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Effect of Hydrogen Peroxide Treatmenton the Nutritional Quality of RapeseedFlour Fed to Weanling Rats1-4G. HARVEY ANDERSON, GRACE S. K. LI,JOHN D. JONES AND FRITZ BENDERDep artm en t o f N utritio n, Sc hoo l of H yg ie ne,Univ er sit y o f T or on to , T or on to , Onta ri o, Can ad a,M 5S 1 A1, a nd F oo d R ese ar ch Ins titu te, A gr ic ultur eC anada, Research Branch, O ttaw a, O ntario, C anada, K 1A OC6

ABSTRACT The effect of treating rapeseed flours with hydrogen peroxide on theglucosinolate content and nutritional value of the protein was examined. Four flourswere prepared from Target variety rapeseed (Brassica napus) by dehulling and de-fatting the seed (sample RF), by heat treating and water washing the dehulled seedprior to defatting (sample W WRF), and by treating a part of samples RF and W WRFw ith solutions of 7 and 3% hydrogen peroxide, respectively. C hem ical analysis show edthat the hydrogen peroxide treatm ent low ered the glucosinolate content of the flour butwas not as effective as the water extraction. The hydrogen peroxide treatment alsooxidized m ethionine to its sulfoxide and sulfone and cysteine to cysteic acid. In the firstexperiment, weanling rats were fed for 3 weeks diets in which casein or each of theflour preparations provided 5, 10, or 20% protein. R ats fed the high glucosinolate-con-taining flour (sample RF) at the 10 or 20% protein level died, while those fed 5%survived but lost w eight. T hose fed the hydrogen peroxide-treated flours survived, butweight gains and food consumption were low compared with the values of the casein-or W WRF-fed groups. Rats fed sample RF exhibited enlarged thyroids. Those fed theperoxide-treated sam ples had high plasm a levels of m ethionine sulfoxide and sulfone.In the second experiment, additions of 0.15 or 0.30% methionine to the 10% proteindiets resulted in increased weight gains of the groups fed the peroxide-treated flours.It was concluded that the hydrogen peroxide treatment was effective in reducing theglucosinolate content of the rapeseed flour. However, the production of the oxidizedsu lfu r am in o ac id s, in p artic ular m ethio nin e su lfo ne, re du ced co nside rab ly the nu tritio nalvalue of the protein. J. Nutr. 105: 317-325, 1975.INDEXING KEY WORDS rapeseed hydrogen peroxide glucosinolate m eth io nin e su lfon e m eth ion in e s ulfo xid e

Rapeseed protein has been shown to be glucosinolates by fungi (3), heavy metalsa protein of high nutritional quality and (4), heat (5), extraction with water (1,therefore of great potential value in human 6-8), or diffusion extraction with ethanolicfood formulations (1, 2). Unfortunately, Na9H (?ii None / these methods is en-rapeseed contains a number of thyroid- tirely satisfactory; they result in a loss ofactive glucosinolates. These compounds Receivedfor publication August i, 1974.., , r , i i 1 Publication number 223 from the Food Researchmust be removed from the protein-contain- institute.in l i. i_ 2 Address reprint requests to : Dr. J . D. Jones Fooding meal, flour, concentrate, Or isolate be- Research Institute, Agriculture Canada, Researchfore they can be used without lim itation in ^SSnSSTt^SSSlSA "Research Grant ecoianimal diets or utilized as a source of pro-tein for human consumption. Canada.ff..,. ^ A resume of part of this study was presented atMany methods or detoxifying rapeseed g annual meeting of the Canadian Federation of, . J i i i- j . ,. r tu Biological Societies, Hamilton, Ontario, June 1!)74,have been used including destruction or the Proceedings17,38. (Atstr.)31 7

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31 8 ANDERSON, LI, JONES AND BENDERbiological value of the protein in some instances as well as being too cumbersomeand expensive for large-scale industrialapplication.The lability of sulfur compounds in thepresence of strong oxidizing agents suggested that a hydrogen peroxide treatm entof rapeseed m eal or flour preparations m ayoffer a sim ple, effective m eans of destroying the sulfur-containing glucosinolates.T his paper reports the effects of such treatment on the glucosinolate content and thenutritional quality of Target variety (highglucosinolate) rapeseed flour in the dietsof w eanling rats.

