Characteristics of divalent minerals extracted from liver of Japanese common squid Todarodes pacificus under various experimental conditions

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1 Fish Si (213) 79: DOI 1.17/s ORIGINAL ARTICLE Chemistry n Biohemistry Chrteristis of ivlent minerls extrte from liver of Jpnese ommon squi Toroes pifius uner vrious experimentl onitions Joko Sntoso Yuk Ishizuk Yumiko Yoshie-Strk Reeive: 23 My 212 / Aepte: 26 Otoer 212 / Pulishe online: 18 Jnury 213 Ó The Jpnese Soiety of Fisheries Siene 213 Astrt In reltion to the prolem of how to utilize the wste from squi proessing, we etermine the proximte n minerl ompositions of liver of the Jpnese ommon squi Toroes pifius, n hrterize the ehvior of ivlent minerls, fousing in prtiulr on mium removl. The squi liver ontine ft (44. g/1 g ry mtter), protein (13.5 g/1 g ry mtter), n sh (2.11 g/ 1 g ry mtter). It lso ontine the mrominerls soium, potssium, mgnesium, n lium, s well s the tre minerls iron, zin, mium, n opper. Low- n high-ph tretments (ph 2 n 12) remove the mium entirely, ut the tre minerls zin, iron, n opper, s well s solule protein, whih re ll importnt for proper funtioning of the humn orgnism, were lso remove in lrge quntities. High perentges of solule minerls n solule protein were foun in the low moleulr weight (MW \ 1,) frtion. The soluility of zin n opper were strongly relte to the soluility of mium, ut there ws no reltionship etween the soluilities of mgnesium n mium. On the other hn, while tretment with either.9 % NCl or 1 mm phosphte uffer (ph J. Sntoso Deprtment of Aquti Prouts Tehnology, Fulty of Fisheries n Mrine Siene, Bogor Agriulturl University, Cmpus IPB Drmg, Bogor 166, Inonesi Y. Ishizuk Deprtment of Foo Siene n Tehnology, Fulty of Mrine Siene, Tokyo University of Mrine Siene n Tehnology, Konn Minto-ku, Tokyo , Jpn Y. Yoshie-Strk (&) Deprtment of Foo Life Siene, Fulty of Life Siene, Toyo University, Itkur, Gunm , Jpn e-mil: yumiko_y@toyo.jp 7.4) lso remove the mium ompletely, it left some useful ompouns (tre minerls n solule protein) in the soli prt, so these tretments my represent pproprite methos for removing mium from squi proessing wste. Keywors Cmium Liver of squi Low moleulr weight Minerls Soluility Solule protein Introution Cephlopos inluing uttlefish, squi, n otopuses re importnt mrine resoures, s they re rih in tste n hve few ineile prts [1]. They hve eome the preferre sefoo in Jpn, n re eten rw (e.g., in sushi n sshimi), ooke (e.g., grille n oile), n s vrious proesse prouts (e.g., s rie sheets n from ns). Although ephlopos hve only few ineile prts, wste prts from the proessing of this sefoo, suh s heptopnres, liver, mntle, n igestive glns, hve eome serious prolem ue to high onentrtions of hevy metls in them, espeilly mium. The use of iomss n orgni wstes s potentil soures of mterils n energy hs reently inrese ue to the proution of novel vlule ompouns n the introution of new environmentl poliies [2]. Squi wste ontins importnt sustnes suh s minerls [3 6], proteinse enzyme [7 9], n orgni is [3]. Therefore, there is high possiility tht vlule resoures n e extrte from squi wste suh s squi liver. Cephlopos re well known to hve the ility to umulte not only the elements tht re essentil for their metolism ut lso nonessentil elements in high onentrtions [1 13]. Cmium is toxi hevy metl tht

