Department of Food Chemistry (Chairman: Prof. Dr. G. J A N f t E K , DrSc.) and Department of Dairy DrSc.), and Fat Technology (Chairman: Prof. Dr. J . DOLEZALEK, Prague Institute of Chemical Technology, Prague, CSSR

Reactions of Oxidized Lipids with Protein. Part XII. Interactions of Polar Groups of Lipids with Nonlipidic Substances1 J. POKORSP, V. KOCOUREK~ and J. ZAJfC

Contrary t o fresh lipids, oxidized lipids form chloroform-insoluble, but methanol-soluble compounds with cellulose. The amount of chloroform-insolublelipids is significantly higher in mixtures containing cellulose impregnated with albumin. Polar groups of oxidized Lipids formed nonextractable compounds with protein more readily than polar groups of a monoglyceride. Compounds insoluble either in chloroform and in chloroform-methanol or methanol were formed in contact of oxidized lipids with protein, contrary to mixtures of lipids with sole cellnlose. The formation of these nonextractable compounds is due both to the interaction of protein with hydroperoxides and with non-peroxidic oxidation products. Oxidized lipids form insoluble compounds with proteins, particularly with albumin [I] or with et al. [3] assumed the existence of only physical forces between the lipid and the casein [z]. NAFAYAN protein moieties which is in agreement with the observation [4]that insoluble complexes are formed by interaction of oxidized lipids with polyamide and formaldehyde-treated polyamide. The formation of covalent-bonded compounds is possible as well, e.g. through free radicals [5] produced by decomposition of hydroperoxides. The amount of insoluble lipoprotein compounds in model mixtures was proportional t o the amount of hydroperoxides decomposed during heating or storage [6].Covalent imino derivatives are produced by interactions of carbonyl compounds with free amino groups of protein [7].The reaction can proceed with aldehydic lipid oxidation products under conditions common during the technological processing of food [S].

In this study, the formation of insoluble lipoprotein compounds containing only physical bonds (mixtures of oxidized lipids with cellulose) is compared with the formation of complexes containing both physical and covalent bonds (mixtures of oxidized lipids with protein-impregnated cellulose). Very mild reaction conditions (low temperature and short reaction time) werde chosen in order to restrict secondary reactions.

Experimental Material Sunflower seed oil (SO) was plant-scale refined. Thermically treated sunflower seed oil (TOSO) was produced by heating SO in a 50 mm layer under free access of air a t 180 "C for 40 h (polymer content 40.4%). Monoglyceride concentrate (MG) was produced by glycerolysis of peanut oil followed by

f

Part XI. Nahrung 19,K 7 (1975). Present address: Institute of Antibiotics and Biotransformations, Roztoky u Prahy, CSSR.

49 Die Nahrung, 20. Jhg., Heft

7

708

POKORi%??/KOCOUREK/ZAJ fC

molecular distillation of the product (composition : triacylglycerols 0.8y0, diacylglycerols 4.5%, I-monoacylglycerols 89.7%, a-monoacylglycerols I . I % , free fatty acids 1.8y;. free glycerol 2.1%) Ethyl linoleate (EL) was produced by Koch-Light Laboratories, Ltd., Colnbrwk, England, oxidized ethyl linoleate (OEL) was prepared by autoxidation of E L in a 8 mm layer at 60 "C for 80 h. Some properties of these substances are summarized in Table I. Egg albumin was produced by Difco Laboratories, Inc., Detroit, Michigan : microcrystalline cellulose LK (particle size 80-120 pm) was manufactured by Lachema n.p., Brno, Czechoslovakia. Table I Properties of lipidic materials Substance

so TOSO

MG EL OEL

EL OEL

Linoleic acid

Iodine value

0.07 I .4 3.5

122.8 79.7

14.6

54.8

1.1

11.2

-a

20.4

I .4*

70.4

10.8

I .2

0 s

0.3

173.0 147.5

1.2

Notes : SO TOSO

YC

Peroxide value Walk1

lcid value

[%I 30.1 93.4b

93

- refined sunflower seed oil; - thermically treated sunflowerseed oil: - monoglyceride concentrate; - ethyl linoleate; - oxidized ethyl linoleate;