M ATERIALS AND M ETHODST arget variety rapeseed ( B rassica napus)was cracked in an 8-inch disc mill6 andthen air classified (10) into a hull-rich(30% ) and dehulled seed fractions (70% ).Part of the dehulled seed fraction washexane extracted for 24 hours (sam ple R F).The recovered defatted meats were mixedwith 1% (w/v) hydrogen peroxide (HoO ;.)solution ( m eats-to-H2O 2 ratio of 1:6 w /w )and stirred for 1 hour. The slurry was

dried to 1% moisture at 48in a fluid beddrier e (sam ple R F H 2O 2). A nother portionof the dehulled seed fraction was pouredw ith stirring into boiling w ater (m eats-to-water ratio of 1:5), and boiling was maintained for 2 m inutes w ith stirring. A n equalvolume of water at ambient temperaturewas added, and the mixture was stirred fora further 30 m inutes, the temperature ofthe mass being maintained at 20. Theslurry was filtered through a vibratoryseive7 ( horizontal seive, 16 inches, 80m esh ). The solids w ere further stirred w ithwater at 20for 30 minutes (1:10 w/w )and separated as before. The final residuecollected on the screen was dried at 60,hexane extracted, and pulverized to produce a flour (sample WW RF). A portionof the dehulled, solvent-extracted, w ater-washed flour was treated with 3% H2O2in a sim ilar manner to the treatment of themeats (sample WWRF H,O2). The process and identification of the rapeseedpreparations are sum marized in figure 1.5Baue r B ro s. L td ., Sp ri ng fi el d, I II . a lm lc Eng in ee rin g Co. L td ., C rewe , L an ca sh ir e,Un ited Kingdom.' Se pa ra to r Engine er in g, L td ., Mont re al , Canada .

T arg et v arie ty rap es ee d-a ir c la s si fi ed

d ehul le d s ee d f ra ct io n70%)

defatted

7% H2O 2 w as he d

sample RF H 2O2

hea t t re at edw at er w as he d\IdefattedIsam ple WWRFi3% H2O 2 w ash ed

if

hu ll -r ich f rac tion(30%)

sam ple WWRF H2O2Fig. 1 Summary of process used to produce the rapeseed flour preparations.

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QUALIT Y O F HYDROGEN PE ROXID E-T RE AT ED RAP ESE ED FL OUR 319Two experim ents were conducted. Tox-icity testing and protein efficiency ratingwere determ ined in experiment 1 according to the method of Campbell (11), except that only six rats per group and a

3-week study period were used. Twelvegroups of six w eanling rats 8 were fed dietscontaining 5, 10, or 20% crude protein( N x 6.25) from each of the rapeseed preparations. In addition, another three groupsof six rats each were fed 5, 10, or 20 %p ro te in from cas ein .9In experiment 2, nine groups of six ratseach were fed 10% protein diets fromsamples RF H2O2, WWRF, or WWRFH2O2 when 0, 0.15, or 0.30% methioninewas added to the diet. A lso, three groupsof six rats each were fed casein at the 10%-protein level in diets containing 0, 0.15, or0.30 % added methionine. The basal dietused was as previously described (12). A lladditions of the protein sources w ere m adeby reducing the cornstarch component ofthe basal diet10 by the appropriate am ount.The rats were housed in individual hanging wire-mesh galvanized cages in aroom m aintained at 25 and w ith a 12-hour(8 AM to 8 PM ) light-dark cycle. A ll dietsand tap water were fed ad libitum . Foodintakes and weight gains were recordedweekly.At the end of the study, the rats wereplaced under ether anesthesia and exsanguinated from the abdom inal vena cavausing a heparinized syringe. The thyroidsw ere rem oved and w eighed. B lood sam pleswere immediately centrifuged, and theplasm a proteins were precipitated by adding plasm a to 10% sulfosa licylic acid (1:3).The supernatant w as stored at 20 ntilanalysis of a pooled sample from eachgrou p. A nalysis in duplicate w as com pletedwithin 1 week from the time of sample collection.Acid hydrolysates of the rapeseed samples were prepared by incubating 200 mgof each sample in 100 ml 6 N HC1 for 22hours at 110(13). Analysis of these hydrolysates provided estim ates of all am inoacids, including total m ethionine, exceptfor cysteine and tryptophan. Estim ation ofcysteine content of the protein was determ ined on the acid hydrolysate preparedafter the addition of 2 ml of dimethyl sul-

