These COHbvaluesdrasticallyreduceblood-O associated withvaluesof50–80%(Stewart,1975;Weaver, 2009). values reach of breath)CO-drivenhypoxiaaretypicallyseenwhenCOHb 2009). Deleterioussymptoms(e.g.headache,nauseaandshortness as hemoglobinandformingcarboxyhemoglobin(COHb)(Weaver, lowering arterialO *Author ([email protected]) for correspondence Oceanography, SanDiego, California LaJolla, CA92093-0204,USA. University of 1752 Received 1December 2013;Accepted4February2014 2 1 depriving thebodyofoxygen(O Carbon monoxide(CO)isoftenclassifiedasastrictlytoxic gas, Hemoglobin absorptionspectra,Marinemammal,Oxygenstores KEY WORDS:Calculatedaerobicdivelimit,Carbonmonoxide, to hemoglobin,potentiallypreventadequateO concentrations ofcarboxyhemoglobin(COHb),formedbyCObinding organs andtoexertcytoprotectiveeffects. However, high proteins, havebeenshowntomaintainphysiologicalfunctionof generated primarilythroughdegradationofhemefromheme- Low concentrationsofendogenouscarbonmonoxide(CO), Michael S.Tift elephant seal Elevated carboxyhemoglobininamarinemammal,thenorthern RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,1752-1757doi:10.1242/jeb.100677 COHb valuesfromthisstudydecreasetotalbodyO turnover ismuchhigherthaninterrestrialmammals.Themaximum COHb suggeststhatthemagnitudeand/orrateofheme-protein little tonofluctuationinCOHbvalues.Thisconsistentelevation and mean COHbvaluesof7.6±0.2%and7.1±0.3%,respectively(N juveniles andpups(withlowerheme-proteincontents)had The mean(±s.e.m.)valueinadultsealswas8.7±0.3%( maximum COHbvaluewas10.4%oftotalhemoglobinconcentration. positively relatedtothetotalhemoglobinconcentration.The hemoglobin andmyoglobinconcentrations.ThelevelsofCOHbwere elephant seals,aspecieswithlargebloodvolumesandelevated elevated COHbconcentrations.Therefore,wemeasuredin degradation, moreendogenousCOproductionand,consequently, as foundinmarinemammals,arelikelyassociatedwithgreaterheme endogenously producedCO. adaptation associatedwithchronicallyelevatedconcentrationsof cytoprotective effects, mechanismsofactionandevolutionary seal representsanidealmodelforunderstandingthepotential the extremebreath-holdsofelephantseals.We suggesttheelephant protect againstpotentialischemia–reperfusioninjuryassociatedwith However, theconstantpresenceofelevatedCOinbloodmayalso thereby reducingthecalculatedaerobicdivelimitforthisspecies. INTRODUCTION ABSTRACT Department of Biology, of SonomaState University,Department Park, CA94928,USA. Rohnert Center for Marine Biotechnology andBiomedicine, ScrippsInstitutionof N =9, respectively).Serialsamplesoverseveralhoursrevealed ≥ 20% oftotalhemoglobinconcentration,anddeathis 1,2, 2 content. Elevatedheme-proteinconcentrations, *, PaulJ.Ponganis 2 ) bybindingtoheme-proteinssuch 1 2 and DanielE.Crocker delivery totissuesby 2 transport andO 2 stores by7%, N =6), while =9 2 from reducedO concentration, atwhichelevatedCOthenleadstodetrimentaleffects can providebeneficialandtherapeuticeffects uptoaspecific et al.,2010;Prabhakar, 2012).Therefore,lowconcentrationsofCO et al.,1998;KevinandLaffey, 2008;Mustafaetal.,2009;Kajimura inflammation, apoptosisandischemia–reperfusioninjuries(Snyder neurotransmission andinprotectionoftissuescellsagainst endogenously inlowconcentrations,andfunctions mitochondrial respiration.However, COisalsogenerated storage capacity(decreasedarterialO concentrations (25 total bodyO mammalian bloodvolumes (216 1994; Denneryetal.,2001;D.E.C., unpublished). range asthoseseeninhealthy