Effects of oxidized vanaspati ghee on the serum lipids. profile and radical scavenging activity of the in vitro lipids of liver, brain and muscles

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1 DERNEĞİ BİYOKİMY DERGİSİ TÜRK TÜRK BİYOKİMY DERNEĞİ DERGİSİ TÜRK BİYOKİMY DERNEĞİ DERGİSİ ORJİNL Türk Biyokimya Dergisi [Turkish Journal of Biohemistry Turk J Biohem] 212; 37 (4) ; oi: 1.555/tj Researh rtile [raştırma Makalesi] Yayın tarihi 3 ralık, 212 TurkJBiohem.om [Pulishe online 3 Deemer, 212] Effets of oxiize vanaspati ghee on the serum lipis 1976 profile an raial savenging ativity of the in vitro lipis of liver, rain an musles [Oksie vanaspati ghee yağının serum lipi profili ve karaiğer, eyin ve kas lipilerinin raikal temizleyii aktivitesi üzerine in vitro etkisi]* 1. ÖRNEK 2. ÖRNEK 1976 lam Ze, rif Mehmoo Department of Biotehnology, University of Malakan, Chakara, Pakistan. BSTRCT Ojetives: The aim of the stuy is to etermine the effets of thermal oxiation of vanaspati ghee on the serum lipis profile an raial savenging ativity of the experimental animals. Methos: Vanaspati Ghee was thermally oxiize at 13 C for 9, 18, 27, 36 an 45 h respetively. The oxiize ghee was fe to the raits. The serum lipis profile was etermine using stanar protools an kits. Raial savenging ativity was etermine with the help of 2,2-iphenyl-1-pirylhyrazyl free raials. Results: It was foun that oxiation inrease formation of hyroperoxies an erease the level of raial savenging ativity. Thermally oxiize ghee was supplie to the raits for one week. The serum lipi profiles showe a ose epenent inrease in triglyerie, total holesterol an LDL-holesterol. No statistially signifiant inrease was oserve in the HDL-holesterol with inrease of oxiation time. Gluose an oy weight ereases signifiantly an was highly orrelate to the serum lipis profile. Raial savenging assay of the lipis extrate from the liver, rain an musles tissues ereases signifiantly. Conlusion: These results showe that thermal oxiation proue oxiize ompouns an the amount of oxiation inrease with inrease of treatment time. The oxiize ghee was prouing signifiant effets in the serum lipis profile as well as peroxiation in ifferent tissue. Therefore oxiation for longer time shoul e avoie. Key Wors: Vanaspati ghee, thermal oxiation, rait, serum lipis profile, raial savenging ativity Conflit of Interest: There is no onflit of interest among the authors to the present stuy, as well as to any person or institution or ageny. Yazışma resi [Corresponene ress] lam Ze Department of Biotehnology, University of Malakan, Chakara, Pakistan Tel: ext lamze1@yahoo.om * Translate y [Çeviri] Dr. Özlem Dalmızrak Registere: 19 Marh 212; epte: 6 ugust 212 [Kayıt Tarihi : 19 Mart 212; Kaul Tarihi : 6 ğustos 212] ÖZET maç: Çalışmanın amaı termal olarak oksie eilmiş olan vanaspati ghee yağının serum lipi profili ve eney hayvanlarına raikal temizleyii aktivite üzerine olan etkisini elirlemektir. Yöntem: Vanaspati ghee yağı termal olarak 13 C e 9, 18, 27, 36 ve 45 saat tutularak oksitlenmiştir. Tavşanlar oksie eilmiş olan yağ ile eslenmişlerir. Serum lipi profili stanart yöntemler ve kitler kullanılarak tayin eilmiştir. Raikal temizleyii aktivite 2,2-ifenil-1-pikrilhirazil serest raikallerinin yarımıyla elirlenmiştir. Bulgular: Oksiayonun hiroperoksit oluşumunu arttırığı ve raikal temizleyii aktiviteyi üşürüğü ulunmuştur. Termal olarak oksitlenen yağ tavşanlara ir hafta oyuna verilmiştir. Trigliseri, total kolesterol ve LDL-kolesterolü içeren serum lipi profiline oza ağlı olarak artış gözlenmiştir. HDL-kolesterol ile oksiasyon zamanı arasına istatistiksel olarak anlamlı ir fark ulunmamıştır. Glukoz ve vüut ağırlığı serum lipi profile ile ilişkili olarak elirgin üşüş göstermiştir. Karaiğer, eyin ve kas okularınan ekstrakte eilen lipilerin raikal temizleyii aktivitesi elirgin şeklile azalmıştır. Sonuç: Bulgular termal oksiasyonun oksie ileşikler ürettiğini ve oksiasyon miktarının muamele süresine parallel olarak arttığını göstermekteir. Oksie yağ serum lipi profilini ve farklı okularaki peroksiasyonu elirgin ir şekile etkilemekteir. Bu neenle uzun süreli oksiasyonan kaçınılmalıır. nahtar Kelimeler: Vanaspati ghee yağı, termal oksiasyon, tavşan, serum lipi profili, raikal temizleyii aktivite Çıkar Çatışması: Yazarlar arasına çıkar çatışması ulunmamaktaır ISSN X (eletroni) (printe)