I

2.130

I

-a

5.1 41.2

I.OC

1

0.gc

1.3d

a

-

b

-

-

i t could not be determined because of interfering substances: total octadienoic acids : petroleum ether procedure; diethyl ether procedure

(20

niin reaction time at ambient temperature)

c d

AnalyticaE Methods The peroxide value was determined iodometrically

{g], the iodine value after HANUS with 500% excess of the reagent [IO], the content of oxidized fatty

acids by thin layer chromatography [IIJ, the content of polymers by gel chromatography [IZ], the content of partial glycerol esters after QUINLIN and WEISER[13], the percentage of a-monoacylglycerols in presence of I-monoacylglycerols after treatment with perchloric acid [14]. The unsaponifiables were determined by the standard procedure [IS]. the linoleic acid content by ultraviolett spectrometry [15). Infrared spectra were measured with use of Perkin-Elmer Infracord 337 spectrophotometer in tetrachloromethane solutions. The thin layer chromatography was carried out on precoated wide-pore silica gel Silufol UV,,@ layers (SklLrny Kavalier n.p., Votice, CSSR), washed with the solvent mixture and activated a t I Z O O C for I h. A 300 p g sample was applied in a 20 mm band, and developed with a mixture of petroleum ether - diethyl ether - acetic acid (50:50:1 v/v/v) in case of oils, and benzene - aceacetic acid (95:5 :I v/v/v) i n case of ethyl linoleate. The running distance was 140mm, the tone detection was carried out with standard ethanolic solution of molybdophosphoric acid.

-

Procedure The solution of 3 g of egg albumin in IOO ml of water was mixed with 20 g of niicrocrystalline cellulose and left t o swell for 16 h a t ambient temperature, The resulting dispersion was homogenized with 700 mg of lipids for 5 min using a tissue homogenizer RT-z (ERlA, Odessa, USSR),speed 2. The resulting mixture was left t o repose for 10min t o allow the formation of physical bonds. The mixture was extracted as follows: The mixture was homogenized with IZO ml of chloroform for 5 min, the solvent was filtered off and the extraction repeated-6 times. The extracted material was reextracted with a mixture of chloroform and methanol ( 2 : I v/v) or with methanol (in case of EL and OEL) i n the same way (4 subsequent extractions). The solvent was distilled off in a rotary evaporator at 60 O C under reduced pressure, the dry residue was reextracted with dry chloroform, the solution filtered, and the solvent was again removed in a rotary evaporator a t 60 "C under reduced pressure.

709

Reactions of oxidized lipids with protein

Six subsequent extractions were sufficient for obtaining quantitative results both in case of chloroform and in that of chloroform-methanol (Fig. I). The results obtained by direct chloroform-methanol extraction were hlgher than the sum of extracts obtained by subsequent chloroform and chloroform-methanol extractions, obviously because of losses of volatile substances during the removal of solvents. In case of MG,a part of losses was caused by incomplete extraction of glycerol. In case of OEL, a part of weight losses was due to the decomposition of hydroperoxides into volatile substances.

3456 Fig.

I.

1 r221456 123056 E

Course of extraction of lipids from model mixtures.

P - p.c. iipids extracted; E - number of extractions; mixture of lipids with cellulose: A

- extraction with chloroform; B - extraction with chloroform - methanol; mixture of lipids with albumin-impregnated cellulose: C extraction with chloroform; D extraction with chloroform - methanol

-

-

Results The chloroform extraction of lipids from their mixtures with cellulose impregnated with albumin was never complete, and additional lipids were obtained by a subsequent extraction with chloroform-methanol or with methanol, however, some lipids reTable 2 Effect of albumin on the content of extractable lipids Lipidic substances

so TOS(3 MG

EL OEL

j j

Mixtures with cellulose impregnated with albumin

I

97.2

I l -

I

B 93.4

92.5

76.2

96.0

71.8 44.8

I

Mixtures with sole cellulose

C I

I

953 90.2

87.1

1

94.7 89.8

i I

96.2 95.0

Notes: A - extracted six times with chloroform - methanol; B - extracted six times with chloroform; C - extracted six times with chloroform and then six times with chloroform - methanol (SO, TOSO, MG) or with methanol (EL, OEL) 49'

