Valtolina et al. BMC Veterinary Research (2017) 13:231 DOI /s y

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1 Vltolin et l. BMC Veterinry Reserh (2017) 13:231 DOI /s y RESEARCH ARTICLE Open Aess Sex speifi differenes in hepti nd plsm lipid profiles in helthy ts pre nd post spying nd neutering: reltionship with feline hepti lipidosis Chir Vltolin 1*, Arie B. Vndrger 2, Roert P. Fvier 1, Midin Tuohethuntil 2, Anne Kummeling 1, Iselle Jeusette 3, Jn Rothuizen 1 nd Joris H. Roen 1 Astrt Bkground: A link etween lipid metolism nd disese hs een reognized in ts. Sine hepti lipidosis is frequent disorder in ts, the im of the urrent study ws to evlute liver nd plsm lipid dimorphism in helthy ts nd the effets of gondetomy on lipid profiling. From six femle nd six mle ts plsm nd liver lipid profiles efore nd fter spying/neutering were ssessed nd ompred to five ts (three neutered mle nd two spyed femle) dignosed with hepti lipidosis. Results: Intt femle ts hd signifintly lower level of plsm triylglyerides (TAG) nd higher liver level of the long hin polyunsturted ftty id rhidoni id (AA) ompred to their neutered stte. Both mle nd femle ts with lipidosis hd higher liver, ut not plsm TAG level nd n inresed level of plsm nd liver sphingomyelin ompred to the helthy ts. Conlusion: Although lipid dimorphism in helthy ts resemles tht of other speies, intt femle ts show differenes in metoli onfigurtion tht ould predispose them to develop hepti lipidosis. The inresed sphingomyelin levels in ts with lipidosis ould suggest potentil role in the pthogenesis of hepti lipidosis in ts. Keywords: Lipid dimorphism, Cts, Feline hepti lipidosis, Sex hormones, Sphingomyelin Bkground In humn mediine, the study nd evlution of lipid metolites with the use of lipid profiling or lipidomis hs eome ruil in order to understnd the role of lipids in the pthophysiology of lipid-relted diseses [1, 2]. The role of lipids in the development nd progression of numerous diseses in mn, suh s dietes mellitus, oesity, non-loholi ftty liver disese (NAFLD), Alzheimer nd rterioslerosis is well reognised [3 5]. The reognition of sex speifi geneti dimorphism in lipid metolism hs helped to identify risk ftors nd to develop more speifi nd trgeted therpies [6, 7]. * Correspondene:.vltolin@uu.nl 1 Deprtment of Clinil Sienes of Compnion Animls, Fulty of Veterinry Mediine, Utreht University, Yleln 108, 3584 CM Utreht, The Netherlnds Full list of uthor informtion is ville t the end of the rtile Pre-menopusl women re onsidered to possess more fvourle (less pro-ogultory nd less pro-therogeni) plsm lipid profile ompred to men nd postmenopusl women, with lower plsm levels of triylglyeride (TAG), totl holesterol (TC), nd low density lipoprotein-holesterol (LDL-C), nd higher levels of high density lipoprotein-holesterol (HDL-C) [8 11]. Prllels in lipid profiles etween speies hve een demonstrted. The essentil ftty id (FA) profile is known to vry onsiderly etween mles nd femles in humns, mie nd rts, inditing tht sex lipid dimorphism ours in different speies [12 20]. When ompred to their mle ounterprt, premenopusl women possess higher perentge of the long hin polyunsturted ftty ids (LCPUFA) rhidoni id (AA 20:4 n-6) nd dooshexenoi id (DHA, 22:6 n-3) in plsm nd liver [20 22]. Also in rts, LCPUFA levels in plsm nd liver The Author(s) Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( pplies to the dt mde ville in this rtile, unless otherwise stted.