2 294 Fish Si (213) 79: n umulte in the internl orgns of ephlopos, suh s the migut gln, liver, gills, n kiney. In the Jpnese ommon squi Toroes pifius, the onentrtion of mium in liver ws reporte to e mg/kg fresh weight [14], while nother report mentione tht the mium onentrtions in liver were n mg/kg ry weight in femles n mles, respetively [6]. The livers of the squi speies Loligo oplesens, Ommstrephes rtrmii, n Sympletoteuthis oulniensis ontine 85., 782, n 287 mg of mium/ kg ry weight, respetively [15]. The mium ontent of squi liver epens on ftors suh s the onition of the environment or wter s well s the sex, size, n ge of the squi. However, s mentione ove, squi liver lso ontins some useful ompouns. Therefore, there is the potentil to tke vntge of fvorle onstituents y eliminting omponents tht re hrmful, suh s mium. There hve een mny ttempts to reue the mium ontents of intestinl orgns of ephlopos vi severl tretments, inluing physil, miroiologil, n hemil methos s well s omintions of them [2, 16 18]. However, the tretments n lso ffet other omponents tht re enefiil to the humn orgnism, espeilly minerls tht hve the sme vlene s mium, s well s protein n ft. Therefore, efore pplying ny tretment to ephlopo proessing wste, it is very importnt to know the hemil profile of the wste n the soluilities of minerls present in it, fousing in prtiulr on the effetive removl of mium. A lrge mount of solvent my e require to flush out mium, whih my then eome miumontining wste solvent. Ghimire et l. [18] reporte the removl of toxi minerls y pssing the wste solvent through gel. As first step in the frtiontion of useful ompouns from wste smple, we fouse on the soli prt of the smple. We will ress the wste solvent in the next step of our reserh in this re. Severl stuies hve revele tht the ontents, soluilities, n ioessiilities of minerls in ephlopo proessing wste re influene y severl ftors, suh s the ph [19, 2], the presene of other ompouns tht t s helting gents with enhning or n inhiiting tions [21, 22], n the tretment provie (suh s heting or ooking) [19, 23, 24]. Moreover, tre minerls in ephlopos n lso e oun with protein in low, intermeite, or high moleulr weight frtions [5, 25]. In orer to etermine how to est utilize squi wste, we etermine the proximte n minerl ompositions of liver of the Jpnese ommon squi Toroes pifius n evlute the soluilities of ivlent minerls n solule protein uner ifferent onitions, fousing in prtiulr on the removl of mium. Mterils n methos Chemils n regents The hemils n regents use in this experiment (nitri i, stnr minerl solution, hyrohlori i, soium hlorie, n soium hyroxie) were of nlytil gre n otine from Wko Pure Chemil Inustries Lt. (Osk, Jpn). A Bio-R protein ssy kit ws otine from Bio-R (Rokfor, IL, USA). Bovine serum lumin ws otine from Sigm Alrih Jpn Co. (Tokyo, Jpn). Colletion of the squi n squi liver pretretment Jpnese ommon squi Toroes pifius were pture off Kyonn (Tokyo By, Jpn) in Septemer 28. Twelve iniviuls with mntle lengths of 2 3 m n weights of 2 5 g were use in this experiment. Squi livers were tken from fresh squi. They were wshe with tp wter, wipe with pper towels, mine to otin homogente using foo proessor (MK-K75, Mtsushit Eletri, Co., Osk, Jpn), n store t -3 C until use. All experiments were performe within week fter fresh liver proessing. Proximte nlysis Squi liver (wet smple) ws foun to onsist of moisture, sh, ft, n protein (nitrogen onversion ftor of 6.25) using metho from the AOAC [26]. Totl minerl nlysis As isusse in our previous report [27], the smples (of squi liver) were trete with the wet shing metho using nitri i. The estroye smples were issolve in 1 % hyrohlori i n then nlyze using n tomi sorption spetrophotometer (moel AA-66, Shimzu Co., Kyoto, Jpn). Soium (N, etetion limit.5 ppm), potssium (K, etetion limit.5 ppm), mgnesium (Mg, etetion limit.1 ppm), lium (C, etetion limit.4 ppm), iron (Fe, etetion limit.6 ppm), zin (Zn, etetion limit.5 ppm), mium (C, etetion limit.7 ppm), n opper (Cu, etetion limit.4 ppm) were mesure. Stnr solutions were prepre in 1 % hyrohlori i. All glsswre n plsti ottles use were ippe in ontminon (Wko Pure Chemil Inustries, Lt., Osk, Jpn) for t lest 2 h n then rinse with Milli-Q wter to remove ontminnts.