710

POKORN~/I(OCOUREK/ZAJ fC

Table 3 Content of polar groups in extracts from mixtures with albumin-impregnated cellulose -______ I Content of polar groups Lipidic substance [ A ~ ~ x~ lo3] : A ~ ~ ~

EL OEL

I

I

1.15

2.67

2;

I

1.52

3.44

Notes: A - original material; B - chloroform extract from a mixture with albumin-impregnated cellulose ; C - chloroform-methanolic extract obtained after a previous chloroform extraction

C

i

f

f

J

X

L

M

Fig. 2. Selectivity of extraction of lipids from model mixtures. chloroform extract; C - methanolic extract: oxidized Ethyl linoleate (EL): A - original E L ; B ethyl linoleate (OEL): D - original OEL; E - chloroform extract; F - niethanolic extract: oxichloroform extract; J - chloroform dized sunflower seed oil (TOSO): G - original TOSO;H methanolic extract; monoglyceride concentrate (MG): K - original MG: L - chloroform extract; M - chloroform - methanolic extract; extraction from mixtures with albumin-impregnated cellulose; separation conditions: Silufol UV,,, layers; A-E: developing with benzene - acetone - acetic acid (95 : 5 : I v/v/v) ; G- ilf : developing with petroleum ether - diethyl ether - acetic acid (50 :50 : I v/v/v); detection with molybdophosphoric acid

-

-

711

Reactions of oxidized lipids with protein

mained even then bound in the extracted material (Table 2). The nonextractable lipidic fraction was significantly lower in control mixtures containing nonimpregnated cellulose in case of oxidized lipids but was nearly identical in case of fresh or less oxidized lipids. The extraction with chloroform was selective in case of mixtures with oxidized lipids because the highly polar and/or polymeric fraction (remaining on the start on thin layer chromatograms) was absent in the chloroform extract but was present in significant amounts in the chloroform-methanolic extract (Fig. 2). The chloroform extract contained a lesser amount of hydrogen bonded groups than the methanolic extract (Fig. 3 and Table 3). Discussion Cellulosewas less active in binding polar lipids into chloroform-insolublecompounds than cellulose impregnated with albumin (Table 2 ) , while there were nearly no chloroform-insoluble fractions in mixtures with a fresh oil in either case. Obviously,

'9-

Fig. j. Infrared spectra of lipid extracts. T - transmittance [%I; i'-waYe number [cm-lj ; lipidic substance: I - ethyl linoieate(EL); 11 - oxidized ethyl linoleate (OEL); sample: A - original ester; extract from a mixture with albumin-impregnated cellulose: B - nith chloroform; C - with methanol after a previous extraction with chloroform

hydroxyl groups present in cellulose form less strong hydrogen bonds than various other functional groups of a protein molecule. This behaviour is in agreement with the observation of NARAYAN et al. [3] that the blocking of hydroxyl groups by acetylation of the nonlipidic material does not much affect the formation of insoluble compounds. Relatively intensive formation of chloroform-insoluble compounds in mixtures of cellulose with oxidized ethyl linoleate may be due to an intermolecular hydrogen bonding [16]between hydroperoxide groups of the lipid and hydroxyl groups of cellulose. The chloroform-insoluble methanol-soluble lipid fraction was expected to possess a higher polarity in comparison with the chloroform-soluble fraction because of