humans(ThorsonandLeBoeuf, [ measured inthebloodofadultandjuvenilenorthernelephant seals human plasma.Similarly, bilirubinandbiliverdinhavebeen plasma wasalsoelevatedapproximatelythreefoldtofourfoldthat of smokers (Pugh,1959).BilirubinconcentrationinWeddell seal were oversixtimesthevaluesseeninbloodofhuman non- 1959). ThisstudyfoundmeanCOlevelsinWeddell sealbloodthat CO inthebloodofWeddell seals( an earlystudybyPugh,therewasunexpectedfindingofelevated investigating elevatedendogenousCOproduction.Forexample, in (Ponganis etal.,2011), potentiallyrepresentanexcellentmodelfor blood volumes,hemoglobincontentandmyoglobinconcentrations endogenous COproduction,marinemammals,whichhaveelevated inflammation andcancer)(MotterliniOtterbein,2010). (i.e. sepsis,organ transplants,heartfailure,hypertension, use ofCO-releasingpharmaceuticalsforfutureclinicalapplications properties thathavestimulatedinvestigationofCOtherapyandthe et al.,1987;Barañano2002;Mustafa2009).Itisthese apoptosis anddownregulationoftheinflammatoryresponse(Stocker properties, attenuationofischemia/reperfusioninjury, inhibitionof multitude ofbeneficialeffects including:vasodilatation,antioxidative products (CO)ofhemedegradationhavebeenshowntoa biliverdin reductase.Theseproducts(biliverdinandbilirubin)by- Tenhunen etal.,1968).Biliverdinisthenreducedtobilirubinvia production offreeiron,COandbiliverdin(Coburnetal.,1963; enzymatically degradedbyheme-oxygenase,resultinginequimolar breakdown ofheme-proteinsreleasesheme,whichisthen proteins (e.g.hemoglobin,myoglobinandcytochrome breakdown ofheme,whichisanessentialcomponentmanyheme- in thebodywhichcanbeviewedastherapeutic. understanding thebiologicaleffects ofspecificCOconcentrations a newfunctionalperspectiveandemphasizetheimportanceof transport O Mirounga angustirostris Northern elephantsealshave someofthehighestknown Considering thatheme-proteinsaretheprimarysourceof Ironically, theprimarysourceofendogenousCOcomesfrom 2 2 2 store (Ponganis, 2011). ThiselevatedbloodO or associatecloselywithaerobicrespiration.The 2 delivery. TheserelativelyrecentfindingsgiveCO
g
dl − 1 ) innature,accountingforover 70% oftheir (Gill 1866)],yetthevaluesarein thesame Leptonychotes weddelli
ml 2 kg content), thuslimiting − 1 ) andhemoglobin 2 ) (Pugh, c storage ) that
The Journal of Experimental Biology a decreaseinbloodO and delayedremovalofCOcouldelevateCOHbcontent,leadingto removal ofCO.Therefore,increasedendogenousCOproduction air-breathing marinediversintroducesapotentiallimitationfor intermittent breathingpatterns(sleepapneaand/ordiving)ofmany animals spendineupnea. These lifehistorybehaviorsdrasticallylimittheamountoftime sleep apnea(BlackwellandLeBoeuf,1993;Robinsonetal.,2012). underwater, andwhenonlandwillspendalarge portionoftimein spends 9–10 damage fromoxidativestress.Additionally, atypicalelephantseal potential forexposureoftissuestoreactiveoxygenspeciesand reperfusion oftissueswithoxygenatedblood,therebyincreasingthe hyperventilation, tachycardiaandvasodilation,whichresultsin The briefperiodsofeupneafollowingbreath-holdsinclude reaching aslow10%(Stockardetal.,2007;Meir2009). and routinehypoxemiawitharterialhemoglobinsaturations peripheral ischemia(Andrewsetal.,1997;Ponganis2006)] these sealsareaccompaniedbythediveresponse[bradycardiaand eupneic periods(BlackwellandLeBoeuf,1993).Breath-holdsof sleep apneaeventsonland,whicharealsousuallyfollowedbybrief for theirextendedandrepeatedbreath-holds(upto25 (Robinson etal.,2012).Additionally, theseanimalsarewellknown