2 Vltolin et l. BMC Veterinry Reserh (2017) 13:231 Pge 2 of 9 re higher in intt femle rts ompred to mles, while this differene disppers fter gondetomy [18, 20]. In mn, mie nd rts LCPUFA re synthesised from the essentil FAs linolei id (LA 18:2 n-6) nd α- linoleni id (ALA, 18:3 n-3) y the sequentil tivities of desturses (Δ5, Δ6) nd elongses [23, 24]. Oestrogen hs een shown to stimulte the FA desturse tivities [20, 25 29] nd is lso known to inrese the tivity of the hormone sensitive lipses, resulting in n inresed level of irulting FA [28, 29]. Furthermore, oestrogen ppers to e involved in lowering the plsm onentrtion of TAG nd very low density lipoprotein (VLDL) triglyerides in premenopusl femles [17, 30, 31]. In ts, the link etween lipid metolism nd speifi diseses hs lso een knowledged. Conditions like Niemnn-Pik in Simese ts, oesity, dietes mellitus, nd hepti lipidosis (HL) hve een ssoited with n ltered lipid metolism in ts [32, 33]. Hepti lipidosis is onsidered one of the most ommon liver diseses in ts nd is hrterised y severe umultion of triglyerides in the liver, resulting in n impired liver funtion [34, 35]. The pthogenesis of HL in ts is still not ompletely eluidted. The strit rnivorous diet nd the unique protein nd lipid metolism of ts hve een indited s importnt predisposing ftors in ts [36]. Cts, s other strit rnivores, possess unique lipid metolism, hrterised y their limited ility, unlike other mmmls, to synthesise AA from LA nd eiospentenoi id (EPA) nd DHA from ALA due to limited desturses Δ5 ndδ6 tivities [37, 38]. It hs een reported tht feline HL hs no reed, ge or sex predisposition [34, 35], lthough in one study femle ts hve een reported to e more ffeted thn their mle ounterprts [39]. This seems to e in ontrst with wht is known in other speies, where femles, espeilly the premenopusl womn nd intt femle rts nd mie, seem to e more proteted from lipid relted disese thn the postmenopusl/spyed femle nd their mle ounterprts. If however, s hs een suggested efore, femle ts re more suseptile to the development of HL ompred to mles, the nswer ould lso e found in unique sex relted lipid profiling. The im of the urrent study is to perform lipid profiling in intt nd spyed/neutered helthy ts fed with ommon ommeril diet nd to evlute the effet of gondetomy on the liver nd plsm lipid profiles. Additionlly, we ompre lipid profiles from helthy ts to those in ts ffeted y HL in n ttempt to eluidte possile predisposing ftors in the development of HL. Methods This study ws pproved y the Committee for the Ethil Cre of Animls of the Utreht University. Helthy ts The helthy ts nd the study design, diet, lood olletion, hormone mesurements, nesthesi nd surgil proedure, nd liver iopsy tking hve een desried previously [40]. Plsm nd pre nd post spying/neutering liver iopsies otined in this study [40] were lso used for nlysis. In summry, six intt femles nd six intt mles lient-owned ts dmitted to the Deprtment of Clinil Siene of Compnion Animls of the Fulty of Veterinry Mediine, Utreht University (DCSCA) for spying/neutering were onsidered for the study. Following onfirmtion of good helth ts were neutered nd spyed nd liver iopsy tken. The helth sttus s well s lood prmeters were evluted gin 4 weeks lter during the seond dmission to the hospitl nd efore the dditionl liver iopsy ws performed. A study diet ws fed to ll helthy ts reruited in the study for totl of 8 weeks, strting four weeks efore the first dmission to the hospitl nd terminting fter the seond lood nd liver tissue smpling 4 weeks fter the spying/neutering surgery. For nlysis nd interprettion of the results, helthy ts were divided in four groups: intt mles (efore neutering, n = 6), intt femles (efore spying, n = 6), spyed femles (fter spying, n = 6), neutered mles (fter neutering, n = 6). Cts with hepti lipidosis Cts dmitted from Jnury 2013 to Jnury 2014 to the DCSCA with suspeted HL sed on linil symptoms (norexi, vomiting nd iterus) were onsidered for this study. In ll ts ultrsonogrphy showed diffuse ehodensity of the liver omptile with HL. Hepti lipidosis ws onfirmed sed on ytologil evlution of fine needle spirtes of the liver. Only ts tht were euthnized due to deteriortion despite intense tretment nd/or finnil onstrints of the owner were enrolled in the study. Owners onsent ws sked to e signed prior to onsidertion of the ts for the study. In ontrst to the helthy ts, ts with HL did not reeive ny speil diet. Plsm tht remined from heprinised lood olleted during hospitlistion for dignosti nd linil monitoring ws freshly stored t -70 C in ryogeni vils (Corning In., Corning NY, USA) for lipidomis nlysis. In ts with HL two wedge liver iopsies were immeditely olleted post-mortem vi lprotomy. As reported efore (Vltolin et l.) one smple ws fixted in 4% neutrl-uffered formlin nd the other liver iopsy ws rinsed in norml sline (NCl 0.9%), rpidly frozen in liquid nitrogen nd then stored t 80 C. The ltter ws used for the lipidomis nlysis.