2 Introution Vegetale oils an fats are main omponents of our iet. The major omposition of these lipis is the triaylglyerols, small amount of holesterol, an various natural antioxiants. During ooking or frying of foos, these triaylglyerols an holesterols forme oxiize ompouns like hyroperoxies, epoxies, hyroxie [1]. These oxiize ompouns proue signifiant hanges in the omposition an properties of fats or oils. For example Betar an Narayanan showe that evelopment of peroxies an epoxies took plae an inrease uring heating of ghee at various temperatures [2]. The hyroperoxies are usually oxiize or egrae to a variety of oxiation prouts. During ooking or frying of foos, these oxiize ompouns are asore into the foos matrix an thus enters human oy. It was estalishe that oxiize fats an oils are toxi [3]. The oxiize fats are signifiane to human health, eause some of these oxiation prouts may e arinogeni [4-5]. These oxiize ompouns proue in the ietary oxiize oils were also foun to enhane the growth of hepato-arinoma [6]. Thus oxiation an formation of oxiation ompouns in oils or fats is hot topi from the inustries to the onsumer. Vanaspati ghee is a vegetale oil-ase prout, wiely use in most of the Southeast sia, an Mile Eastern ountries for ooking an frying of foos an vegetales. In ertain ountries, vegetale ghee may e formulate for general purpose appliations, ooking, frying, an aking [7]. Vanaspati ghee is a omplex lipi onsists of triaylglyerols aout 98% of the total material. Bhanger an Farooq stuie the fatty ai (F) omposition an ontents of trans-unsaturate F (TF) in 34 vanaspati ghee, 11 shortenings an 11 margarine samples from Pakistan [8]. The authors showe that vanaspati ghee ontains , , %; vegetale shortenings , , %; an margarines , , %, of saturate F, is monounsaturate F, an is polyunsaturate fatty ais (PUF), respetively. These authors also showe higher amounts (14.2 to 34.3%) of TF. Ghee also ontains small amount (.15 to.31%) holesterol. Vegetale ghee is also oxiize y heating [9], as well as fluoresent light [1]. However there is lak of literature regaring the effets of thermally oxiize vanaspati ghee on the lipi profile. To the est of our knowlege this paper presents the prime ata on the effets of thermally oxiize vanaspati ghee on the serum lipis an raial savenging potential of the lipis from the liver, rain an musles of the experimental animals. Materials an Methos Materials Fresh vanaspati ghee was otaine from the loal market. Gluose an holesterol was from Sigma lrih (Germany). ll other hemials an reagents were of merian Chemial Soiety (CS) grae from Sigma lrih US or otherwise mentione. Thermal oxiation Vanaspati Ghee was thermally oxiize on hot plat at 13 ± 5 C, for five onseutive ays for 9 h. t the en of eah ay, a representative ghee sample was taken an store in refrigerator at -2 C. Thus the ghee sample was oxiize for 9, 18, 27, 36 an 45 orresponing to eah ay of oxiation. Peroxie value The peroxie value (POV) of ontrol an oxiize ghee samples were etermine using the merian Oil Chemists Soiety (OCS) offiial metho (metho C 8-9) an expresse as meq O 2 /kg of fat [11]. ll samples were measure in tripliate or otherwise mentione. Experimental animals Raits of the loal Himalayan strain were selete for the stuy eause of the highly seletive an evelope organ system an were easily availale animals for these experiments. Raits were groupe y ranom istriution into five groups an eah group has 9 animals irrespetive of the gener. They