intervals duringforagingtripslastingupto8 Boeuf, 1994),adult elephantsealtHbwassignificantly higherthan including humans(Zijlstraetal., 2000)(Fig. identical tothecattle,sheepand multipleothermammalianspecies, elephant sealhemoglobinvarieties hadabsorptionpeaksthatwere evaluation ofthehemoglobin propertiesinelephantseals.All sheep weremeasuredtovalidate theuseofco-oximeterfor oxyhemoglobin (O Spectrophotometric absorptionpeaksofdeoxyhemoglobin(HHb), to 25 capacity contributestotheirabilityperformrepetitivedivesof20 RESEARCH ARTICLE be highestinadults. volumes inadultsversusyoungerseals,theamountofCOHbwould hemoglobin concentration(tHb),and(2)becauseoflarger blood that (1)theamountofCOHbwouldcorrelatewith total differences inCOHbvalues associatedwithage.We hypothesized (Thorson andLeBoeuf,1994;Tift etal.,2013),weinvestigated natural ontogeneticincreaseinbothhemoglobinandmyoglobin values againsthemoglobinconcentrations.Further, becauseofa of COHbinthebloodnorthernelephantsealsandcomparedthese in aspecieswithexceptionallylarge O behaviors. reperfusion injuryassociatedwiththesenaturalbreath-holding elevated endogenousCOmayprovideprotectionagainstpotential duration foraerobicmetabolismduringadive.Ontheotherhand, RESULTS Considering endogenousCOisexpelledviarespiration,the Similar toresultsfoundina previous study(ThorsonandLe To addressthepotentialforelevatedendogenousCOproduction O H deoxyhemoglobin carboxyhemoglobin carbonmonoxide O HHb calculatedaerobicdivelimit COHb CO cADL H totalhemoglobinconcentration tHb List of abbreviations List of 2 2 boxyhemoglobin Hb min durationtodepths>500 months peryearatseawithover90%ofitstime oxygen 2 Hb) andCOHbfromelephantseals,cattle 2 stores andthereforepotentiallylimitingthe
m withonly2–3 2 stores, wemeasuredlevels
1).
months induration
min surface min) during of thefirsthumanclinicalpharmaceuticalinvestigationsusing COHb) wasalsocomparableto12%COHbvaluesmeasuredinone maximum COHbvaluefoundinanadultelephantseal(10.4% patient withhemolyticanemia(Hampson,2007)(Fig. endogenous values(9.7%COHb)foundinacriticallyillhuman per day(Lawetal.,1997)andcomparabletothehighestrecorded Peaks forO (HHb) andcarboxyhemoglobin(COHb)innorthernelephantseals. of 21.7±0.8and19.6±0.8 the meanCOHbvalue( COHb remainedelevatedandshowednosignificantvariationfrom Hampson (Hampson, 2007).Values aremeans+s.e.m. 1997). HumanCOHb valuesofCOHbforhemolyticanemia arefrom values ofCOHbforsmokingandnon-smoking arefromLawetal.(Lawal., smokers andnon-smokerselephant sealadultsandpups. 2.Mean carboxyhemoglobin(COHb)valuesinthebloodofhuman Fig. ( age andtHbexplained80%ofthevarianceinCOHbvalues 2).Together, both juveniles(7.6±0.2%)andpups(7.1±0.3%;Fig. that ofpups,butnotjuveniles( 1.Absorptionspectraforoxyhemoglobin(O Fig. study arehigherthanvaluesinhumansthatsmoke The valuesofCOHbfoundinnorthernelephantsealsfromthis adults was24.0±1.0 569 DISCUSSION (from Zijlstraetal.,2000). (hemolytic anemia) F Human (smoker) Adults hadsignificantlyhigherCOHbvalues(8.7±0.3%)than 3,20
Absorbance nm) matchthoseofothermammalianspecies(human,cow, sheep) (non-smoker) 0.1 0.2 0.3 0.4 Seal juvenile =30, 0 450 500550 Seal adult Seal pup Human Human 2 P Hb (542and577 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.100677 000;Fg 3).Overthecourseofseveralhours, <0.0001; Fig. 0
g
dl
F − 2 1 1,801
, whilejuvenilesandpupshadtHbvalues g nm), HHb(555
dl Wavelength (nm) =0.81, − 1 , respectively. F 2,21
4 6 COHb (%) P =6.5, 03;Fg 4). =0.37; Fig.