foxide to the 6 N HC1 to convert all of thecysteine to cysteic acid ( 1 4 ).T o obtain estim ates of tryptophan, m eth ionine sulfone and methionine sulfoxidebase hydrolysates w ere prepared. Tw o hundred m illigram s of sam ple w as hydrolyzedwith barium hydroxide according to theprocedure of Kohler et al. (15). Methionine content of the sam ples w as estim atedby subtracting the methionine found asm ethionine sulfoxide and m ethionine sulfone by base hydrolysis from the totalmethionine recovered as methionine, methionine su lfone, and m ethio nine sulfo xidedur ing ac id hydr olys is .A ll am ino acid analyses w ere conductedon an automatic amino acid analyzer.11The basic amino acids were eluted from aPA-35 resin (5.5 cm height) with 0.25 Nsodium citrate buffer, pH 5.25, at a colum ntem perature of 55 .The tim e for the basicrun was 1 hour with a buffer flow rate of70 ml/hour. Neutral and acid amino acidswere eluted from a UR-30 resin (56 cmheight) with 0.20 N sodium citrate buffersof pH 3.25 and 4.30. The buffer changeoccurred at 90 minutes in the 188-minuterun. Flow rate of the buffer w as 70 m l/hourand the column temperature was maintained at 55 .The nitrogen contents of the sam ples anddiets were determ ined by the sem i-m icro-K jeldahl m ethod using a m ercury catalyst( 16, 17 ). G lucosinolate analysis of therapeseed preparations was performed according to the method of Youngs andW etter (18).The results w ere analyzed using Tukey'sm ultiple t test (19 ).

RESULTSThe protein and glucosinolate contentsof the rapeseed sam ples are shown in table1. Treating the dehulled seed fraction w ith7% H 2O 2 m arkedly reduced the glucosinolate content. However, water washing ofRF was m ore effective, and the glucosinolate content of WWRF was reduced furtherbv treatm ent with 3% H2O 2.8 W i star st rain , W o od lyn L ab oratory L td ., G uelp h,

Ontario .9 C asein , h igh p rotein 88.5% p rotein as su p p liedb asis. T ek lad M ills, D iv ision of A R S /S pr agu e-D aw ley .M a d iso n. W is.10Com posed of (in % ) : corn starch , 87.7 : corn oil,10.0; v itam in m ix , 2.5; m in eral m ix . 3.8 (12).11Beck man Sp in co m od el 121C , B eck man In st rum en ts I nc., Palo A lto, C alif .

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32 0 ANDERSON, LI, JONES AND BENDERTABLE 1

G lu co si no la te a nd p ro te in c on te nt s o f t herapeseed preparations1GlucosinolatesSampleRF

RF H,02WWRFWWRF H2O2Is oth io -

cyanates*Oxazolidi-ethionemg/a4.0812.771.08 1.150.40 0.630.26 0.33Protein%

47.248.862.262.41 M eans of four analyses (air-dry basis),e qui va le nt s o f n -bu ty l i ao th io cy ana te . 2 Reported as

Although the m ethionine, half cystineand tryptophan contents of samples RFand WWRF were sim ilar, treatment ofthese samples with hydrogen peroxidecaused the formation of methionine sulfoxide, m ethionine sulfon e, and cysteic acid,and, in sample RF, destroyed some of thetryptophan (table 2). The oxidative effectof 3% H;,O2 on WWRF was perhapsgreater than the effect of 1% H2O .2 on RF,because the latter treatm ent w as on coarsermaterial lower in protein. None of theother am ino acids w as affected by the treatment.

