glycerol (F(2,38) = 4.75, p = 0.02) and cerebrosides (F(2,38) = 13.36,p < 0.001) differed significantly among groups. Summer controlshad a greater proportion of triacylglycerol than winter controls

Fig. 5. Change in the ratio of the low energy OH stretching peak (3300) to the highenergy OH stretching peak (3500) in response to changes in hydration expressed ashumidity exposure. Standard error bars are based on the mean at each hydrationlevel.

(p = 0.02), and winter controls had a higher proportion ofcerebrosides than all other groups (p < 0.001; Fig. 7).

At thermoneutral temperatures of 25 and 30 �C, the amount(p < 0.001, R2 > 0.33) and proportion (p < 0.04, R2 > 0.12) of triacyl-glycerol correlated positively with CWL, and were the only lipidclass to correlate with CWL. At 35 �C, the amount of triacylglycerolremained positively correlated with CWL (p = 0.003), and theamount of ceramide III (p = 0.01) also positively correlated withCWL. The amount (p = 0.03, R2 = 0.47) and proportion (p = 0.02) ofcholesterol ester and the proportion of cerebrosides (p = 0.02,R2 = 0.25) negatively correlated with CWL at this temperature. At40 �C, the amount of ceramides I and II were positively correlatedwith CWL (p = 0.002 and p = 0.006, respectively, R2 = 0.34).

In our analyses using PCA of the amount of each lipid class, twoaxes accounted for 60.29% of the variance. A plot of scores for each

Lipid ClassTriacylglycerol

Cholesterol

Ceramide III

Cerebroside

Amou

nt o

f Lip

id (m

g/g

SC

)

0

20

40

60Summ er Winter Acclimated

cd cd

e

ff

g

h

gh

a

bb

Fig. 6. Differences in amounts of each lipid class for which significant differenceswere observed between groups. Data for warm and cold-acclimated birds arecombined. For each class of lipids, significant differences are indicated by letters.Standard error bars are shown.

Lipid ClassTriacylglycerol Cerebroside

Prop

ortio

n of

Tot

al L

ipid

s

0.00

0.05

0.10

0.15

0.20

0.25

0.30Summ er Winter Accli mated

a

b

ab

c

dd

Fig. 7. Differences in proportions of triacylglycerol and cerebrosides betweengroups. Data for warm and cold-acclimated birds are combined. For each lipid class,significant differences are indicated by letters. Standard error bars are shown.

54 A.M. Champagne et al. / Chemistry and Physics of Lipids 195 (2016) 47–57

treatment group along these axes showed that principal compo-nent 2 (PC2) separated summer birds from winter birds, but did notseparate acclimation groups, and principal component 1 (PC1) didnot separate any groups (Fig. 8b). A plot of the eigenvector loadingsshowed a separation of lipid classes into two distinct groups.Although every lipid class loaded positively on PC1, cholesterolester, triacylglycerol, free fatty acids, cholesterol, and ceramide Iloaded positively on PC2, whereas fatty acid methyl esters,ceramides II and III, and cerebrosides all loaded negatively(Fig. 8a). Thus, with the exception of fatty acid methyl esters, PC

Fig. 8. (A) Eigenvector loadings of both principal components for cholesterol ester(Filled square), fatty acid methyl ester (filled diamond), triacylglycerol (filled right-side-up triangle), free fatty acids (open diamond), cholesterol (open square),ceramide I (filled circle), ceramide II (open circle), ceramide III (filled upside-downtriangle), and cerebroside (open right-side-up triangle). (B) Principal componentscores for summer controls (filled triangles), winter controls (open triangles),warm-acclimated (filled diamonds), and cold-acclimated birds (open circles).