nm) andCOHb(539 600 P =0.0066). MeantHbin 2 Hb), deoxyhemoglobin
650 ≥
40 cigarettes 8 COHb HHb O 2 Hb
Human 2). The
1753 700
10
The Journal of Experimental Biology 1754 RESEARCH ARTICLE of heme-proteinsorerythrocytes),asisfoundinhumanpatientswith suggest: (1)morerapidturnoverofhemestores(i.e.shorterhalf-life 5). adult humans(Fig. approximately fourand16timesgreater, respectively, thanthosein of hemefrombothhemoglobinandmyoglobinstoresaloneare Thorson andLeBoeuf,1994).Inelephantseals,themolarequivalents hemoglobin concentrations(Pugh,1959;Ponganisetal.,1993; increased myoglobincontent,bloodvolume,hematocritand be attributedprimarilytotheelevatedhemestoresassociatedwith endogenous COvaluesinnorthernelephantsealsfromthisstudycan adverse effects comparedwithcontrollevels. These valuesinhealthyhumanswerewelltoleratedandshowedno inhaled COasatherapeuticagent(MotterliniandOtterbein,2010). ( (COHb=–1.31+0.42tHb), andthe95%confidenceintervalsareinblack age classesofnorthernelephantseals. percent carboxyhemoglobin(COHb)inthebloodfromthreedifferent Relationship betweenthetotalconcentrationofhemoglobinand 3. Fig. F 3,20
COHb (%) As originallysuggestedinWeddell seals,webelievethatthehigh
10 12 14 16 10 12 14 16 COHb (%) =30.1, P 10 12 0 2 4 6 8 0 2 4 6 8 2 4 6 8 8 121620 8 14
<0.0001, 10 Pups Juveniles Adults
16
12 r 2 =0.80). The levelsofCOHbmeasuredinelephantseals
18
14 Hemoglobin (gdl
20
16 10 12 14 16 10 12 14 16 0 2 4 6 8 0 2 4 6 8 The regressionlineisinred 7 9 9 11
22 –1 ) Time ofday(h)
11 11 24
13 151719
13
26
15
17
28 10 12 14 16 10 12 14 16 0 2 4 6 8 0 2 4 6 8 10 1214161820 9 11 maintain lowerlevelsofthosemetabolites. heme contentandturnoverintheseanimalsisadequateto or recyclingofbilirubinandbiliverdinrelativetoabsolutetotalbody 2013; D.E.C.,unpublished).Onepossibilityisthattheclearancerate classes ofelephantseals(CostaandOrtiz,1982;Champagneetal., 1984), yettherearerelativelylowlevelsofbilirubinfoundinallage also beenfoundinsomeneonatemarinemammals(Dieraufetal., heme stores(Denneryetal.,2001).Neonatalhyperbilirubinemiahas common occurrenceinhumaninfants,whichhavemuchlower neonatal hyperbilirubinemiamightbeexpectedbecauseofthe animals. Thisisespeciallyinterestinginelephantsealpups,where unpublished) toaccompanythehighbloodCOlevelsseeninthese elevation inthehemoglobinbreakdownproductbilirubin(D.E.C., may alsocontributetothebuild-upofendogenousCO. (Andrews etal.,2000)andthus,thisnaturalbreath-holdingbehavior elephant sealwillspendamajorityofitslifeinanapneicstate produced CO.Betweenthesetwobreath-holdbehaviors,atypical therefore, decreaseoratleastdelaytheexhalationofendogenously land (sleepapnea)limitthetimeelephantsealsspendineupnea,and the naturalandrepetitivebreath-holdsbothatsea(diving)on could elevatetheproductionrateofendogenousCO.Additionally, marine mammalsisunknown,butanincreasedhemeturnoverrate endogenous COproduction. of heme-oxygenase,theenzymeresponsibleforhemedegradationand concentrations and/orwithanincreaseintheactivityorconcentration degradation couldbeassociatedsimplywiththeelevatedheme mammals, or(3)acombinationofboth.A greatermagnitudeofheme magnitude ofhemedegradationthanhasbeenmeasuredinother hemolytic anemia(Coburnetal.,1966;Hampson,2007),(2)agreater The conceptsofincreasedO (Kooyman, 1989;ButlerandJones,1997;Ponganisetal.,2003).Total dive performanceandforagingecologyinapexmarinepredators aerobic divingunderliecurrentinterpretationsofphysiology, Effect of elevated COonO of Effect Unlike Weddell seals(Pugh,1959),elephantdonothavean Currently, thelife-spanofheme-proteins anderythrocytesin