3 Vltolin et l. BMC Veterinry Reserh (2017) 13:231 Pge 3 of 9 Lipidomis nlysis For the lipidomi nlyses the liver iopsies of the helthy ts pre- nd post spying/neutering nd of the ts with HL were re-suspended in 350 μl of uffer A (250 mm surose 0.2 mm EDTA, 5 mm DTT, nd 10 mm Tris/HCl ph 8.0) nd homogenized y mehnil disruption with pestle tightly fitting to n Eppendorf tue followed y sonition (10 s, mplitude 10 μm). Protein ws determined in the homogentes y the BCA method Piere BCA protein ssy kit (Thermo sientifi, Rokford, IL, USA) with BSA s stndrd. To 100 μl of homogenized liver iopsy or 100 μl of plsm, 200 pmol of tri-pentdenoylglyerol (TAG 45:0) nd 100 pmol of di-linolenoyl phosphtidylholine (PC (18:3, 18:3)) were dded s internl stndrds. Susequently, lipids were extrted nd seprted in neutrl nd phospholipid frtion y frtiontion on sili-g olumn s desried [41]. In short, lipid extrts were dissolved in methnol/hloroform (1/9, v/v) nd loded on top of the sili olumn. Neutrl lipids were eluted with two volumes etone, dried under nitrogen gs nd stored t 20 C. In the neutrl frtion, TAG speies, holesterol, holesterol esters (CE), nd retinyl esters (RE) were determined y HPLC-MS s desried [41]. Typilly, just efore HPLC-MS nlysis, the neutrl lipid frtion ws reonstituted in methnol/hloroform (1/1, v/v) nd seprted on Lihrospher RP18-e olumn (5 μm, mm; Merk, Drmstdt, Germny). A grdient ws generted from etonitrile to etone/hloroform 85/15, v/v, t onstnt flow rte of 1 ml/min. Mss spetrometry of lipids ws performed using Atmospheri Pressure Chemil Ioniztion (APCI) on Biosystems API-4000 Q-trp (MDS Siex, Conord, Cnd). The system ws ontrolled y Anlyst version softwre (MDS Siex, Conord, ON, Cnd) nd operted in positive ion mode. Full sn runs spetr were otined from m/z Totl TAG ws determined y quntitting ll ions etween m/z 530 nd 1050 with retention time of TAG speies nd orreted for the presene of seond nd third isotope peks. The perentge of TAG 56:6/7 ws determined y quntittion of the ions with m/z 905 nd 907. TAG unst/st rtio ws lulted fter quntitition of the ions with m/z of 853 (TAG52:5), 879 (TAG54:6), 859 (TAG 52:2) nd 885 (TAG54:3). In the phospholipid frtion, sphingomyelin (SM) nd PC speies were determined s desried [42]. The phospholipid frtion ws dissolved in hloroform/methnol (1:1, v/ v), nd PC nd SM moleulr speies were seprted LiChrospher 100 RP18-e olumn (Merk), with moile phse of etonitrile/methnol/triethylmine (25:24:1, v/v/ v). Identifition of moleulr speies ws performed y on-line tndem mss spetrometry in the positive-ion mode on n API 4000 Q Trp mss spetrometer fitted with n eletrospry ioniztion soure (Siex) y preursor sns of 184 m/z (holine hed group). Dt nlysis ws performed using Anlyst softwre (MDS Siex, Conord, ON, Cnd) nd lirtion urves of ll lipid lsses were estlished under similr onditions s the smples. Sttistil nlysis Helthy ts, pre versus post spying/neutering The outome vriles TAG, TAG unst/st, TAG56:6/7, holesterol, holesterol ester, totl PC, PC speies (% of totl PC) nd SM were respetively nlysed using liner mixed model with the t identifition s rndom effet to tke repeted oservtions within the sujet into ount. Time (efore/fter spying/neutering) nd gender (mle/femle) nd the intertion etween time nd gender were used s explntory vriles. The Akike Informtion Criterion (AIC) ws used to selet the est model. Residuls plots