3 Fish Si (213) 79: Determintion of solule minerls n solule protein t ifferent ph vlues Following the metho esrie in our previous report [27], fresh squi liver smples (1 g) were lene in tue with 3 ml of ilute hyrohlori i t ph 2 5, or Milli-Q wter, or soium hyroxie solution t ph 7 12 t 5, 1, rpm for 3 min using n Ultr-Turrx (T-25, Jnke n Kunkel, IKA-Lortehnik GmH Co., Stufen, Germny) to otin the solule frtion. The smple suspensions were entrifuge n the resulting superntnts were e-ftte using hloroform. The minerls n protein in the e-ftte lyer were then mesure. The minerls were mesure using n tomi sorption spetrophotometer (Shimzu AA-66). The soluility of eh minerl ws lulte using the eqution: soluility (%) = (solule minerl mg/g)/(totl minerl mg/g) 9 1. The solule protein in the e-ftte lyer ws etermine using the Brfor protein ssy (protein ssy kit, Bio-R, Rokfor, IL, USA). Bovine serum lumin ws use s stnr. The soluility of the protein ws lulte using the formul: soluility (%) = (solule protein g/1 g)/(totl protein g/1 g) 9 1. Determintion of minerls n protein in the whole extrt n in the low moleulr weight frtion The solule minerls n solule protein in oth the whole extrt n the low moleulr weight frtion from squi liver were otine s follows. Fresh squi liver smples (1 g) were lene in tue with 3 ml of.9 % NCl or 1 mm phosphte uffer (ph 7.4) t 5, 1, rpm for 3 min using n Ultr-Turrx (T-25) to otin the solule frtion. Next, the smples were entrifuge t 5,59g n 4 C for 1 min (Kuot 6, Kuot Corportion, Fujiok, Jpn). The ollete superntnts were e-ftte using hloroform to otin the e-ftte lyer. The e-ftte lyer ws then psse through n ultrfilter (Avnte Q1 13D, Toyo Roshi Co. Lt., Tokyo, Jpn) with moleulr weight ut-off 1, to otin the low moleulr weight frtion. The minerls n protein in oth the e-ftte lyer n the low moleulr weight frtion were mesure. The methos use to etermine the minerls n protein n to lulte their soluilities were the sme s those mentione ove. Sttistil nlysis The results re presente s the men ± SD (n = 3 8). ANOVA ws use to hek for signifint ifferenes (p \.5) [28]. Results Proximte n minerl ompositions of the squi liver Tle 1 shows the proximte n minerl ompositions of the squi liver. The squi liver smple h protein, ft, n sh ontents of 7.69 g/1 g fresh weight, 25. g/1 g fresh weight, n 1.2 g/1 g fresh weight, respetively. After onverting them to 1 g ry mtter smples, the ontents of protein, ft, n sh were foun to e high: 13.5 g/1 g ry mtter, 44. g/1 g ry mtter, n 2.11 g/1 g ry mtter, respetively. Potssium ws foun to e the mjor mrominerl in squi liver (3.75 mg/g ry mtter), followe y soium (2.69 mg/g ry mtter), mgnesium (.58 mg/g ry mtter), n lium (.13 mg/ g ry mtter). The tre minerls present, in orer of onentrtion (highest first), were iron, opper, zin, n mium, with vlues of 162 lg/g ry mtter, 133 lg/g ry mtter, 66.3 lg/g ry mtter, n 33. lg/g ry mtter, respetively. Mngnese ws not etete (i.e., its ontent ws less thn.5 ppm, the etetion limit of AAS) n phosphorus ws not etermine. When the vlues from the Foo Composition Dtse ( esse 2 Otoer 212) were onverte to mg or lg/g ry mtter, the eile prt (musle) of Jpnese ommon squi ws foun to hve soium, potssium, mgnesium, lium, iron, zin, n opper ontents of 14.3 mg/g, 12.8 mg/g, 2.57 mg/g,.6 mg/g, 4.76 lg/g, 71.4 lg/g, n 16.1 lg/g, respetively. In omprison to these vlues, liver of Jpnese ommon squi h muh lower ontents of soium, Tle 1 Proximte omposition n minerl ontents of liver of the Jpnese ommon squi Toroes pifius Vlues (g/1 g) Fresh weight Dry mtter Composition Moisture 43.2 ± 3.84 Crue protein 7.69 ± ±.9 Totl ft 25. ± ± 4.61 Ash 1.2 ± ±.25 Crohyrte (y ifferene) 22.9 ± ± 7.99 Minerl ontents Soium (mg/g ry mtter) 2.69 ±.14 Potssium (mg/g ry mtter) 3.75 ±.24 Mgnesium (mg/g ry mtter).58 ±.5 Clium (mg/g ry mtter).13 ±.1 Iron (lg/g ry mtter) 162 ± 22.9 Zin (lg/g ry mtter) 66.3 ± 21. Cmium (lg/g ry mtter) 33. ± 4.7 Copper (lg/g ry mtter) 133 ± 32.6