13 151719 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.100677 2 storage, alowdivingmetabolicrate,and 2 lines represent95%confidenceintervals. regression lineforthedataandblack red linesrepresentthebest-fitlinear from sixjuvenileelephantseals. carboxyhemoglobin (COHb)values Time courseof 4. Fig. stores andO stores 2 delivery The
The Journal of Experimental Biology O the rateofbreath-holdO included inthebloodO Sylven, 1981;Hassricketal.,2010;Ponganis2011). 1968; Oscaietal.,Simpson1970;Bryden,1972;Möllerand from previousstudies(Kendrewetal.,1958;Hill1962;Åkeson blood volumes,musclemass,hemoglobinandmyoglobinconcentrationsare perfusion, andactivation ofthesympatheticnervous systemduring The routinehypoxemia,hypercarbia, reductioninperipheral body O calculated usingahemoglobinmolecularweightof65,000 northern elephantsealsandhumans. blood (hemoglobin)and(B)muscle(myoglobin)oxygenstoresin 5.Mass-specificmolarequivalentsofhemecalculatedfrom(A) Fig. RESEARCH ARTICLE O potential forendogenousCOto reducethemagnitudeoftotalbody in elephantsealsfromthisstudyshouldheightenawarenessof the of thepredictedcADL of30 elephant sealsareactually22–23 reduction mighthelpexplainwhythemajorityofdivesinfemale [~30 consideration inthecalculationofacADL ofadultelephantseals calculation, whichisperformedbydividingtotalbodyO 1997; KooymanandPonganis,1998;Costaetal.,2001).Forthis lactate occurs(KooymanandKooyman,1995;ButlerJones, duration ofadiveorbreath-holdbeforepost-diveriseinplasma aerobic divelimit(cADL),whichisapredictionofthemaximum pinniped diver, effectively reducingitstotalbodyO 10% oftotalhemoglobincontentcanbeboundtoCOinthepremier myoglobin molecularweightof17,000 mammals elevated CO inmarine benefitsof Potential therapeutic 2 2 When theseCO-relatedreductionsinO store insomespecies).However, thepresent studyshowsthatover stores calculatedinotherspecies.
min (Hassricketal.,2010)],thereisreductionof~2 Mass-specific molar equivalents of Mass-specific molar equivalents of –1 –1
2 heme from myoglobin (mol kg ) heme from hemoglobin (mol kg ) 0.0002 0.0004 0.0006 0.0008 0.0010 0.0012 0.0014 stores (lung,bloodandmuscle)areusedtocalculatethe 10 0 0 2 4 6 8 B A 2 2 store (whichcanmakeup70%ofthetotal lpatsa Human Elephant seal lpatsa Human Elephant seal consumption, allmeasuredhemoglobinis
min. TheelevatedCOHbvaluesseen
min (Robinsonetal.,2012)instead
g
mol Molar hemeequivalentswere –1 . Dataforspecies-specific 2 stores aretakeninto
2 g
stores by~7%. mol –1 2
and min. This stores by Motterlini andOtterbein,2010).Thus,althoughCOdecreases O anti-oxidative responses(Wu andWang, 2005;Ryter etal.,2007; oxidative stress.Theseincludeanti-inflammatory, anti-apoptosisand therapeutic effects intheavoidance ofre-perfusioninjuryand and biliverdin)arealsoassociatedwithawidearrayofpotential (Andersen etal.,2009). activity, whichcouldreducevasodilatorycapacityinthefetus potentially COexposure)andendothelialnitricoxidesynthase this suggestionisthenegativerelationshipbetweensmoking (and continuously divingfemaleelephantseals.However, complicating flow andplacentalgrowthduringtheat-seapregnanciesof the elevatedCOexposureinsealsmayhelpoptimizeuterineblood Recently demonstratedinthepregnantmouse(Venditti etal.,2013), with thediveresponseinaphocidseal(Ligginsetal.,1980). maintained duringextremeperipheralvasoconstrictionassociated