were used to ssess the vlidity of the model y visul inspetion of the QQ-plot of residuls for normlity nd stterplots of residuls versus predited vlues for onstnt vrine. Some outome vriles (liver TAG st/unst; plsm PC 34:1) were log trnsformed to meet the model ssumptions normlity nd onstnt vrine. In this model we lso ould tke the orreltion etween oservtions into ount. If dt were not normlly distriuted, neither fter log trnsformtion (liver TAG 56:6/7; liver PC 38:6; plsm TAG st/unst), nonprmetri Kruskll nd Wllis test ws pplied to ssess the differene in mens etween groups of omined gender nd time (premle, prefemle, postmle en postfemle respetively) nd non-prmetri Wiloxon test ws pplied to ssess the differene within genders etween oth times. Unfortuntely this model ws not le to tke the orreltion etween oservtions into ount. In order to identify ftors involved in the vrition in liver TAG levels in the helthy ts Person orreltion nlysis ws performed. Helthy ts versus ts with hepti lipidosis Comprison etween the men of the respetive outomes of the helthy ts fter spying/neutering nd ts with HL were lulted with the independent Student s T-test. P-vlue <0.05 ws used to ssess sttistil signifine. Results re expressed s men nd stndrd devition (SD), if not stted otherwise. Dt nlysis ws performed using IBM SPSS 22 sttisti softwre (IBM Corportion Armonk, NY, USA). Results Animl s hrteriztion A desription of the helthy ts hs een previously reported [40]. In summry, six helthy femles (20.5 ( months); 3.0 ( ) kg; (medin (rnge)) nd six helthy mles (7.0 ( ) months; 4.0 ( ) kg; (medin (rnge)) were enrolled in this study Results of the

4 Vltolin et l. BMC Veterinry Reserh (2017) 13:231 Pge 4 of 9 CBC, iohemistry nd ogultion profile of ll 12 ts were within referene intervls. Both oestrogen in the femle group (P = 0.041) nd testosterone in the mle group (P = 0.001) dropped signifintly onfirming suessful gondetomy [40]. Histologil evlution of the liver iopsies performed in the helthy ts (pre nd post spying/neutering) t 4 weeks nd t 8 weeks fter the eginning of dministrtion of the diet, reveled no histologil hnges omptile with HL or ny other pthologil hnge in ny of the helthy ts [40]. Two spyed femles (50.0 nd 52.0 months; 3.5 kg nd 4.5 kg, respetively), nd three neutered mles (38.0, 40.0, nd 48.0 months; 3.0, 4.0, nd 4.3 kg, respetively) with HL were enrolled in this study. During period of one month to three dys prior to dmittne ts showed linil signs onsisting of, ut not limited to, lethrgy, norexi, vomiting, nd weight loss. Dignosis of HL during life ws onfirmed with ytologil exmintion of liver fine needle spirtes performed y diplomte in Europen College of Veterinry Clinil Pthology. Lipid profiles Helthy ts, pre- nd post-spying/neutering Plsm level of TAG ws lmost one-nd--hlf-fold lower (P = 0.017) in intt femle ts ompred to intt mle ts (Tle 1). There ws lso signifint differene of the plsm level of TAG etween mles pre nd post neutering, where neutered mle ts hd higher plsm levels of TAG ompred to the intt mles (P = 0.033). In ontrst, intt femle ts hd two-fold higher TAG levels in the liver ompred to intt mles (Tle 1). Due to lrge vrition, this differene did not reh sttistil signifine (P = 0.17). After strtion the TAG liver levels in the femle ts dropped, ut were still higher (ut not signifint), ompred to the TAG levels in strted mles (Tle 1). The