4 296 Fish Si (213) 79: potssium, n mgnesium, wheres it h muh higher ontents of iron n opper. Soluilities of ivlent minerls n protein t vrious ph vlues In orer to unerstn the ehvior of ivlent minerls n protein t ifferent ph vlues, we evlute their soluilities, s presente in Fig. 1. De-ftte squi liver ws eompose y wet shing n the reoveries of the minerls were evlute. The reovery of protein ws mesure y the Kjelhl metho [26]. Almost ll (nerly 1 %) of the minerls n protein remine in the squi liver fter e-ftting. For exmple, the ontent of mium in the e-oile frtion ws 33 lg/g ry mtter, n nerly 1 % of it ws issolve t ph 12. Protein soluility ws lulte to e 1 % se on protein ontent of 13.5 g/1 g ry mtter. The soluilities of ivlent minerls (zin, iron, n mium) showe the sme pttern. They tene to e more solule t low n high ph. There ws no suh pttern for the soluility of mgnesium, whih remine onstnt t *6 % t ll ph vlues. Low-pH tretment (ph 2) remove the mium entirely, while * % of the protein remine. However, importnt tre minerls (zin, iron, n opper) were lso remove in lrge quntities. In the high-ph tretment (ph 12), ll of the mium ws remove n \4 % of the protein ws left. Moreover, high-ph tretment resulte in the removl of [6 % of the tre minerls opper, zin, n iron. The soluilities of ivlent minerls (opper, iron, zin, n mium), espeilly uner lkline onitions, tene to e positively orrelte with the soluility of protein. This inites tht these minerls my in to wter-solule protein, forming protein minerl omplexes. Correltions of the soluilities of ivlent minerls with the soluilities of mium n protein Sine there is ompetition mong ivlent minerls n protein in terms of soluility, it is importnt to evlute the orreltions mong their soluilities. The orreltions of the soluilities of ivlent minerls with the soluility of mium (Fig. 2) n with the soluility of protein (Fig. 3) t ifferent ph vlues re epite in the figures. Cmium soluility showe strong reltionships with the soluilities of zin n opper (r =.764 n r =.6528, respetively), Fig. 1 The soluilities of mgnesium (), zin (), mium (), opper (), iron (e), n protein (f) extrte from the liver of Jpnese ommon squi Toroes pifius t vrious ph vlues. Columns with ifferent letters ove them on the plot re signifintly ifferent (p \.5; eh olumn n r ove the olumn show the men n SD, respetively; n = 3) Perent solule Mg Perent solule Zn () Mgnesium () Zin Perent solule Fe Perent solule Cu () Copper (e) Iron Perent solule C () Cmium Perent solule protein (f) Solule protein

5 Fish Si (213) 79: wheres iron n mium exhiite moertely strong reltionship (r =.4795) n there ws no reltionship etween mgnesium n mium (r =.574). These results suggest tht zin, opper, iron, n mium show similr soluility ehvior, s inite y their similr soluility ptterns (Fig. 1). A positive orreltion etween the soluilities of ivlent minerls n the soluility of protein t ifferent ph vlues (2 12) ws oserve, s shown in Fig. 3. The highest soluility orreltions were foun for the minerls mium (r =.5113), iron (r =.583), n zin (r =.4399). There ws negtive orreltion etween the soluilities of mgnesium n solule protein (r = -.515). In the se of mium, the orreltion eme more positive uner lkline onitions (r =.671). These results inite tht iron, mium, n zin re extrte with protein t ll ph vlues; in prtiulr, mium tens to e extrte with protein t high ph. Inresing the ph my hnge the protein onformtion to one tht is more solule (Fig. 1). This soluilize protein is then more likely to form omplex with mium through minerl protein ining, thus inresing the soluility of mium t higher ph. Soluilities of ivlent minerls n protein in e-ftte extrt n in the low moleulr weight frtion The ivlent minerls n protein showe the sme soluility ptterns in.9 % NCl n 1 mm phosphte uffer (ph 7.4); see Fig. 4. Among the five minerls, mium ws the most solule in e-ftte extrt n in the low moleulr weight frtion in oth.9 % NCl n 1 mm phosphte uffer, followe y opper, mgnesium, iron, n zin. Protein ws less solule thn the ivlent minerls. All of the mium ws in solule form in oth.9 % NCl n 1 mm phosphte uffer. This mens tht it is possile to remove mium ompletely using.9 % NCl or 1 mm phosphte uffer. After extrtion with.9 % NCl or 1 mm phosphte uffer, high perentges (25 7 %) of the zin, opper, n iron were still present, n 75 % of the protein remine. After pplying.9 % NCl or 1 mm phosphte uffer, *2 % of the protein ws still present in the low moleulr weight frtion. Disussion Proximte n minerl ompositions Severl experiments onerning the minerl ontent of squi liver hve een onute; however, they fouse solely on tre minerls (they i not nlyze the mrominerl profile or the proximte omposition). In the Jpnese ommon squi T. pifius, the tre minerls opper, zin, n mium were present in liver t levels of 1 () Mgnesium, ph () Copper, ph 2 12 Perent solule Mg r =.574 Perent solule Cu r = Perent solule C Perent solule C 1 () Zin, ph () Iron, ph 2 12 Perent solule Zn r =.764 Perent solule Fe r = Perent solule C Perent solule C Fig. 2 Correltions of the soluility of mium with the soluilities of other ivlent minerls mgnesium (), zin (), opper (), n iron () extrte from liver of Jpnese ommon squi Toroes pifius (n = 33)

6 298 Fish Si (213) 79: Fig. 3 Correltions of the soluility of protein with the soluilities of mgnesium (), zin (), opper (), iron (), mium t ph 2 12 (e), n mium t ph 7 12 (f), s extrte from liver of Jpnese ommon squi Toroes pifius ( e n = 33, f n = 18) Perent solule Mg () Mgnesium, ph R = Perent solule Fe () Iron, ph R = () Zin, ph (e) Cmium, ph 2-12 Perent solule Zn R =.4399 Perent solule C R = () Copper, ph (f) mium, ph 7-12 Perent solule Cu R =.391 Perent solule C R = Perent solule protein Perent solule protein , 31 89, n mg/kg fresh weight, respetively [14]. Kim et l. [6] lso mesure the tre minerl ontents in liver of the squi T. pifius from offshore Koren wters. In squi with meium mntle lengths ( m), the ontents of zin, opper, n mium in liver were 221, 989, n 198 lg/g ry mtter, respetively. In squi with long mntle lengths ( m), the ontents of zin, opper, n mium in liver were signifintly lower thn those seen in squi with meium mntle lengths: 194, 793, n 155 lg/g ry mtter, respetively. In nother squi speies, Nototorus gouli, Finger n Smith [4] reporte tht the liver ontine opper (66.7 mg/kg fresh weight), zin (152.6 mg/kg fresh weight), n mium (6.7 mg/kg fresh weight). Furthermore, Crig n Overnell [5] nlyze the minerls in the liver of the squi Loligo foresi. They reporte tht the liver ontine iron, zin, opper, n mium t levels of 52.2, 43.6, 11, n 1.5 mg/kg fresh weight, respetively. This wie rnge of minerl ontents seen in livers in ifferent stuies my e relte to ftors suh s speies, geogrphil origin, sesonl vrition, sex, ge, size, n environmentl n physiologil vritions. However, these experiments lso show tht squi liver, sefoo proessing wste prout, ontins highly nutritionl omponents tht oul e utilize further. Severl stuies hve shown tht squi proessing wste lso ontins importnt sustnes suh s minerls [3 6], proteinse enzyme [7 9], n orgni is [3].

7 Fish Si (213) 79: Perent soluility Perent solulility ().9% NCl () Phosphte uffer t ph 7.4 Soluilities of ivlent minerls n protein t vrious ph vlues Mg Zn C Cu Fe SP Fig. 4 The soluilities of mgnesium, zin, mium, opper, iron, n protein (SP) extrte from liver of Jpnese ommon squi Toroes pifius using.9% NCl () n 1 mm phosphte uffer, ph 7.4 (). Open rs whole extrt, fille rs low moleulr weight frtion. Columns with ifferent letters ove them on the plot re signifintly ifferent (p \.5; eh olumn n r ove the olumn show the men n SD, respetively; n = 3) The solvent/liver rtio ffets the perentges of minerls n protein extrte. In our preliminry experiments, we teste vrious solvent/liver rtios from 2 to 1, n ultimtely eie to use solvent/liver rtio of 3. Ghimire et l. [18] use rtio of 1, similr rtio to tht hosen y us. The soluilities of ivlent minerls (opper, iron, zin, n mium) t high ph tene to e positively orrelte with protein soluility. This inites tht minerl protein omplex formtion my our. Minerls n hnge their hemil form uring n/or fter proessing or n intert with other ompouns. As onsequene, their soluilities n inrese or erese epening on the tretment pplie, whih n influene their hemil form. Minerls in squi liver n hnge their hemil form epening on the ph employe, so