2 separates the most polar lipids from less polar lipids. Combiningthe treatment group plot with the lipid plot suggests that winterbirds have more polar lipids in their SC, whereas summer birdshave more nonpolar lipids, and acclimated groups show no clearpattern. Because the interaction of these lipid classes may affectCWL at different temperatures, we performed stepwise regressionfor both principal components against CWL at each temperature.We found that PC2 was positively correlated with CWL at 25(R2 = 0.20, p = 0.005) and 30 �C (R2 = 0.17, p = 0.01), and at 35 �C,PC1 and PC2 both positively correlated with CWL (R2 = 0.17,p = 0.04 for both). At 40 �C, neither principal component correlatedwith CWL (p = 0.09).

4. Discussion

We have shown that water loss through the SC in birdsincreased as Ta increased, and the rate of CWL and its relationshipwith temperature depended on the environment to which birdswere acclimated. The increase in CWL as Ta increased wasaccompanied by an increase in the number of gauche defects inthe SC, suggesting that lipid packing phases at least partiallydetermine the rate of CWL. Additionally, lipid classes present in theSC varied as a function of treatment group, and the strength ofhydrogen bonding between lipid head groups and water moleculesvaried as a function of hydration, suggesting that these variablesalso play a role in regulating CWL. Our results also suggest afundamental difference in the arrangement of lipids, especiallycerebrosides, in the avian SC when compared with the mammalianSC.

CWL and RWL remained constant in house sparrows between25 and 30 �C, but both increased at 35 �C and again at 40 �C, andRWL became the primary avenue of evaporative water loss at highTa, a pattern consistent with results found in larks from mesicenvironments (Tieleman and Williams, 2002). Summer birds hadhigher rates of CWL than other groups at all temperatures, whichmay reflect a mechanism by which birds use CWL for thermoreg-ulation in environments that are relatively warm, but not waterlimited (Muñoz-Garcia and Williams, 2007). In contrast, winterbirds had lower rates of CWL than summer birds at all temper-atures and both acclimation groups at high temperatures,suggesting that birds acclimated to cold, dry environmentsattempt to minimize CWL as either a water or heat conservingmechanism. We found similar differences between groups whenwe measured CWL of dead birds, as summer controls had higherrates of CWL than other groups at all Ta values, and winter controlshad lower rates of CWL than other groups at all Ta. However, in allgroups, CWL was lower in dead birds than in live birds, and livebirds increased CWL at a greater rate as Ta increased. These dataenforce the idea that some of the water barrier properties of the SCmay be under biological control and cannot be attributed solely todiffusion processes (Elias, 2004; Ro and Williams, 2010). Addition-ally, other factors that influence CWL may be controlled in livebirds, such as increased hydrostatic pressure from dermal capillarybeds (Ophir et al., 2002), or even an increase in evaporative waterloss from the cloaca (Hoffman et al., 2007). We found nodifferences in CWL between warm and cold acclimated treatmentgroups, a pattern that persisted for all aspects of the study,including FTIR and lipid composition data. Because the winter andsummer groups exhibited differences in CWL and SC lipidcomposition, and because previous studies have found that birdscan change CWL and SC lipid composition in response to changes inhumidity (Muñoz-Garcia et al., 2008a), this pattern suggests thateither the difference in temperature between treatments was notextreme enough to produce acclimatory changes in the SC of thesebirds, or humidity, rather than temperature, is the environmentalcue that regulates acclimation in the SC of birds.

A.M. Champagne et al. / Chemistry and Physics of Lipids 195 (2016) 47–57 55

In parallel with increases in CWL as Ta increased, the number ofgauche defects present in SC lipids also increased, as the CH2 Sstretching peak increased in frequency from roughly 2850.0 cm�1