al., 2008;Meiret2009). tissue vasoconstrictionduringbreath-holdinginseals(Ponganiset contribute tovascularregulationforcopingwithhypoxemiaand pre-eclampsia (Wikström etal.,2010).Thus,elevatedCOmayalso been showntobeanassociateddecreasedriskofhypertensionand and lowbirthweights(CnattingiusLambe,2002),therehas during pregnancyisassociatedwithnumerousadverseoutcomes hypertension (Herreraetal.,2008).Andlastly, althoughsmoking pulmonary COproductionisassociatedwithdecreased example, inthellama( vasodilation anddecreasehypertensioninseveralspecies.For Additionally, lowconcentrationsofCOareknowntopromote perfusion associatedwithbreath-holds(Ponganisetal.,2008). reperfusion injury, associatedwiththemarkedchangesinperipheral seals couldprovideprotectionagainstpotentialischemia– Therefore, theelevatedlevelsofendogenousCOfoundinelephant being appliedinclinicalinvestigations(Ozakietal.,2012). these injuriesduringischemia–reperfusionevents,andiscurrently exposure tolowlevelsofCOsignificantlydecreasestherisk apoptosis, intenseinflammationandthrombogenesis.However, associated withischemia–reperfusioninjurytypicallyinclude transplantation (CardenandGranger, 2000).Deleteriouseffects of clinicalscenarios,includingheartattacks,strokesandorgan seals, mayalsocontributetotheprotectionagainsttheseconditions. associated by-productssuchasendogenousCOfoundinelephant al., 2011). We proposethatthelevelsofhemedegradation,and al., 2002;Olsonet2010;Tift etal.,2011; Vázquez-Medinaet concentrations andantioxidantenzymeactivities(Zenteno-Savínet pulmonary vasodilatationandelevationsinbothantioxidant potentially decreasesuchrisksinmarinemammalsincludehypoxic Ponganis, 1998;Zenteno-Savínetal.,2002).Adaptationsthat damage (Kooyman,1989;ButlerandJones,1997;Kooyman injury, reactiveoxygenspecies formation,andsubsequenttissue risks ofsystemicandpulmonaryhypertension,ischemia–reperfusion both divingandsleepapneainelephantsealspotentiallyincreasethe potential ofcarbon monoxide,deep-divingseals maybecomea (Krogh, 1929).With thegrowingknowledgeontherapeutic fundamentals andextremesof thisuniquephysiologicalsystem represent anexcellentopportunity tostudyandunderstandthe CO andCOHbconcentrations foundindeep-divingphocidseals apoptosis andoxidativestress. in divinganimalsandotherexposedtoinflammation, storage, ithasthepotentialtooffer severalotherbeneficialeffects Exposure tootherheme-proteinbreakdownproducts(bilirubin While themechanismisstillunknown,placentalbloodflow Ischemia–reperfusion injuryiscommonlyseenduringavariety We concludewiththesuggestionthatelevatedendogenous The JournalofExperimentalBiology(2014)doi:10.1242/jeb.100677 Lama glama)athighaltitude,elevated 1755 2
The Journal of Experimental Biology absorption peaksforoxygenatedhemoglobin(O sheep fromZijlstraetal.(Zijlstraal.,2000).Isolatedhemoglobin compared withhemoglobinabsorptionpeakvaluesforhuman,cattleand Hemostat Laboratories,Dixon,CA,USA)werespectrophotometrically (Na environment. ForHHb,anadditionof3 an airtightblood-gassyringe,therebyminimizingexposuretotheambient of distilledH times with0.9%NaClsolution.Erythrocyteswerethenlysed1volume Lakes, NJ,USA).Plasmawasdiscardedanderythrocyteswerewashedthree a BayerRapidlab 845bloodgasanalyzerwith co-oximeter (Siemens ice untilanalysis (<3 intravenous ketamineinjections(0.5 spectrophotometer cuvettes(light-path=1.0 Vacutainer (~3 drugs fromFortDodgeLabs,Dodge, IA,USA).Venous bloodsamples immobilization fortheintravenous