they oul intert with other ompouns, suh s proteins. Sntoso et l. [19] reporte tht the soluility of mgnesium n lium from sewee inrese signifintly fter oiling it in eti i solution rther thn oiling it in wter or slt solution. It ws lso foun tht the presene of orgni i inreses the soluilities of minerls [2, 29, 3]. Minerls in solule form were lso foun in high onentrtions in ii foos suh s yoghurt [31]. In our experiment, ivlent minerls tene to e more solule uner oth ii n lkline onitions. We presume tht i or lkli tretment n hnge the onformtions of the minerls, mking them more solule. One possile wy of improving minerl soluility is to in the minerl with protein, estlishing solule minerl protein omplex. In this se, the solule protein of squi liver ts s soluility enhner. Clyesle [22] efine n enhner s moleulr speies tht ins with minerls to form ompouns tht re solule n n e sore y muosl ells. An enhner my unergo levge to relese the minerl in solule form, or its thermoynmi onstnts my le to the trnsfer of the minerl to muosl eptor. Correltions of the soluilities of ivlent minerls with the soluilities of mium n protein Among the three tre minerls zin, opper, n iron, zin showe the highest orreltion with mium, whih my relte to its funtion in metllothioneins. Roesiji [32] propose moel for ouple metllothionein inution n the resue of trget ligns ompromise y inpproprite metl ining. Aoring to this hypothesis, the roles of mium n zin re interhngele. When nonessentil metls suh s mium, merury, or silver enter ell, there is inevitly ompetition etween them n other minerls like opper n zin for intrellulr ligns, suh s metlloproteins [33]. Therefore, mium n isple zin s well s lium [34]. Reeves n Chney [35] reporte tht mium ioessiility ws lso relte to the ingestion of other minerls suh s lium, zin, n iron. Copper n zin re essentil tre metls tht re require y wie vriety of metlepenent enzymes. Cephlopos use the opper-ontining protein hemoynin s respirtory pigment, so opper is require in lrge onentrtions [36]. Therefore, opper n zin re oun to either the prtiulte frtion or very low moleulr weight speies [5]. The orreltions of the soluilities of ivlent minerls with protein soluility t ifferent ph vlues inite tht zin, iron, n mium were extrte with protein t ll ph vlues. However, t high ph, mium tene to e extrte with protein more thn t low ph. As shown in Fig. 1, the perent of solule C ws lmost the sme s the perent of solule protein t high ph. We ssume tht the proteins, in their metl-ining forms, issolve in the liqui phse t high ph. Minerl-ining proteins known s methllothioneins re ssoite with the mehnism of metl etoxifition in quti invertertes [33, 37]. Tre minerls (opper, zin, n mium) were foun to in strongly with squi protein, espeilly in the low moleulr weight frtion [5]. Rimuno et l. [25] reporte tht the

8 3 Fish Si (213) 79: strongly positive orreltions mong mium, zin, opper, n low moleulr weight protein point to the presene of metlloproteins with high ffinities for these elements. Methllothioneins re low moleulr weight proteins tht n e inue y free ytosoli metl ions, espeilly mium, opper, zin, n merury, n re involve in efene ginst metl toxiity [4, 38, 39]. Soluilities of ivlent minerls n protein in the e-ftte extrt n the low moleulr weight frtion Although the use of 1 % soium hlorie n oiling tretment n inrese the soluilities of minerls from shellfish [4], this tretment ereses the soluilities of minerls from sewees [19]. This suggests tht the soluilities of minerls epen on the tretment n the type of mterils use. Aquti orgnisms n remove nonessentil metls suh s mium, merury, n silver using metlining sites of low moleulr mss, suh s those on metllothioneins, s prt of the norml metolism of the orgnism [33, 39]. Thus, lmost ll of the soluilize minerls from squi liver were foun in the low moleulr weight frtion (moleulr weight = MW \ 1,). Low, intermeite, or high moleulr weight proteins hve een lso foun to t s potentil ining sites for tre metls, inluing mium [4, 5]. In the present experiment, mong the four tre minerls zin, mium, opper, n iron, signifintly higher