to 2850.5 cm�1 between 25 and 50 �C. This frequency rangeindicates the beginning of a transition from the gel phase to theliquid crystalline phase (Casal and Mantsch, 1984; Gay et al., 1994),suggesting that lipids in bird SC may be composed primarily of amixture of these two phases, with very few lipids arranged in theorthorhombic phase. This prevalence of more disordered phasescould help explain why birds have higher rates of CWL thanmammals, which generally have a high proportion of SC lipids inthe orthorhombic phase (Muñoz-Garcia et al., 2008b; Damien andBoncheva, 2010). However, analysis of the CH2 scissoring androcking bands at �1470 cm�1 and 720 cm�1, respectively, of theFTIR spectrum could better discern the exact phase of these lipids(Lewis and McElhaney, 2007). Regardless of the exact phasebehavior, the presence of more gauche defects at higher temper-atures may partially explain the increase in CWL observed in birdsas Ta increases. Indeed, CWL of dead birds, in which skintemperature matched Ta, positively correlated with the frequencyof the CH2 S stretching peak, and thus with the number of gaucheconformers. However, the relative number of gauche conformersexplained a relatively small portion of the variation in CWL, andchanges in the relative number of gauche conformers did not varyamong treatment groups, whereas CWL did. These factors indicatesome degree of uncoupling between lipid phase behavior and CWL,even in dead birds where factors such as perfusion of dermalcapillary beds or cloacal evaporation can be ruled out as factors (Roand Williams, 2010; Ophir et al., 2002; Hoffman et al., 2007).

Because CWL did not correlate tightly with lipid phase behavior,we hypothesized that interactions between lipid head groups andwater molecules may account for differences in CWL betweengroups. Water in the SC forms hydrogen bonds with lipid headgroups, and these hydrogen bonds contribute to the ability of theSC to hold water, thus improving barrier function (Imokawa et al.,1986; Pieper et al., 2003). In studies on pig skin, when the SC is lessthan 6% water by weight (w/w), all water molecules are thought toform strong hydrogen bonds with lipid head groups to form aprimary solvation shell of water. When the SC is between 6 and 12%w/w, additional water molecules form weaker hydrogen bondswith water in the primary solvation shell and with adjacent watermolecules to form a secondary solvation shell. Above 12% w/w,additional water molecules are thought to collect in thecorneocytes in a weak hydrogen bonding structure resemblingbulk water (Pieper et al., 2003). As we hydrated our samples, weobserved an overall decrease in hydrogen bonding strength, asevidenced by increases in the frequency of the OH stretching peaksat �3300 and �3500 cm�1. However, although the ratio of thesetwo peaks decreased slightly after the SC was exposed to 55% RH,and thus hydrated to roughly 4% w/w, it remained unchanged afterthe SC was exposed to 100% RH to hydrate to approximately 35% w/w. These data indicate that even at 35% w/w, at least some addedwater continues to exhibit strong hydrogen bonding, and there isno evidence of bulk water forming in the corneocytes of avian SC.This contrast in water binding properties with mammalian SCcould indicate that more water binds with lipid head groups in theinterlamellar spaces, and less water hydrates the corneocytes,which are smaller in birds than in mammals (Menon et al., 1986).

Because water molecules appear to interact with lipid headgroups in avian SC more than in human SC, the composition of lipidclasses and their associated head groups may affect the rate of CWLin birds, as lipids with more polar head groups are better able tobind with water than more nonpolar lipids (Imokawa et al., 1986).Indeed, treatment groups differed in the amounts and proportionsof lipid classes in the SC, and these differences in lipid classes wereassociated with differences in CWL. The most striking difference

between groups was a greater amount and proportion oftriacylglycerol and a greater amount of cholesterol in the SC ofsummer birds than in winter birds. Winter birds on the other handhad a greater proportion of cerebrosides than summer birds. Theamount and proportion of triacylglycerol positively correlatedwith CWL and the proportion of cerebrosides negatively correlatedwith CWL at 35 �C. Furthermore, PCA indicated that as a whole,winter birds had greater amounts of more polar lipids whereassummer birds had more nonpolar lipids, and these polar lipidswere negatively correlated with CWL. These data suggest amechanism by which polar lipids, especially cerebrosides, formstrong hydrogen bonds with water molecules to increase theoverall viscosity of the water, which may slow the rate ofpermeation through the SC (Comesaña et al., 2003; Clementet al., 2012; Champagne et al., 2012, 2015). In addition to loweringCWL, these interactions between polar lipid head groups and watermay ensure proper hydration in the SC in dry environments in amechanism similar to water-binding free amino acids in mammalSC (Scott and Harding, 1986), and this function may provide analternative explanation to the prevalence of polar lipids in birdscaught in a dry winter environment.