were plae in same approve animal house faility an fe fresh iets aily. The raits ha free aess to foo an water throughout the stuy. Before starting the treatments, all animals were plae one week ahea to get familiarize with the environment. nimal feeing Raits were lassifie into five treatment groups i.e. 9, 18, 27, 36 an 45 h, whih esignate the iniviual oxiize ghee samples. Eah single treate group was further lassifie into three su-groups esignate as, B an C, whih orrespons to.5, 1. an 1.5 g/ oy wt of aily oses of the oxiize vegetale ghee. Raits were fe for one week an two ays later they were slaughtere. The loo samples were ollete in falon tues an entrifuge for 1 min to otain serum for further analysis. Weight hange The hange in the weight of whole rait was measure with igital platform sale analytial alane (Marino, China) with 3 kg apaity of weighing. Extration of lipis The tissues of eah iniviual rait were taken an store in formalin. Lipis have een extrate from the selete tissues (liver, rain & musles) aoring to the proeure of Folh et al. with little moifiation [12]. Briefly after homogenization of tissues, hloroformmethanol (2:1) mixture was ae. The sample was kept on shaker for 98 h at 3 rpm. To the extrate mixture 1% KCl were ae, washe an filtere. The solvent was evaporate using rotary evaporator. nalytial parameters Biohemial parameters like holesterol, HDLholesterol, LDL-holesterol an gluose were Turk J Biohem, 212; 37 (4) ; Ze an Mehmoo

3 measure using HUMN (Germany) kits, while for total triglyeries DiaSys (Germany) kit was use. The quantifiation was arrie out using six point stanar aliration urves with help of Shimazu UVvis-17 spetrophotometer (Shimazu, Japan). Raial savenging assay Raial savenging assay (RS) of the oxiize ghee, serum an lipis samples extrate from the tissues aoring to the proeure of Lee et al. with some moifiations [13]. Briefly, an ethyl aetate solution of 2,2-iphenyl-1-pirylhyrazyl (DPPH) raials was freshly prepare at a onentration of.1 mm. Five milliliters of.1 mm DPPH solution were mixe with 56 µl oil samples in a 3 ml serum ottle. fter 3 min in ark, the asorane of the sample mixture was measure at 515 nm using a UVvis-17 spetrophotometer (Shimazu, Japan). The RS towar DPPH raials was estimate from the ifferenes in asorane of the DPPH solution with or without sample (ontrol), an the perentage of RS was alulate from the following equation: Raial savenging ativity (%) = (-s/) 1 Statistial analysis ll samples were measure in tripliate or otherwise mentione. Data were analyze y one-way analysis of variane (NOV) an Holm-Siak metho of multiple omparison metho at α =.5 using SigmaPlot for winows version 11. (Systat Software, In, 28). Results an Disussion Deep fat frying is a traitional metho of ooking an is popular throughout the worl for preparation an manufature of nearly all foos [14]. In restaurants, fast foo hains as well as in inustrial frying operations suh as potato hips, instant nooles, fat frying is the main proeure. In Pakistan, eep ghee proesse foo prouts are one of the key hoie of the onsumer from the street foos to the restaurants ue to high prie of vegetale oils. During frying, the ghee is expose ontinuously or repeately to elevate temperatures in the presene of air, moisture an foos. The oxiize ghee or oils thus enters the foo matrix an proue toxi effets [1]. However ue to the asene of literature an knowlege, these foos are eoming one of the most serious health risk fator of the ountry as well as aross the ghee using ountries. This stuy will not ompletely iagnose the relation of risk, ut may serve the preliminary soure of knowlege in this regar. Hyroperoxies ontents Fatty ais or triaylglyerols are oxiize y thermal oxiation to form hyroperoxie. These hyroperoxies are the further oxiize or egrae to seonary oxiation prouts of various hain lengths (1, 15). Peroxie value is the measure of the hyroperoxies