perentge of mium ws foun in the low moleulr weight frtion. A similr result ws lso reporte y Rimuno et l. [25]: mium extrte from the igestive gln of Otopus vulgris exhiite strong ssoition with low moleulr weight protein (LMW; 11, 6, D) n weker ssoition with high moleulr weight protein (HMW; 144, 13, D). Reltively high levels of mgnesium were lso foun in low moleulr weight frtions. This result is in or with tht of stuy onute y Suzuki et l. [4], in whih high perentge of the mgnesium extrte from shellfish ws foun in the low moleulr weight frtion. However, in sewees, high perentges of solule minerls were foun in the high moleulr weight frtion (MW [ 2,) [19], euse sewees ontin high levels of ietry fier n low levels of protein [41]. The ietry fier in sewee ts s n inhiitor of minerl soluiliztion (n inhiitor is moleulr speies tht forms n insolule ompoun tht nnot e sore or leve to relese the minerl in solule form [22]). Aoring to the results of our stuy, liver of the Jpnese ommon squi Toroes pifius ontins high levels of nutritionl ompouns: ft (44. g/1 g ry mtter) n protein (13.5 g/1 g ry mtter). Low- n high-ph (ph 2 n 12) tretments re le to remove mium entirely, lthough high perentges of solule minerls n protein were foun in the low moleulr weight (MW \ 1,) frtion. The soluilities of zin n opper were strongly relte to the soluility of mium, ut there ws no reltionship etween the soluilities of mgnesium n mium. In terms of methos for removing mium from squi liver, tretment with.9 % NCl or with 1 mm phosphte uffer t ph 7.4 hs severl vntges: these tretments re more eonomil, environmentlly frienly, n leve more useful ompouns in the liver thn tretment with hyrohlorie i or soium hyroxie. When we filtere the solvent-trete liver, tretment with.9 % NCl or 1 mm phosphte uffer t ph 7.4 gve 2 25 mg (ry sis) of e-oile soli power from 1 g of fresh liver. These two tretments my therefore e pproprite methos for removing mium from squi liver. Referenes 1. Sikorski ZE, Koloziejsk I (1986) The omposition n properties of squi met. Foo Chem 2: Tvkoli O, Yoshi H (28) Applition of su-ritil wter tehnology for reovery of hevy metl ions from the wstes of Jpnese sllop Ptinopeten yessoensis. Si Totl Environ 398: Kni Y, Yoshikw N, Ok S, Ae H (27) Comprison of extrtive omponents in musle n liver of three Loliginie squis with those of one Ommstrephie speies. Fish Si 73: Finger JM, Smith JD (1987) Moleulr ssoition of Cu, Zn, C n 21 Po in the igestive gln of the squi Nototorus gouli. Mr Biol 95: Crig S, Overnell J (23) Metls in squi, Loligo foresi, ults, eggs n hthlings. No eviene for role for Cu- or Znmetllothionein. Comp Biohem Physiol C 134: Kim GB, Kng MR, Kim JW (28) Speifi umultion of hevy metls in squi ollete from offshore Koren wters: preliminry results for offshore iomonitoring n foo sfety ssessment. Fish Si 74: Kishimur H, Seki H, Hyshi K (21) Isoltion n hrteristi of trypsin inhiitor from the heptopnres of squi (Toroes pifius). Comp Biohem Physiol B 13: Komi T, Kwt C, Amno M, Lee BR, Ihishim E (24) Torepsin, new thepsin D from heptopnres of Jpnese ommon squi (Toroes pifius). Comp Biohem Physiol B 137: Crens-Lopez JL, Hr NF (29) Ientifition of ysteine proteinse from Jumo squi (Dosiius gigs) heptopnres s thepsin L. Foo Chem 112: Bustmnte P, González AF, Roh F, Mirmn P, Guerr A (28) Metl n metlloi onentrtions in the gint squi Arhiteuthis ux from Ierin wters. Mr Environ Res 66: Rimuno J, Vle C, Cnário J, Brno V, Mour I (21) Reltionship etween merury, methyl-merury n selenium in tissues of Otopus vulgris from Portuguese Cost. Environ Pollut 158:294 21

