DIETARY FOOD FORTIFIED WITH OROTIC ACID AND LIVER FUNCTION

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1 MAKARA, SAINS, VOL. 15, NO. 2, NOVEMBER 211: DIETARY FOOD FORTIFIED WITH OROTIC ACID AND LIVER FUNCTION Yohnes Bung Deprtment of Chemistry, Fculty of Science nd Engineering, Nus Cendn University, Kupng 851, Indonesi E-mil: Astrct The effects of dietry food fortified with orotic cid (1.%) on liver function were studied in rts. The rts fed with orotic cid promoted liver triglyceride content mrkedly, tht ws 5-fold higher thn tht of the control. The liver mlondildehyde (MDA) content incresed y 1%, ut the gluthtion peroxidse (GSH-Px) ctivity decresed y 5%. The serum sprtte minotrnsferse (AST) nd lnine minotrnsferse (ALT) ctivities incresed y 25% nd 3%, respectively. Therefore, the decresed GSH-Px ctivity ws ssocited with the promotions of AST, ALT, nd the liver MDA levels. In conclusion: dietry orotic cid promotes lipid peroxidtion ut reduces the rte of the ntioxidnt enzyme. Therefore, dietry food fortified with orotic cid ttenutes the liver function. Keywords: niml model, liver function, stetoheptitis 1. Introduction Liver is known s the centrl orgn of metolism providing numerous metolic intermedites/precursors used in vrious enzymtic pthwys throughout the ody. The gluconeogenetic metolism, for exmple, occurs in liver, ut its precursors cn come from extrheptic tissue nd its product, glucose, cn e used to provide cellulr energy metolism throughout the ody. Hence, the glucose, in the liver, cn e synthesized from glucogenic mino cids from extr heptic tissue, such s muscle. This mechnism is useful when glucose is the primry energy resource molecule. The energy needed y the some cells of humn ody, such s rin nd erythrocytes, for exmple, comes only from glucose. Furthermore, in poison, the liver provides specific metolic pthwy to detoxify the toxins known s glucuronidtion pthwy [1]. The glucuronidtion pthwy plys role in removing nonpolr molecules (toxins) nd excreting them into the urine. The liver is lso known s the min orgn of lipid metolism [2], minly in nolic metolism. The liver converts glucose to ftty cid s well s to ft (triglyceride). Overll, liver is viscerl orgn regulting metolic functions throughout the ody. Most of the chemicls grouped s nutrients cn enter nd e known y the cellulr metolic pthwys. Although ech nutrient hs mjor functions in prticulr metolic rections, most of those nutrients re interchngle. The glucose, for exmple, is the min source of energy for the metolic system; however, it cn lso serve s structurl molecule [1]. The nutrients cn only e metolized optimlly in certin quntity. Nutrients, therefore, cn e ctegorized into mcro- nd micro-nutrients. The mcronutrients re needed in lrge mounts y the metolic systems. The micronutrients, however, re needed in smll mounts. Severl micronutrients, such s minerls, vitmins, the intermediry metolic system, s well s orotic cid, ply essentil roles in homeosttic mechnisms. The orotic cid is well known s n intermedite of pyrimidine nucleotides iosynthesis. Severl studies hve reported tht dietry orotic cid tend to induce ftty cid iosynthesis ccompnied with promotion of triglyceride iosynthesis in liver cells [3-4]. The promotion of triglyceride iosynthesis which is not equivlent with the secretion nd the degrdtion cuses heptic stetosis [3-5]. The ppernce of the heptic stetosis is often ccompnied with n increse of free rdicl production tht is not equivlent with their rte of use y the defense system. The ltter condition cn reduce the liver function. Biomrkers indicting decrese of liver function hve een reported elsewhere [6-9]. Severl of them re the finl products of lipid peroxidtion known s mlondildehyde (MDA), sprtte minotrnsferse (AST), nd lnine minotrnsferse (ALT), nd the gluthtion peroxidse (GSH-Px). The AST nd ALT re two enzymes found in serum, in which their incresing level s well s MDA indicte injury to the liver cells nd vice vers. Our previous studies [3-5] found tht dietry orotic cid induced ftty liver; however, the effects of liver function 11