Person orreltion nlysis demonstrted the highest rnked orreltions for liver TAG levels with PC 36:4 in oth the liver nd plsm (r =+0.62;P =0.001forliver PC36:4, nd r =+0.540;P = for plsm PC36:4). A signifint positive orreltion (r) of liver TAG levels with liver holesterol esters ws lso oserved (r = +0.59; P = 0.003), ut no signifint orreltion ws found etween plsm nd liver TAG levels (r = 0.19; P =0.39). The totl levels of PC in plsm nd liver did not differ signifintly etween mle nd femle ts, oth efore nd fter spying /neutering (Tle 1). The overll speies profile of PC in plsm ws similr to tht in liver tissue (Fig. 1). The most undnt PC speies were PC34:2 nd PC36:2, oth predominntly ontining linolei id (18:2) in omintion with plmiti id (16:0) (PC34:2) or steri id (18:0) (PC36:2). There were differenes in PC speies profiles etween the mle nd femle ts efore Tle 1 Effet of sex hormones nd spying/neutering on liver nd plsm lipids of helthy ts (mens ± SD) Liver Plsm Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 Totl TAG 1 Mle intt Mle neutered Femle intt Femle spyed Mle intt Mle neutered Femle intt Femle spyed (nmol/mg prot) 51 ± ± ± ± 52 (mmol/l) 0.22 ± ± ± ± 0.05 TAG 56:6/7 2 (%) 0.6 ± ± ± ± ± ± ± ± 0.2 TAG unst/st ± ± ± ± ± ± ± ± 0.4 Cholesterol (rel. Units) 13.1 ± ± ± ± ± ± ± ± 0.6 Cholesterol ester (rel. Units) Totl PC ± ± ± ± ± 8 62 ± ± 6 59 ± 3 (nmol/mg prot) 24.2 ± ± ± ± 2.3 (mmol/l) 0.16 ± ± ± ± 0.03 SM 5 16:0 (% of PC) 3.8 ± ± ± ± ± ± ± ± TAG = triylglyerol 2. TAG 56:6/7 = TAG ontining reltive long unstured yl hins 3. TAG unst/st = the rtio etween TAG with 5 or 6 doule onds versus 2 or 3 doule onds 4. PC = phosphtidylholine 5. SM = sphingomyelin (SM) P < 0.05 mle vs femle (gender) = P < 0.05 intt vs spyed/neutered (time) = P < 0.05 intertion etween gender nd time

5 Vltolin et l. BMC Veterinry Reserh (2017) 13:231 Pge 5 of 9 30 liver M intt F intt M neuter F spyed undne (% of totl PC) Reltive 5 0 PC34:1 PC34:2 PC36:4 PC36:1 PC36:2 PC36:3 PC38:4 PC38:6 PC40:6 30 plsm M intt F intt M neuter F spyed PC) totl 25 (% of 20 undne Reltive 5 0 PC34:1 PC34:2 PC36:4 PC36:1 PC36:2 PC36:3 PC38:4 PC38:6 PC40:6 Fig. 1 Effet of sex (gender), spying/neutering (time) nd their intertion (gender x time) on speies distriution of phosphtidylholine (mens ± SD). The mjor phosphtidylholine (PC) speies were determined in liver iopsies () ndplsm(). PC36:4 nd PC38:4 ontin rhidoni id (20:4). PC38:6 nd PC40:6 ontin dooshexenoi id (22:6). = P <0.05mlevsfemle(gender),=P < 0.05 intt vs spyed/neutered (time), = P <0.05 intertion etween gender nd time nd fter spying/neutering (Fig. 1). The most signifint differenes in PC speies etween femle nd mle ts were with those with n AA (20:4). The sum of the perentge of PC ontining LCPUFA AA, PC36:4 nd PC38:4, in intt femle ts were signifintly (P < 0.01 nd P < 0.002, respetively) higher in oth plsm (21.8 ± 1.6%) nd liver (22.2 ± 2.3%), ompred to mle (plsm: 18.2 ± 1.7%; liver: 17.1 ± 1.4%) (Figs. 1 nd 2). These perentges were lso higher in oth plsm nd liver of intt femle ts were ompred to spyed femle ts (plsm:19.5 ± 2.7%; 20.2 ± 1.2% liver), however the differenes were not sttistilly signifint (P < 0.08 nd P < 0.1 respetively) (Figs. 1 nd 2). The lower levels of AA-ontining PC speies in intt mle ts oinided with higher levels of PC34:1 nd PC36:3, predominntly ontining non-pufas (16:0, 18:1, nd 18:2). Phosphtidylholine