9 Fish Si (213) 79: Kojinovi J, Jkson CH, Cherel Y, Jkson GD, Bustmnte P (211) Multi-elementl onentrtions in the tissues of the oeni squi Toroes filippov from Tsmni n the southern Inin Oen. Eotoxiol Environ Sf 74: Piere GJ, Stowsser G, Hstie LC, Bustmnte P (28) Geogrphi, sesonl n ontogeneti vrition in mium n merury onentrtions in squi (Cephlopo: Teuthoie) from UK wters. Eotoxiol Environ Sf 7: Tnk T, Hyshi Y, Sihizw M (1983) Suellulr istriution n ining of hevy metl in the untrete liver of the squi; omprison with t from the livers of mium n silverexpose rts. Experienti 39: Mrtin JH, Flegl AR (1975) High opper onentrtion in squi livers in ssoition with elevte of silver, mium n zin. Mr Biol 3: Or T, Swy T, Hokri K, Umehr Y, Mizukmi M, Tomit F (1999) Removl of mium from sllop heptopnres y miroil proesses. Biosi Biotehnol Biohem 63(3): Ren H, Okmoto Y, Ji H, Fuku R, Koyshi A, Goto S, Eno H, Hyshi T (28) Removl of mium from sllop proessing wste y wshing with wek i solution n utiliztion of useful onstituents for orgni fertilizer mnufturing. Fish Si 74: Ghimire KN, Ki H, Inoue K, Ohto K, Kwkit H, Hr H, Morit M (28) Hevy metl removl from ontminte sllop wste for fee n fertilizer pplition. Biores Teh 99: Sntoso J, Gunji S, Yoshie-Strk Y, Suzuki T (26) Minerl ontent of Inonesin sewees n minerl soluility ffete y si ooking. Foo Si Tehnol Res 12(1): Yoshie Y, Suzuki T, Pnolf T, Clyesle FM (1999) Soluility of iron n zin in selete sefoos uner simulte gstrointestinl onitions. Foo Si Tehnol Res 5: Vitli D, Drgojević I, Šeečić B (28) Bioessiility of C, Mg, Mn, n Cu from whole grin te-isuits: impts of protein, phyti i n polyphenols. Foo Chem 11: Clyesle FM (1988) Minerls: their hemistry n fte in foo. In: Smith KT (e) Tre minerls in foos. Mrel Dekker In., New York, pp Ersoy B, Ozeren A (29) The effet of ooking methos on minerl n vitmin ontents of Afrin tfish. Foo Chem 115: Greffeuille V, Kyoe APP, Ir-Verniere C, Gnimi M, Rohette I, Mouquet-Rivier C (211) Chnges in iron, zin n helting gents uring tritionl Afrin proessing of mize: effet of iron ontmintion on ioessiility. Foo Chem 26: Rimuno J, Vle C, Durte R, Mour I (21) Assoition of Zn, Cu, C n P with protein frtions n su-ellulr prtitioning in the igestive gln of Otopus vulgris living in hitts with ifferent metl levels. Chemosphere 81: Assoition of Offiil Anlytil Chemists (AOAC) (22) Offiil methos of nlysis of the Assoition of Offiil Anlytil of Chemists. The Assoition of Offiil Anlytil Chemists, In., Arlington 27. Sntoso J, Ishizuk Y, Yoshie-Strk Y (212) Chrteristi of minerls extrte from mi-gut gln of Jpnese sllops Ptinopeten yessoensis in vrious ph vlues. Fish Si 78: Hoherg Y (1988) A shrper Bonferroni proeure for multiple tests of signifine. Biometrik 75: Yoshie Y, Suzuki T, Clyesle FM (1997) Iron soluility from sefoos with e iron n orgni is uner simulte gstrointestinl onitions. J Foo Qul 2: Ekhlom P, Virkki L, Ylinen M, Johnsson L (23) The effet of phyti i n some nturl helting gents on the soluility of minerl elements in ot rn. Foo Chem : e l Fuente MA, Montes F, Guerrero G, Jurez M (23) Totl n solule ontents of lium, mgnesium, phosphorus n zin in yoghurt. Foo Chem : Roesiji G (1996) Metllothionein n its role in toxi metl regultion. Comp Biohem Physiol C 113(2): Amir JC, Amir-Triquet C, Brk S, Pellerin J, Rinow PS (26) Metllothioneins in quti inverterte: their role in metl etoxifition n their use s iomrker. Aqut Toxiol 76: Ggnon E, Hontel A, Jumrie C (27) Reiprol inhiition of C n Cu uptke in isolte he kiney ells of rinow trout (Onorhynhus mykiss). Toxiol In Vitro 21: Reeves PG, Chney RL (28) Biovilility s n issue in risk ssessment n mngement of foo mium. A review. Si Totl Environ 398: Deleir W, Vlemink A, Geli P, vn Grieken R (1978) Determintion of protein-oun opper n zin in some orgns of the uttlefish Sepi offiinlis L. Comp Biohem Physiol B 6: Virengo A, Nott JA (1993) Mehnism of hevy metl tion homeostsis in mrine invertertes. Comp Biohem Physiol C 14: Kägi JHR, Shäffer A (1988) Biohemistry of metllothionein. Biohemistry 27(23): Erk M, Ruus A, Ingerigtsen K, Hylln K (25) Cmium umultion n C-ining proteins in mrine invertertes riotrer stuy. Chemosphere 61: Suzuki T, Yoshie Y, Horii A (2) Soluility of minerls in shellfish y heting with slt wter. In: Crmn O, Sulistiono, Purynto A, Suzuki T, Wtne S, Arimoto T (es) Proeeings of the JSPS-DGHE Interntionl Symposium on Fisheries Siene in Tropil Are. TUF Interntionl JSPS Projet, Tokyo, pp Sntoso J, Yoshie Y, Suzuki T (22) The istriution n profile of nutrients n tehins of some Inonesin sewees. Fish Si 68(Suppl. 2):

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