catheterization oftheextraduralvein(all 1756 RESEARCH ARTICLE (1 April–May. Pups weresampledinFebruary–Marchandjuveniles sampled fromJanuarytoFebruaryatthebeginningoftheirbreedingseason. and adults( classes: pups(<3 enables referencetotheanimals’ ages.Subjectsweredividedintothreeage with flipper-tags (JumboRototag,Dalton,UK)followingweaning,which County, CA,USA.Everyyear, asubsetofpupsatthereservearemarked season (December–April)fromAñoNuevoStateReserve,SanMateo We sampled24northernelephantsealsthroughoutthe2009winterbreeding below), sheep(n juvenile northernelephantseals( humans andotherterrestrialanimals.Hemoglobinfromthebloodofhealthy hemoglobin absorptionspectraofelephantsealsweresimilartothose the determinationofelephantsealhemoglobinvalues,weconfirmedthat oximeter (SiemensMedicalDiagnostics,Bayer, Tarrytown, NY, USA)for To validatetheuseofaBayerRapidlab845bloodgasanalyzerwithco- gasotransmitter. exposure toelevatedconcentrationsofthisendogenouslyproduced mechanisms ofactionandevolutionaryadaptationfromlong-term valuable modeltoinvestigatethepotentialcytoprotectiveeffects, were thenimmediatelycapped,sealedwithParafilm introducing 100%nitrogen(N prevent O for upto2 collected andcentrifugedat8000 stromata andabottomlayerofhemoglobinlysate.Thewas 16 h. Thisresultedinatoplayeroftoluene,middleerythrocyte were filledwith2.5 Elephant seal COHb absorption spectrum Elephant sealCOHbabsorption MATERIALS ANDMETHODS Study siteandsubjects Clara, CA,USA)inthespectralrangeof450–700 with aCary60UV-Vis spectrophotometer(AgilentTechnologies, Santa analyzed. 158 2000). Briefly, bloodwascollectedintochilledvacutainerscontaining similar tothatdescribedpreviously(Kreutzeretal.,1993;Zijlstra hemoglobin (HHb)andCOHbweremeasuredcomparedusingmethods different hemoglobinforms(HHb,O placed insideatonometerwhererespectivegaseswereintroduced.The Q, Millipore,Billerica,MA,USA).Thedilutederythrolysatewasthen then filteredthroughsterilegauzeanddilutedwithdeionizedwater(Milli- Animals werechemicallyimmobilizedwithanintramuscularinjection Absorbance measurementsweremadeatroomtemperature(20–25°C) gkg mg 2 ml) werecollectedintolithium heparinizedbloodtubes(BD USP units(equivalentto158 S 2 O 4 ) per2 − 2 1 ® h atarateof30–40 ) ofTelazol (tiletamine/zolazepam HCl)andsubsequent or COfrombinding.Oncethelysatewastransferred,cuvettes , FisherScientific,FranklinLakes, NJ,USA)andchilledover ˃ 2 3 O and0.4volumesoftoluenelefttositat4°Cforup
years old; ml ofbuffered erythrolysatewasincludedfortonometryto =1) andcattle( months old;n
ml oferythrolysatecollectedfromthetonometerusing
h). Standardblood gasanalysiswasperformedusing n =6). Adultanimalswereallfemale,and 2 ), O
ml n =9), juveniles(3 =1) (sheepandcattlebloodobtainedfrom min 2
g
and CO,respectively, intothetonometer IU) ofsodiumheparin(BD,Franklin n for 20 =2, seebloodcollectiontechnique, −
2 1 mg . ForO Hb andCOHb)werepreparedby kg
min, andtheerythrolysatewas cm) coveredwithParafilm
− mg ofsodiumhydrosulfite 1 2 ) asnecessarytomaintain Hb andCOHb,polystyrene months–3