712

POKORN P/KOCOURE K/ZA J f C

greater affinity toward hydrogen bonding. The experimental evidence (Fig. 3 and Table3) supports this assumption as the methanolic extracts contain higher percentages of hydroxyl groups than the corresponding chloroform-soluble extracts. The formation of hydrogen-bonded lipoprotein compounds by interaction of oxidized lipids with protein is, however, not due exclusively to the presence of hydroxyl groups.in the lipid moiety. A monoglyceride emulsifier (MG) formed markedly lower amounts of chloroform-insoluble compounds (based on the number of polar groups per ester group) than oxidized lipids in spite of its high content of polar groups (about four times more than in oxidized ethyl linoleate OEL). The steric structure of the molecule and the influence of adjacent reactive groups may play here their role. A small fraction of oxidized lipids could not be extracted from mixtures containing albumin even with methanol or chloroform-methanol while the extraction was nearly quantitative in case of mixture of lipids with cellulose not containing albumin (Tab. 2 ) . This difference is probably due to the formation of covalent compounds between oxidized lipids and protein [6, 171. The reaction is, a t least partially, initiated by the catalytic effect of free amino groups [IS] present in the protein molecule on the hydroperoxide decomposition. The interaction of oxidized ethyl linoleate with protein caused losses of available lysine [IS, 201. We have reported earlier [6] that the amount of nonextractable lipids in lipid-protein-mixtures was proportional to the amount of peroxides decomposed during heating of the mixture. The reaction of hydroperoxides with amines proceeds rapidly even at room temperature [ZI]. The occurrence of such a reaction is evident from Fig. 2. The spot corresponding to lipid hydroperoxides disappeared or diminished by contact with protein while another spot appeared on the start. Such a spot has been shown [ 2 2 ] to originate from an interactionof oxidized lipids with free aminogroups of protein and is due to nitrogencontaining oligomers. The methanol-insoluble fraction may not be produced solely by reaction of protein with lipid hydroperoxides. Malondialdehyde [23, 241 or alkanals [8, 251 or other carbonyl oxidation products are possible precursors as well. Therefore, thermically

0

41

92

Fig. 4. Effect of total oxidation products on the amount of lipids nonextractable with chloroform but extractable with methanol. D - difference between the content of lipids extractable with chloroform - methanol and extractable with chloroform (expressedin p.c. of total lipids) ; P - number of oxidized fatty acids (determined by TLC) per one ester group (determined by IR)

Reactions of oxidized lipids with protein

713

treated oils (e.g. frying oils), containing nearly no peroxides, formed insoluble lipoprotein derivatives with similar ease as substances rich in hydroperoxides (Fig. 4). KAJIIUOTO et al. [z6] found similar relations a t higher temperatures and after longer reaction times. For this reason, the content of total oxidized lipids is a better criterion of the tendency to form insoluble lipoprotein compounds than the content of peroxides. Zusammenfassung

J. POKORN~, V. KOCOUREK und J. Z A J ~ CReaktionen : oxydierter Lipide rnit Proteinen. 12. Mitt. Wechselwirkungen der polaren Gruppen der Lipide rnit nichtlipidischen Substanzen Im Gegensatz zu unveranderten Lipiden bilden oxydierte Lipide chloroform-unl6sliche,aber methanol-18sliche Verbindungen mit Cellulose. Die Menge an chloroform-unltklichen Lipiden ist in Gemischen. die mit Albumin impriignierk Cellulose enthalten, signifikant heher. Polare Gruppen der oxydierten Lipide bilden nichtextrahierbare Verbindungen rnit Proteinen Vie1 leichter als die polaren Grnppen eines Mono-Glycerins. Verbindungen, die weder in Chloroform noch in Chloroform-Methanol oder in Mkthanolliislich sind, werden beim Kontakt oxydierfm Lipide mit Protein gebildet, im Gegensatz w Gemischen von Lipiden mit reiner Cellulose. Die Bildung dieser nichtextrahierbaren Verbindungen ist sowohl auf die Wechselwirkung des Proteins rnit Hydroperoxiden a l s auf die rnit nichtperoxidischen Oxydationsprodukten zunlckzuflihren.