2 12 MAKARA, SAINS, VOL. 15, NO. 2, NOVEMBER 211: induced y orotic cid hve not een elucidted yet. Therefore, the present study ws conducted to elucidte the effects of dietry orotic cid on liver function using Sprgue-Dwley (SD) rts s niml model. 2. Methods Animls, diets, nd experimentl design. All spects of the experiment were conducted ccording to the guidelines provided y the ethicl committee of experimentl niml cre t Sg University (Sg, Jpn). Mle SD rts ged 5 weeks were housed individully in n ir-conditioned room (24 o C) with 12-h light/drk cycle. After 1-week dpttion period, rts were ssigned to two groups (five rts ech). Control diet (s control group) ws prepred ccording to recommendtions of the Americn Institute of Nutrition (AIN), contining (in weight %) 2 of csein, 1 of sfflower oil, 1 of vitmin mixture (AIN-93), 3.5 of minerl mixture (AIN-93),.2 of choline itrtrte,.3 of DL-Methionine, 5 of cellulose, 15 of α- cornstrch, nd sucrose to mke 1. The orotic cid diet (s orotic cid group) ws prepred y supplementing 1.% orotic to the control diet t the expense of sucrose. The nimls received the diets for 1 dys. On dy 11, rts were killed y decpittion fter 9-h strvtion. Livers were excised immeditely, nd serum ws seprted from lood. Anlyses of liver nd serum lipid contents. Liver lipids were extrcted nd the concentrtions of triglyceride nd phospholipids were mesured y the methods used in our previous study [4]. The totl cholesterol in liver tissues nd the serum triglyceride, phospholipids, nd cholesterol contents were mesured using enzyme ssy kits from Wko Pure Chemicls ccording to the mnufcturer s instructions. Assys of MDA contents. The MDA content of the liver tissue ws determined ccording to method reported y Lykkesfeld [1]. The liver protein content of liver tissue ws determined ccording to the Lowry s method s reported in previous study [4]. Assys of the AST nd ALT enzymes. The AST nd ALT ctivities were mesured using enzyme ssy kits ccording to the mnufcturer s instructions. Preprtion of liver sucellulr frctions nd ssy methods of GSH-Px. The mitochondri of liver sucellulr frctions were prepred s previously reported y Ngo et l. [11]. The GSH-Px ctivities were determined ccording to the method reported y Torres et l. [12] with slight dpttion. The regent mixture (regent I) ws 645 μl of phosphte uffer 5 mm contining 5mM EDTA nd M NN 3 (ph 7.); 25 μl of GSH.15 M (regent II), nd mitochondri which contined.24 mg of protein. These regents were llowed to rech equilirium t 2 o C. The rection ws strted y the ddition of 25 μl of H 2 O M nd evluted for 5 min t λ 34nm. The non-enzymtic oxidtion of GSH ws done y replcing mitochondril protein with H 2 O. The differences of the sornce etween the enzymtic nd the nonenzymtic oxidtion of GSH would indicte the rtes of NADPH molecule oxidtion. Sttisticl nlyses. Dt were nlyzed y one-wy nlysis of vrince, nd ll differences were inspected y Duncn s new multiple-rnge test using SPSS sttisticl softwre. 3. Results nd Discussion Dietry food fortified with orotic cid reduces growth of ody ut promotes liver weight. The consequences of food intke generlly induce growth; however, some disese might distur this pthwy. Growth mens the individul orgnism growing orgniclly, gining weight of the ody nd weights of numerous orgn of the ody. The growth dt is found y weighing the odies s well s the orgns, such s liver. The ltertions of those growth fctors indicte the effects of food intke. In order to eliminte the devitions cused y the different quntities of food consumed, the differentition in the mount of food intke is firstly cnceled through pired-fed procedure. The pired-fed food intke implicted similr quntities of mcronutrients, vitmins, nd minerls ingested. The replcement of portion of dietry energy, sucrose (mcronutrient), y orotic cid did not influence the homeosttic metolism ecuse the dietry energy prepred ws in excess. Therefore, the effects of food quntity on the tretment hve een firstly cnceled in the dietry design. Severl growth prmeters determined in present study re descried in Tle 1. The tle shows tht the verge dily food intke etween the groups were lmost similr. However, it ws found tht the finl ody weight of the rts in orotic cid group decresed slightly. Hence, the decresed ody weight s Tle 1. Dietry Orotic Acid Promotes Liver Weight Groups Control Orotic cid Initil BW* (g) 297. ± ± 9. Finl BW* (g) 344.± ±13. Food intke (g/dy) 24.4±.7 24.± 1. Liver weight (g/1g BW) 4.4±.2 5.8±.3 Vlues re expressed s men± SEM of five rts. Clerly define & regrding difference of significnce t p <.5. *BW, ody weight