speies with nother undnt LCPUFA, i.e. DHA (22:6 n-3), PC38:6 nd PC40:6, did not differ etween intt mles nd femles nd strted mles nd femles. After spying/neutering, the levels of PC36:4 dropped signifintly in femle ts nd inresed signifintly in mle ts (Figs. 1 nd 2), resulting in similr levels of the AA-ontining PCs in mle nd femle ts fter spying/neutering. Helthy ts versus ts with hepti lipidosis The ts with HL hd the sme level of plsm TAG s the helthy spyed/neutered ts, ut n lmost ten-fold higher level of TAG in the liver (P = 0.004) (Tle 2). The levels of AA-ontining PC speies (36:4; 38:4), whih

6 Vltolin et l. BMC Veterinry Reserh (2017) 13:231 Pge 6 of Liver PC 36:4 (% of totl PC) 8 6 Liver PC 36:4 (% of totl PC) 8 6 # # 4 M intt M neutered 4 F intt F spyed Fig. 2 Effet of sex nd strtion on plmitoyl-rhidonoyl-phosphtidylholine (PC36:4). The phosphtidylholine (16:0, 20:4) speies (PC36:4) were determined in liver iopsies from 6 ts per group. Signifint differenes were present in liver PC 36:4 etween mle nd femle ts (gender) nd etween mle ts pre nd post neutering nd femle ts pre nd post spying. Notie how in the mle ts, neutering inreses the PC 36:4 liver vlues. In femle ts, spying dereses the PC 36:4 liver vlues. # intt t hd low estrogen level Tle 2 Comprison of liver nd plsm lipids etween helthy nd ts with hepti lipidosis (HL) Liver Plsm Cts spyed/neutered HL ts Cts spyed/neutered HL ts (n = 12) (n =5) (n = 12) (n =5) Totl TAG 1 (nmol/mg prot) 68 ± ± 277 (mmol/l) 0.22 ± ± 0.25 %TAG 56:6/ ± ± ± ± 1.4 TAG unst/st ± ± ± ± 0.7 Totl PC 4 (nmol/mg prot) 24.1 ± ± 5.0 (mmol/l) 0.16 ± ± 0.04 %PC 4 34: ± ± ± ± 1.9 % PC 34:1 8.2 ± ± ± ± 7.0 % PC 36:4 6.6 ± ± ± ± 2.0 % PC 36:3 8.4 ± ± ± ± 2.3 % PC 36: ± ± ± ± 3.0 % PC 36: ± ± ± ± 2.7 % PC 38: ± ± ± ± 3.2 % PC 38:6 3.8 ± ± ± ± 1.5 % PC 40:6 4.0 ± ± ± ± 2.0 SM 16:0 5 (% of PC) 3.8 ± ± ± ± TAG = triylglyerol 2. TAG 56:6/7 = TAG ontining reltive long unstured yl hins 3. TAG unst/st = the rtio etween TAG with 5 or 6 doule onds versus 2 or 3 doule onds 4. PC = phosphtidylholine 5. SM = sphingomyelin (SM) =P < HL vs helthy ts

7 Vltolin et l. BMC Veterinry Reserh (2017) 13:231 Pge 7 of 9 were found to orrelte positively with the liver TAG levels in helthy ts, were not higher in the HL ts nd were even lower for PC 38:4 in the liver (see Tle 2). Sphingomyelin (SM) (16:0) s perentge of totl PC, ws signifintly higher (two-fold) oth in plsm nd in liver when ompred to the helthy ts. Disussion Butterwik et l. hs reported no importnt differenes in plsm lipid nd lipoprotein onentrtions etween sexully intt femles nd mles or etween sexully intt nd strted mles hve een demonstrted [43]. However, s sexul dimorphism in the lipid metolism hs een demonstrted so lerly in different speies, we wnted to explore this further in ts. In reent study we were unle to demonstrte signifint differenes in liver PC levels etween mle nd femle ts pre- nd post- spying/neutering nd lk of evident influene of sex hormones on the PC synthesis y the phosphtidylethnolmine N- methyltrnsferse (PEMT) pthwy [40]. However, other lipid ftors might e involved if potentil sexul dimorphism in ts exists. To evlute if sexul dimorphism ould still e estlished nd if differenes ould e relted to HL, we evluted in this study the lipid profile in ts in more detil. Intt femle ts hve signifintly lower TAG levels in plsm ompred to their mle