forme in fats an oils. Figure 1 shows that peroxie value inrease with inrease of oxiation time with respet to ontrol. The oxiation was statistially signifiant at P <.1. The results are in agreement with Ze & Murkovi who showe that peroxie values inreases with inrease of oxiation time [15]. Similarly hma et al. showe that peroxie value of vegetale ghee inreases with inrease of irraiation oses an fluoresent light respetively [9-1]. This means that thermal stress as well irraiations of vegetale ghee results enhane the formation of hyroperoxies. These hyroperoxies proues seonary oxiation ompouns an thus the quality of the vanaspati ghee ereases an eventually not aeptale to the onsumer. Raial savenging assay of vanaspati ghee The vanaspati ghee manufaturers usually a syntheti vitamin an D, eause uring the ghee manufature a signifiant loss of these natural antioxiants ours. These syntheti antioxiants are important in nutritional as well provie protetion against any possile stress uring storage or ooking [13]. The raial savenging assay is therefore more reasonale parameter to etermine the total antioxiant potential of the ghee. The raial savenging assay of the ontrol (58.%) an oxiize vanaspati ghee shows a signifiant erease an reahe a value of 16.3%. This shows that thermal oxiation release signifiant amount of free raials an loss of antioxiant potential. Figure 1B shows that %RS value was highly orrelate (R 2 =.9445) with the inrease of hyroperoxies ontents. The inrease of hyroperoxies thus ereases the raial savenging ativity. Effets on oy weight Growth is an important parameter for stuying the effet of a speifi iet. Figure 2 showe a signifiant loss of the oy weight with inrease of oxiation time. The loss was statistially signifiant at P <.5 in all three sugroups. Previously it was oserve that vanaspati ghee fe to the rat showe inrease of weight [16]. The present results are the lear piture of the involvement of oxiize ghee in the retention of growth rate an also loss of the oy weight. The main reason may e the oxiation of proteins as well as the isturane of the metaolism of the lipis in the oy. This was onfirme y stuying the orrelation of weight hange, triglyerie an oxiation time as shown in the Figure 2B. It has een oserve that inreasing oxiation time is highly orrelate with hanges in the weight an serum triglyerie ontents. These oxiize lipis ompouns are ontriuting to the enhanement of the free raial reations in the oy an thus hanging the oy weight. Effets on serum lipis The effets of oxiize vanaspati ghee on the serum lipis like triglyeries, holesterol, an high an low ensity lipoproteins were the speial interest of this work. Tale 1 show the effets of thermally oxiize vanaspati ghee on the serum lipis profile of the raits. It has een oserve that at lower feeing ose of, the triglyerie onentrations inreases signifiantly with Turk J Biohem, 212; 37 (4) ; Ze an Mehmoo

4 Figure 1. Figure POV (meq/kg) e Weight Change (g) Group Group B Group C 5 a Time (h) () -6-8 () Time (h) 7 (B) Raial Savenging ssay (%) Triglyeries (mg/l) Weight Change (g) Oxiation Time (h) POV (meq/kg) Figure 1. Charateristis of the thermally oxiize vanaspati ghee, () Peroxie value (meq/kg) of the vanaspati ghee, an (B) Correlation of raial savenging ativity (%) an peroxie value (POV) of the oxiize vanaspati ghee. Figure shows a time epenent inrease of peroxie value, while Figure B shows that erease in the raial savenging is highly orrelate with the inrease in peroxie value. Figure 2. Effets of oxiize vanaspati ghee, () Effets of hange in the oy weight, an (B) Correlation of triglyerie, oy weight hange an oxiation time. There are signifiant hanges in the oy weight of raits with inrease feeing of oxiize samples (Figure ). Figure B shows that the triglyeries are highly orrelate with the hange in the oy weight as well as oxiation time. Tale 1. Raial savenging assay (RS) of the lipis extrate from the liver, rain an musles of the rait. Conentration (mg %) Sample Dose Oxiation Time (h) Control ± ± ± ± ± 7.2 Triglyerie B 59.2 ± ± ± ± ± ± 1.2 C 43.2 ± ± ± ± ± ± ± ± ± ± 7. Total Cholesterol B 66.6 ± ± ± ± ± ± 7.4 C 84.5 ± ± ± ± ± ± ± ± ± ±.9 HDL-holesterol B 45.4 ± ± ± ± ± ± 1.3 C 56.2 ± ± ± ± ± ± ± ± ± ±7.2 LDL-holesterol B 21.5 ± ± ± ± ± ± 7.3 C 28.4 ± ± ± ± ± 4.2 Values are the means of tripliate (n=3) reaings. Turk J Biohem, 212; 37 (4) ; Ze an Mehmoo