3 MAKARA, SAINS, VOL. 15, NO. 2, NOVEMBER 211: ccompnied with n enhncement of the liver weight (p <.5). The weight of the liver ws 32% higher thn tht of the control group. It cn e concluded tht orotic cid diet induces growth of the liver with slight reduction of the ody weight. Dietry orotic cid promoted triglyceride content in liver. Liver cells, unlike the dipose, hve limited cpcity for lipid storge. The lipid compositions contined in liver orgn re reported s triglyceride, phospholipids, nd cholesterol (Fig. 1). Both ltter lipids re involved in structurl lipids, the min lipids of cell memrnes. The deposited lipids in liver tissue re minly triglyceride nd dipose tissue. The enlrgement of liver weight induced y dietry orotic cid (Tle 1) might indicte n excessive ccumultion of triglyceride in tht orgn. As shown in Fig. 1, the liver triglyceride levels of the orotic cid group were 5-fold higher thn tht of the control group. The liver triglyceride contents of the control nd the orotic cid groups involved 7.5% nd 17.9% of the liver weights, respectively. The liver cholesterol lso incresed, ut it filed to rech significnt levels (p <.5). The liver phospholipids, however, decresed significntly (p <.5). Becuse lipid storge in liver tissue contins of minly triglyceride, the enlrgement of the liver weight, therefore, might refer to their triglyceride contents in those orgns. Sherlock nd Dooley [13] report tht the excess ccumultion of lipids in the liver with cut-off level exceeding 5-1% of the liver weight is known s simple ftty liver or heptic stetosis. Bsed on this rule, therefore, dietry orotic cid develops heptic stetosis. Dietry orotic cid promotes lipid peroxidtion ccompnied with reduction of the ntioxidnt defense system. The ftty liver induced y orotic cid is included in the term of non-lcoholic ftty liver. Recently, the non-lcoholic ftty liver is the primry disese of the liver, known s the non-lcoholic ftty liver disese. The ftty liver t the stetosis level does not cuse injury to the liver. However, t further stges, Liver TG (mg/g liver) Liver PL (mg/g liver) Liver Cholesterol (mg/g liver) Control Orotic cid Control Orotic cid Control Orotic cid Vlues re expressed s men± SEM of five rts. They clerly define & regrding difference of significnce t p <.5. Arev. TG, triglyceride; PL, phospholipids. Fig.1. The Effects of Dietry Orotic Acid on Liver Lipid Levels the stetosis genertes stetoheptitis s well s nonlcoholic stetoheptitis (NASH). The NASH indictes the ftty liver ccompnied with the inflmmtion of the tissues [15]. The enhncement of the NASH levels might induce poptosis. This ws ecuse the triglyceride ccumultion ctivtes the mitochondril poptosis [14]. The swellings nd pin re the types of responses of the helthy cells to the inflmmtion, locl cse of results of injuries. The inflmmtion cused y the stetosis is prtilly resulted y the excessive lipid ccumultion in liver cells. The high lipid droplet level in cytosolic cellulr frctions might interrupt its homeosttic cellulr equilirium nd e possile to reduce spce nd pull the neighoring cells to induce inflmmtion. The incresed degrees of inflmmtion re prtilly ssocited with promotion of ftty cid peroxidtion. The peroxidtion of hydrocron chins of the ftty cid on structurl lipids cn occur in overcpcity of ntioxidnt defense systems in consumption of the generted lipid peroxide. Therefore, the increse of uncontrolled lipid peroxidtion of the tissues increses the inflmmtion. The uncontrolled inflmmtion of the liver tissue cn cuse firosis nd even cirrhosis. Hence, the heptic stetosis ccompnied with the inflmmtion hs two ctegories: lcoholic stetoheptitis nd NASH. Severl uthors report tht the stetosis tht ws developed to the stetoheptitis level cn only cuse reductions of the liver functions [11,15-16]. Therefore, the stetosis induced y orotic cid, if it reches stetoheptitis level, is importnt in determining the risk fctors of the inflmmtions. As shown efore, the risk fctors of inflmmtions include the incresed mgnitudes of MDA, reduced GSH-Px levels, nd promoted AST nd ALT levels. The liver s MDA level slightly incresed lthough it filed to rech significnt levels (p <.5; Fig 2A). Becuse MDA is the finl product of the lipid peroxidtion, this result, therefore, indictes tht the lipid peroxidtion is promoted y the orotic cid Lipid peroxidtion (pmol MDA/mg protein) GSH-Px ctivities (nmol/min/mg protein) Control Orotic cid Control Orotic cid (A) (B) Vlues re expressed s men± SEM of five rts. They clerly define & regrding difference of significnce t p <.5 Fig. 2. The liver MDA Contents nd GSH-Px ctivities