ounterprts, similrly to premenopusl women, intt femle rts nd mie [12 15, 18, 19]. However, this differene ws not reversed y spying ut rther enhned due to rise in the plsm TAG level in mle ts fter neutering. Although not signifint possily due to lrge vrition, the liver TAG levels were lower in mle ts ompred to femle ts. The signifintly higher plsm TAG levels in mle ts my e relted to higher rte of polipoprotein B nd VLDL synthesis y heptoytes in mle ts [30, 31], s the TAG levels in plsm most likely reflet the VLDL frtion [44]. Also redued plsm lerne of VLDL triglyeride in mle ts ompred to their femle ounterprts nnot e exluded. Interestingly, in premenopusl women lower onentrtion of VLDL-TAG nd LDL prtiles ompred to men seems to e ssoited with elerted (rther thn redued) VLDL-TAG nd LDL prodution nd inresed plsm lerne [30, 31, 45 48]. Our oservtions wrrnt further studies into gender speifi VLDL seretion/rekdown in ts. The results of the lipidomi nlysis in the ts with HL in this study onfirm findings in previous studies nlysing lipids in liver nd in plsm [49, 50]. We lso oserved tht ts with HL hve signifintly higher TAG levels in their liver. However, in our study ts with HL hd no signifintly different plsm TAG levels ompred to helthy ts, wheres in other studies high plsm TAG levels in ts with HL hve een desried [39, 49, 50]. The seretion of VLDL in HL my prevent lipid umultion in the liver y exporting the surplus TAG nd it n e stimulted y higher levels of TAG in the liver of ts with lipidosis [44]. However, the results in the ts with HL suggest there my e mximum pity of the liver in ts to serete VLDL, so tht no further inrese in plsm VLDL is oserved in ssoition with high liver TAG levels [51]. Intt femle ts hve higher ontent of LCPUFA AA (20:4 n-6) in PC in oth plsm nd liver tissue, when ompred to spyed femle nd the mle ounterprts. This finding is similr to wht hs een reported in premenopusl women, intt femle rts nd mie [12 15, 18, 19]. However, in ontrst to wht hs een reported in these speies, PC levels ontining LCPUFA DHA (22:6 n-3) were not elevted in intt femle ts [18, 21, 52, 53]. Despite this differene, there seems n influene of sex hormones on the profile of PC speies in ts likely s the differenes etween the sexes disppered fter spying/ neutering. In humns, the inresed LCPUFA levels pper to e relted to n oestrogen effet on FA desturse tivity vi inresed expression of Δ5 nd Δ6 desturse [18, 25, 26]. Although ts were elieved to hve low Δ5 ndδ6 desturse tivity [38, 54], in more reent study ts were le to synthetize AA, ut not DHA, from the sustrte γ- linoleni id (18:3 n 6) vi Δ5 desturse, ypssing the Δ6 desturse step [37]. This is supported y findings in this study s the primry inrese in AA ut not DHA levels in the liver of intt femle ts ompred to the mle nd spyed femle ounterprts might lso suggest possile role of Δ5 desturses. In humns nd mie the higher LCPUFA levels my protet ginst the development of HL vi the so-lled fuel prtitioning tion of LCPUFA [55 57]. Long hin polyunsturted ftty ids fvour FA oxidtion over TAG storge nd they diret gluose wy from FA synthesis y filitting glyogen synthesis. However, the n-3 LCPUFA speies (i.e. DHA), rther thn the n-6 LCPUFA (i.e. AA), re minly responsile for these effets vi the tivtion of the peroxisome proliferting reeptors (PPARs) in liver nd dipose tissue [57 60]. The low liver levels