nm. ® 2 Hb), deoxygenated M andimmediately years old;n ® =9) M adn .L n rne,D.N. Granger, and D. L. Carden, ulr .J n oe,D.R. Jones, and P. J. Butler, was determinedat random subjecteffect. Allmeanvaluesarereported±s.e.m.Significance using arepeated-measureslinearmixedmodel,withindividualIDasthe analysis. ChangesinCOHboverthecourseofseveralhourswereevaluated the percentageofCOHbinbloodsamplesusingamultipleregression the effects ofageclass(adult,juvenileandpup)tHbconcentrationon Data wereanalyzedusingthestatisticalprogramJMP 11.0. We examined juvenile elephantsealsforupto12 periods ofapneaandeupnea,weopportunisticallysampledsixthenine COHb andtHbconcentrations. Medical Diagnostics,Bayer, Tarrytown, NY, USA)toobtainfractionsof months. Science Foundation[09-44220toP.J.P.]. DepositedinPMC for releaseafter12 National InstitutesofHealth[NHLBIR01-HL091767toR.M.O.]andthe [N000141010514 toP.J.P.]. GraduatestudentsupportforM.S.T. wasprovidedby Funding forthisprojectwasprovidedbytheOffice ofNavalResearch and interpretationofresultswereconductedbyM.S.T., P.J.P. andD.E.C. and dataanalysiswereconductedbyM.S.T. andD.E.C.Manuscriptpreparation M.S.T., P.J.P. andD.E.C.designedthestudy. Fieldwork,executionofexperiments The authorsdeclarenocompetingfinancialinterests. P.J.P. toPugh’s 1959findingsandstimulatedthisresearch. Maresh andB.McDonaldformanuscriptreview, andJ.West, whointroduced spectropohotometer aregreatlyappreciated.We alsothankG.Kooyman,J. T. JueandV. AgarwalonabsorptionspectratechniquesanduseofB.Moore’s Champagne andM.Fowlerprovidedvaluableassistanceinthefield.Advicefrom support. E.Ranalli,A.Dallara,V. Farnham,J.Jelincic,Vázquez-Medina,C. We thanktherangersand docentsatAñoNuevoStateReservefortheirlogistical Use CommitteeatSonomaStateUniversity. permit no.14636andwasapprovedbytheInstitutionalAnimalCare to 134samplesperindividual. course of8–12 voluntary sleepapneaandeupnea,withminimaldisturbance.Overthe was attachedtothecatheterallowbloodcollectionduringperiodsof OH, USA).Anextensiontube(76 was affixed tothefurusingLoctiteglue(HenkelCorporation,Westlake, with along-termpolyurethanecatheter(MILA;16 in aholdingcageandtheextraduralveinwaspercutaneouslycatheterized nesn .R,Smne,U,Udjr,N,Alje,C n tne,S. Stender, and C. Aalkjaer, N., Uldbjerg, U., Simonsen, M.R., Andersen, R. Simon, and G. Biörck, A., Åkeson, aaao .E,Ro . ers .D n ndr S.H. Snyder, and C.D. Ferris, M., Rao, D.E., Barañano, Acknowledgements Statistical analysis References Funding Author contributions Competing interests nrw,R . ot,D . eBef .J n oe,D.R. Jones, and B.J. LeBoeuf, D.P., Costa, R.D., Andrews, Costa, G.W., Oliver, P. H., Thorson, J.D., Williams, D.R., Jones, R.D., Andrews, rdn M. Bryden, B. LeBoeuf, and S.B. Blackwell, injury. Physiol. Rev. 276. absolute measurementsandestimates frombonedimensions. spectral analysis. frequencies ofnorthernelephantsealsatseaandonlandrevealedbyheart rate observational studyofhealthysingletonpregnancies. and endothelialnitricoxidesynthaseactivityinumbilicalchorionicvessels: an Smoking cessationearlyinpregnancyandbirthweight,length,headcircumference, muscles. reductase: amajorphysiologiccytoprotectant. sea andrestingonthebeach. B.J. LeBoeuf, and D. P. northern elephantseals, 16098. To determinethevariabilityofCOHbovertimeandbetweenvoluntary This workwasconductedthroughtheNationalMarineFisheriesService J. Pathol. Acta Med.Scand. (1972). Bodysizeandcompositionofelephant seals( The JournalofExperimentalBiology(2014)doi:10.1242/jeb.100677 77, 837-899. h, wecollectedatotalof806bloodsamplesrangingfrom36 190 Respir. Physiol. P , 255-266. <0.05. Mirounga angustirostris 183 (1997). Heartratesofnorthernelephantsealsdivingat J. Exp.Biol. (1997). Physiologyofdivingbirdsand mammals. , 307-316. (1993). Developmentalaspectsofsleep apnoeain 123 (2000). Pathophysiology ofischaemia-reperfusion
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