4 14 MAKARA, SAINS, VOL. 15, NO. 2, NOVEMBER 211: The incresed liver MDA level indictes tht rte of peroxidtions on hydrocron chins of the ftty cids in metolic system ws stimulted y the orotic cid t present concentrtions (1.%). Presumly, the incresed rte of the peroxidtion onto the hydrocron chins of the ftty cids ws relted with the promotions of the ftty cid contents, which re sustrtes of the triglyceride iosynthesis, ecuse the liver triglyceride content incresed (Fig. 1). This is in greement with the dt reported in previous studies [3-5]. It is possile tht in first stge of the peroxidtion, oth enzymtic nd non-enzymtic rections, the mono-dehydrogention occurs first. The mono-dehydrogention rections deliver mono-rdicls of the mcromolecules. Secondly, the interctions etween the two mcromolecule mono rdicls develop swelling s well s inflmmtion in situ. These events cn induce the dmging of the mcromolecules s well s the structurl lipids nd the functionl proteins. The formtion of the mcromoleculr mono-rdicls, prticulrly on the protein enzymes, cn cuse enzyme mlfunction. One of the enzymes tht ply essentil roles to ttenute the peroxidtion is GSH-Px. This enzyme ctlyzes conversion oth of lipid peroxide (ROOH) into lcohol (ROH) nd the hydrogen peroxide (H 2 O 2 ) to generte H 2 O [8,17]. The incresed GSH-Px ctivity promotes levels of the helthy cells nd vice vers. As shown in Fig. 2B, the GSH-Px ctivities decresed significntly (p <.5). This result indictes tht the promotion of lipid peroxidtion (Fig. 2A) is resonle with the decresed GSH-Px ctivities. The decresed GSH-Px ctivities in orotic cid treted rts re in greement with the report of Aoym et l. [18] tht dietry orotic cid reduces superoxide dismutse enzyme ctivities, in which the ltter enzyme converses rective oxygen species to generte O 2 nd H 2 O 2. Overll, the most importnt mechnism of liver cell injury y the orotic cid involves the formtion of the rective free rdicls (GSH-Px decresed significntly) nd susequent lipid peroxidtion (MDA incresed slightly). Dietry orotic cid promotes heptotoxicities. The promotion of MDA level in liver tissue ccompnied with the reduction of the GSH-Px ctivities indictes tht dietry orotic cid induces lipid peroxidtive degrdtion of iomemrnes. Bruss et l. [16] report tht the injuries of the liver cells induce secretions of AST. The AST is known s the enzyme involved in gluconeogenetic pthwys, in which its ctivities re controlled y the glucocorticoid nd glucgon hormones. The ALT is lso one of the enzymes involved in injuries of the liver cells [6,11-12]. Both enzymes cn e determined y their ctivities in loodstrem. The ctivities of the AST nd ALT found in serum incresed significntly (p <.5; Fig.3). This result indictes tht secretion of oth protein enzymes into the AST ctivities (Krment unit) Control Orotic cid (A) ALT ctivities (Krment unit) Control Orotic cid (B) Vlues re expressed s men ±SEM of five rts. Clerly define & regrding difference of significnce t p <.5 Fig. 3. The AST nd ALT Activities loodstrem incresed. This result is in greement with the reports of Edgr et l. [7] tht the AST nd ALT levels in serum incresed in stettoheptis ptients. Therefore, the chnges of these AST nd ALT levels (Fig. 3A-B) re resonle with the ltertions of the MDA nd GSH-Px levels (Fig. 2A-B). In conclusion, these results indicte tht the heptostetosis induced y orotic cid cuses heptotoxicity. 4. Conclusion The promotion of liver MDA levels ccompnied with the reduction of GSH-Px ctivities, one of the ntioxidnt enzymes in defense system, suggests tht dietry orotic cid cuses inflmmtion nd injuries to the liver. Although some other importnt fctors filed to determine in present study (such s tumor necrosis fctor-α, interleukin-1, nd interleukin-6), the injuries of the liver tissue re resonle ecuse the AST nd ALT levels in the serum incresed. The inflmmtion of the liver cells, therefore, might reduce nd even shift the liver function. Acknowledgement The uthor would like to express high pprecition for the suggestions nd the continued encourgement from Dr. Teruyoshi Yngit, professor t Sg University, nd the excellent ssistnce from Dr. Koji Ngo in enzymtic determintions s well s the useful ssistnce from Dr. Yu-Ming Wng in hndling instruments nd nimls. The uthor lso wishes to give credit to the Jpnese Monukgkusho for providing the fund for the reserch. References [1] A.L. Lehninger, D.L. Nelson, M.M. Cox, Principles of Biochemistry, 2 nd ed., Worth Pulishers Inc., New York, 1993, p.113.

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