Pearo~e

References [I] NARAYAN, K. A., and F. A. KUMIEROW, J. Amer. Oil Chemists’ SOC.35, 52-56 (1958);40, 339-342 (1963). [2] POKORN~, J., and G.JANfCEK, Nahrung 12,81-85 (1968). E3] NARAYAN, K. A.. M. SUGAIand F. A. KUMMEROW, J. Amer. Oil Chemists’ Soc. 41,254-259 (1964). [4] P O K O R NJ., ~ ,H.ZWAINand G. JANfEEK, Nahrung 10, 315-320 (1966). [5] ROUBAL, W. T., Lipids 6, 62-64 (1971). M.F.M.M.,J. PoKoRNPand G. J A N f h K , Nahrmg 15, 663-670 (1971). [6] EL-TARRAS, [7] Bow~s,J. H., and C. W. CATER,Biochim. biophysica A d a [Amsterdam] 168,341-348 (1968). [83 P O K O R NJ., ~ ,P.-T. T ~ IN.-T. , LUHNand G. JANfbK, Nahrung 17,621-627 (1973). [g] TOLLENMR, F.D.,Tluszcze Srodki Piorace 2,273-285 (1958). [IO] POKORN~, J., Sb. Vysokk skoly chem.-technol. Raze, Oddil Fak. potravinfikb Technol. 2, 193-208 (1958). [TI] NAUDET,M., and S. BIASINI, Rev. franc. Corps gras 19,307-319 (1972). [12] P O K O R NJ., ~ ,M. K. KUNDU, S. POICORN$, M. BLEHAand J. ~ U P E K Nahrung , 20,157 (1976). [IS] QUINLIN, P.,and W. J. WEISERJr., J. Amer. Oil Chemists’ Soc. 35. 325-327 (1958).

714

POKORN~/KOCOUREK/ZAJ fC

[14] BROKAW, G. Y.,E. S. PERRY and W. C. LYMAN, J. -4mer. Oil Chemists’.Soc. 32,194-196 (1955). [15] IUP.4C: Standard Methodsfor the Analysis of Oils, Fats and Soaps. 5th ed. ButterworthS. Lon-, don 1974.Meth. 11. D.5 and Yeth. 11. D. 17. [16]ZAIKOV, G. E., Z. K. MAIzus and N. 31. EMANIJEL’, Izv. Akad. Nauk SSSR, a i m . 1968, 256- 260. [17] P O K O RJ., N ~Fette, , Seifen, Anstrichmittel 65, 278-284 (1963). [IS] CHALTYKYAN, 0. A., N. M. BEYLERYAN and S. K. GRIGORYANin: Uspekhi Khimii OrganichesEditor). Khimiya, MOSCOu kich Perekisnykh Soedineniy i Autookisleniya (N. N. EMANUEL’, 1969.Pp. 171-176. [IS] HORIGOME, T.,T.YANACIDA and 34.MIURA, Nippon Nogei Kagaku Kaishi 48, 195-197 (1974). [zo] HORIGOME. T.,and M. MIURA, Nippon Nogei Kagaku Kaishi 48, 437-444 (1974). [PI] OKURA,H.,Shikoku Igaku Zasshi 25, 548-551 (1969). [22] POKORNP, J., B. A. EL-ZEANY and G. JANfEEK, Nahrung 17,545-552 (X973). [23] MATSUSHITA,S.i and N. IWAMI, Kyoto Daigaku Shokuryo Kagaku Kenkyusho Hokoku 30, 1-4 (1967). Lipids 8, 194-198 (1973). [24] MALSHET,V. G., and A. L. TAPPEL, [25] MOHAMMAD, A., H . S. OLCOTTand H. FRAENKEL-CONRAT, Arch. Biochem. 24, 270-280 (1949). [ z 6 ] KAJIMOTO, G.,and.H. YOSHIDA, Nippon Nogei Kagaku Kaishi 47, 515-522 (1973).

Doz. Dr. J. P O K O R Department N~, of Food Chemistry, Institute of Chemical Technology, Suchbktarova 1905, 16628 Prague, CSSR Eingegangen 30. 12. 1975