5 relative value of 12%, 14%, an 36% at 18, 27 an 45 h, while no signifiant hange was oserve at 9 an 36 h. While at higher oses suh as B & C, the amount was initially ereases (1 to 16%) an then a signifiant inrease (aout 46 an 2%) our with the inrease of oxiation time. Kumar et al. showe that feeing heate esi ghee prepare from milk signifiantly lowere the triglyeries ontents of the experimental animals [17]. This is ue to the ifferene in the soures of ghee (animal & plant), oxiation level, an animals. Cholesterol is the omponent of ell memrane an soure of vitamin D. The total holesterol ontents inrease signifiantly (P <.5) at the oses of & B with inreasing oxiation time. While in the ase of ose C, it was inrease y 18% initially an reahe an inrease of 35% on 45 h sample. In the ase of HDLholesterol there were inrease in the total amount, ut the inrease was not signifiant in all three oses. While in the ase of LDL-holesterol there was signifiant (P <.5) inrease ue to the inrease of oxiation time. Figure 3 shows that the hyroperoxies are highly orrelate with the LDL-holesterol an HDLholesterol. The inrease of LDL-holesterol was positively orrelate with hyroperoxies. The present results for HDL-holesterol is in aorane with the Kumar et al. who showe that feeing heate ghee at 5% level proue no signifiant hange in the HDLholesterol, while LDL-holesterol was roppe y 32%, whih is inverse to our finings [17]. The main reason may e the presene of trans-fatty ais in the vanaspati ghee [8]. The foos are usually roaste in vanaspati ghee in most of the ases in Pakistan, is thus ausing inrease in the fatty sustanes an oesity. Reently vanaspati ghee was foun to inrease LDL-holesterol an thus onsier harmful [18]. Figure 4. Effets on gluose ontent Sine gluose is one of important arohyrate highly linke with the metaolism of lipis. During glyolysis gluose is onverte to pyruvate. The earoxylation of pyruvate proues aetyl Co. This ompouns has many possile pathways, the most important is the onversion to fatty ais, eventually triglyeries, holesterol an many more lipis sustanes [19]. Thus it is essential to etermine gluose ontent uring the stuy of in-vivo lipi metaolism or lipi relate speies. Figure 4 shows serum gluose onentration was signifiantly negative orrelate with the inrease of oxiation time. The lower gluose onentration an e one of the auses of inrease in the ontents of triaglyeries, holesterol, an LDL-holesterol. Figure 4B shows that erease in gluose is highly orrelate (R 2 =.8827) with the hange in the oy weight. Effets on raial savenging ativity of lipis Free raials an other reative oxygen ontaining ompouns are usually generate y exogenous hemials or enogenous metaoli proesses in foo systems or in the human oy. These raials may ause oxiative amage an onsequently plays a POV (meq/kg) Gluose (mg/l) Weight hange (g) () (B) Time (h) Group Group B Group C Gluose (mg/l) Figure 4. Effets of the thermally oxiize vanaspati ghee, () Effets on serum gluose (mg/l) of the rait, an (B) Correlation of serum gluose (mg/l) an weight hange of the rait inue y oxiize vanaspati ghee. Derease in the oy weight is highly epenent on the erease in the serum gluose ontent. 5 HDL-C (mg/l) LDL-C (mg/l) Figure 3. Correlation of peroxie value (POV) of oxiize ghee, serum HDL-holesterol an LDL-holesterol values. The inrease in the POV values is highly orrelate with the hange in the serum HDL-holesterol as well as LDL-holesterol. Turk J Biohem, 212; 37 (4) ; Ze an Mehmoo