of n-3 LCPUFA in the intt femle ts my suggest less resistne to HL ompred to femles of other speies. In ontrst to wht it would e expeted, in the ts with HL in this study, we did not find diret evidene for n inrese in the n-6/n-3 rtio s indeed, the AA-ontining PC 38:4 in ts with HL ws even signifintly lower ompred to helthy ts nd there ws no differene in n-3 LCPUFA etween HL nd helthy ts. In ts with HL lower perentges of the LCPUFA (i.e. AA) in the liver hve een demonstrted efore [49, 50]. Sphingomyelin, memrne lipid primrily present in the memrnous myelin sheth tht surrounds nerve ell

8 Vltolin et l. BMC Veterinry Reserh (2017) 13:231 Pge 8 of 9 xons, nd its reltion in the development of stetosis hs een oserved in different niml models of oesity nd ftty liver [61, 62]. The inresed synthesis of SM in livers ffeted y NAFLD promotes hepti insulin resistne, heptoyte poptosis, n inresed relese of inflmmtory meditors, nd ermide umultion in peripherl tissues [63]. The inresed SM onentrtions, oth in liver nd in plsm, in our ts with HL wrrnts further study in order to understnd the potentil role of SM in feline HL. Conlusions Although some differenes re found, the sexul dimorphism in the hepti nd plsm lipid profile of helthy ts resemles oservtions in other speies with lower plsm TAG levels nd inresed plsm nd liver AA in intt femles ompred to mles. However, the higher plsm nd liver levels of AA ut not DHA ould predispose intt femle ts for HL. Also spying of femle ts my not inrese the risk for HL. In ontrst to previous studies, the higher liver ut not plsm TAG levels in ts with HL ompred to helthy ts ould suggest mximum pity for liver exretion of VLDL. The inresed liver nd plsm SM onentrtion in ts with HL ompred to helthy ts ould indite novel mehnism in the development of HL in ts. Arevitions AA: Arhidoni id; DHA: Dooshexenoi id; FA: Ftty id; HDL: High density lipoprotein; HL: Hepti lipidosis; LA: Linolei id; LCPUFA: Long hin polyunsturted ftty id; LDL: Low density lipoprotein; NAFLD: Non-loholi ftty liver disese; PC: Phosphtidylholine; PEMT: Phosphtidylethnolmine N- methyltrnsferse; SM: Sphingomyelin; TAG: Triglyeride; TC: Totl holesterol; VLDL: Very low density lipoprotein; Δ: Desturse Aknowledgments We thnk Dr. Hns J.C.M. Vernooij for ssistne with the sttistis. Funding This reserh ws finnilly supported y Affinity Petre, Brelon, Spin. Affinity Petre lso supplied the study of the diet tht ws fed to the ts in this study. Avilility of dt nd mterils The dtsets used nd/or nlysed during the urrent study ville from the orresponding uthor on resonle request. Authors ontriutions CV, RHJ, RPF, JHR, ABV, IJ ontriuted to the development nd writing of the pper. RPF, JR performed the liver iopsies. ABV nd MT performed the lipidomis nd the sttistis. AK performed the surgeries. All uthors red nd pproved the finl mnusript. Competing interest The uthor Iselle Jeusette delres to hve ompeting interest with the mnusript y eing urrently employed y the study sponsor. Ethis pprovl The projet ws pproved y the responsile ethil ommittees for the use of lient owned nimls ording to Duth legisltion. Informed owner onsent ws otined prior to enrolment of ll ts. Consent for pulition Not pplile Pulisher s Note Springer Nture remins neutrl with regrd to jurisditionl lims in pulished mps nd institutionl ffilitions. 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