Our observation that water molecules formed strong hydrogenbonds with lipid head groups also allowed us to determine theposition of cerebrosides within lamellar lipids in birds. If theglucose moieties of cerebrosides are located in the center of lipidtrilayers (Muñoz-Garcia et al., 2008b), then we should haveobserved that as the SC hydrated, water molecules would bind withthe glucose moieties and thus disrupt the adjacent interdigitatinglipid alkyl chains to increase gauche defects (Bach et al., 1982;Golden et al., 1986). Although we observed a small increase in thefrequency of the CH2 S stretching peak when freeze-dried SC wasexposed to 55% RH, the degree of this increase was only 0.14 cm�1.Furthermore, we observed no noticeable difference in thefrequency of the CH2 S stretching peak between freeze-dried SCand SC exposed to 100% RH, indicating that water molecules didnot interfere with lipid alkyl chains while binding with lipidheadgroups and glucose moieties of cerebrosides. This lack ofinterference suggests that cerebrosides are part of lipid bilayers,with glucose moieties located in the hydrophilic space betweenlamellae, where they may play a major role in decreasing CWL bybinding with multiple water molecules (Champagne et al., 2012,2015). The possible arrangement of lipids in bilayers in the avian SCdiffers from the sandwich model proposed for mammalian SC(Bouwstra et al., 2000), and may explain why cerebrosides appearto inhibit water loss in birds while enhancing water loss inmammals (Holleran et al., 1994)

5. Conclusions

We found that CWL increases in house sparrows as Ta increasesabove the thermoneutral zone. Birds that were caught in a warmer,more humid summer environment had higher rates of CWL thanbirds caught in a cooler, less humid winter environment and birdsacclimated in the lab to either cold or warm conditions withintermediate humidity. The winter-caught birds also had lowerCWL than all other groups. This pattern remained similar in deadbirds. These differences between groups may be an acclimatoryresponse to different environmental pressures for water conser-vation and thermoregulation. In parallel with increases in CWLwith increasing Ta, the number of gauche defects in the SC lipidsincreased, indicating that a subset of lipids transitioned from a gelto a more disordered liquid crystalline phase, and these phasetransitions may explain higher rates of CWL at high Ta. However,the appearance of gauche defects in response to changes intemperature was the same for all groups, in contrast to thedifferences we observed in CWL between groups, and suggesting

56 A.M. Champagne et al. / Chemistry and Physics of Lipids 195 (2016) 47–57

that lipid phase behavior does not fully explain the variation inCWL. The remaining variation may be explained by interactionsbetween lipid head groups and water molecules, as lipid headgroups form strong hydrogen bonds with water molecules to slowits permeation through the SC, thus lowering CWL. As we addedwater to the SC, the water interacted with polar lipid headgroups,and more polar lipids were more common in the SC of winter-caught birds. Lipid phase behavior and the interactions betweenwater and lipid molecules in the SC of birds could be furtherstudied by additional methods in physical chemistry, includingLangmuir film studies on lipid phase behavior under differentpacking conditions, and accompanying studies using infrared andRaman techniques to study interactions between water, lipid headgroups, and lipid alkyl chains under these same conditions. Thesetests will allow us to better understand the mechanisms by whichlipids serve as a barrier to water loss at different temperatures.Understanding these mechanisms will be critical to predicting theresponse of birds to climate change (Williams et al., 2012) andsuggesting potential avenues of research for practitioners studyingGaucher disease.

Acknowledgements

We thank members of the Allen and Williams labs for theirvaluable feedback throughout this study. We also thank membersof the Ohio State University Department of Chemistry machineshop for designing our temperature controlled sample holder forFTIR. Finally, we thank the managers at OSU Waterman Farm fortheir assistance in obtaining House Sparrows. This research wassupported by grant NSF-CHE 1111762 to H.C.A and grant 2008469of the U.S.–Israel Binational National Science Foundation to J.B.W.The authors declare no competing interests.

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