6 signifiant pathologial role in some hroni iseases like atheroslerosis, aner an arthritis [2-21]. In orer to overome this, ingestion of foos ontaining antioxiants suh as asori ai, toopherols an arotenois may reue the oxiative amage. However, thermal oxiation was foun to egrae an estroy these antioxiants [22-24], thus ereasing the raial savenging ativity. Tale 2 shows the effets of thermally oxiize vanaspati ghee on the raial savenging ativity assay of the lipis extrate from tissues of the liver, rain an leg musles. Signifiant reution in the RS value has een oserve in the liver tissue with inrease of oxiation time from 9 h to the 45 h. In the ase of rain an musles tissues, a small initial reution of aout 2-6% an 3-6% respetively in the RS was oserve. Further erease was oserve in oth ases with inrease of oxiation time. The results showe that oxiize lipis reahe liver easily an affet its antioxiant potential than rain an musle. This also shows that the erease ours in the antioxiants level or the inrease of reative speies in these tissues may e the main ause of reution in the RS values. The reution in RS is therefore one of the alarming point. Conlusion The effets of thermally oxiize vanaspati ghee on the serum lipi profile an antioxiant ativity of the lipis extrate from the ifferent tissues were oserve. Ghee was thermally oxiize for 9 to 45 h. It was foun that thermal oxiation inrease the formation of hyroperoxies an erease the level of raial savenging ativity. Thermally oxiize ghee was given to the raits for one week. There were signifiant hanges in the lipis profile of the animals. Triglyeries, holesterol an LDL-holesterol were inrease with inreasing oxiation time. No statistially signifiant hanges were oserve in the HDL-holesterol with inrease of oxiation time. Gluose an oy weight ereases signifiantly an was highly orrelate to the serum lipis profile. Raial savenging assay of the lipis extrate from the liver, rain an musles tissues ereases signifiantly. These results showe that thermal oxiation an use of thermally oxiize vanaspati ghee are harmful an it is suggeste that longer oxiation shoul e avoie. knowlegment We are grateful of yaz li Khan, ssistant Professor, Wahee ur Rehman, BS stuent an Hussain Mohamma, La ssistant, Department of Biotehnology, for their help uring animal stuy an analysis. The finanial support for this stuy is kinly provie y the University of Malakan (grant vie letter # UOM/Fin/11/227). Referenes [1] Kamal-Elin, Makinen M, Lampi. The hallenging ontriution of hyroperoxies to the lipi oxiation mehanism. In (E. Kamal-Elin ) Lipi Oxiation Pathways 23; pp. 1-35, OCS Press, Champaign, Illinois. [2] Betor BS, Narayanan KM. Effet of thermal oxiation on the physio-hemial onstants of ghee. Inian J Dairy Si 1974; 27(2):9-93. [3] Frankel EN, Smith LM, Hamlin CL, Creveling RK, Cliffor J. Ourrene of yli fatty ai monomers in frying oils use for fast foos. J m Oil Chem So 1984; 61(1):87-9. [4] Yang CM, Kenall CW, Stamp D, Meline, rher MC, et al. Thermally oxiize ietary fat an olon arinogenesis in roents. Nutr Caner 1998; 3(1): [5] Jurek D, Uilova N, Jozkowiz, Nohl H, Marian B, et al. Dietary lipi hyroperoxies inue expression of vasular enothelial growth fator (VEGF) in human oloretal tumor ells. FSEB J 25; 19(1): [6] Rohr-Uilova NV, Stolze K, Sagmeister S, Nohl H, Shulte-Hermann R, et al. Lipi hyroperoxies from proesse ietary oils enhane growth of hepato-arinoma ells. Mol Nutr Foo Res Tale 2. Raial savenging assay (RS) of the lipis extrate from the liver, rain an musles of the rait. Raial Savenging ssay (%) Sample Dose Oxiation Time (h) Control Liver B 42.7 a e C e 15.7 f Brain e 22.2 B 44.4 a e C 42. a e Musles a B 45.8 a 41.7 a e C e Different letters (a-f) in the row represent signifiane at p <.5. Turk J Biohem, 212; 37 (4) ; Ze an Mehmoo

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