TABLE 2Su lfu r a min o a cid a nd try pto ph an co nten ts o fthe rapeseed prepara tions1

RFRFHiOjWWRFWWRFjO,mg/16gNMethionineMethioninesulfoxideMethioninesulfoneHalf

cystineCysteicacidTryptophan2.000.080.102.420.071.700.191.051.001.680.611.341.820.130.041R es ul te a re means o f d up li ca te a na ly se s.Experiment 1. All of the rats fed 10 and20% protein diets containing sample RFdied during week 1 of the experiment,probably due to the high glucosinolate and

m yrosinase contents of this preparation.When this material was treated with 1%HO,he rats survived, but weight gainswere negative and unrelated to dietaryprotein level (table 3). Rats fed the heat-treated water-extracted detoxified flour( WWRF ) ate as much and grew as well asthe c asein contro ls. H ow ev er, w hen WWRFwas treated with 3% H2O2, weight gainsTABLE 3E xp erim en t l: w eig ht g ain , fo od co nsu mp tio n, a nd p ro tein e fficien cy r ating d ata

ProteinourceCasein

RFRF H202WWRFWWRF2O2Casein

RFRF H202WWRFWWRF202Casein

RFRF HzOsWWRFWWRF H 02 532 .8

4.12i-8.3 1 .2*-7.3 0.4"5 3.5 2 .3 ei4.0 .3"165.113.92"L67.3 S.O *93.2 6.0ct2 10 .1 4 .0 L105.4 .5eL3.930 . 1 7 ^-2 .5 2 0 .39 6-1 .5 8 0 .0 5r i5 .08 0 .15"0.68 0.23

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32 2 A NDERSO N, LI, JONES A ND BENDERTABLE 6

E xp er im en t 2 : w eig ht g ain , f oo d c on sump ti on , a nd p ro te in e ff ic ie nc y r at in g d ataD ie ta ry pro te insource

D ie ta ry m eth io nio e a dd ed0. 0 0.15 0 .30

CaseinRF HjO jVVWRF\VWRF H20 ,CaseinRF H 202WWRFWWRF H ,02

Weig ht g ain ,1 g /3 w k84. 0 7 .82" '--6.2 0.5r'-9 7.5 4.0 "L2 2.0 1.4 "226.2 16.8L8 1.2 3AcL233 .2 9. 5L118.9 4 .9bL

1 15 .2 13.7 1 04 .5 76.8 4.4'"0.8"4.6i3.7"

i'o od co nsump tio n g /S w k257.8 8 .8' -107.6 3 .9"247 .3 4. 9l209.5 10.2"""

Protein e ff ic iency ra ting

116.7 7 .441.3 3.2 "105.2 5 .9"-79.7 4.9*'"268.6 13.7" '-138.8 5 .6""219.1 16.9L190.8 10.56"'

CaseinRF H2O2WWRFWWRF HZO23 .6 9

0.99^0.770.07at4.180.03"-1.850.09''-4.470.04m1.270.074.220.12L3.680.09

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QUALIT Y O F HYDROGEN P EROX ID E-T RE AT ED RAP ESE ED FL OUR 323TABLE 7E xp er im en t 2 : e ffe ct o f r ap es ee d p re pa ra ti on s o n t hy ro id s iz e

D ie ta ry me th io ni ne a dd ed (%)sourceCasein

RF HsOjWVVRFWWRF H2O20 .0 6.9 50.52'22.302.02cL10.61 0.64"10.740.51tL0.15ma/

100 g bodyt8.210.55a/-21.362.19"9.29 0.797.87 0.560.308.140.59i

22.90 2.7410.601.21L8.330.56i'">1Me an8EM .Me an s w ith d if fe re nt s up er sc ri pts w it hin e ac h c olumn ( su pe rs cr ip ts a a nd 6 ) a nd w it hin e ac h r ow ( su pe rs cr ip tsL a nd m ) a re sig nifica ntly d iffer en t (P < 0 .0 5).

Plasm a m ethionine sulfoxide increased inthe supplem ented groups fed the peroxide-tre ate d flo urs. S im ila rly , th e le ve l o f p la sm amethionine sulfone increased but only inthe R F H 2O 2 group.DISCUSSIONIt is clear from the amino acid analysisof the chem ically detoxified rapeseed flourpreparation that the hydrogen peroxidetreatm ent oxidized not only the glucosino-lates but also the sulfur amino acids.Chemical treatment was effective in reducing the glucosinolate level, but not aseffective as the water washing, a methodsho wn previously to p roduce a hig h q uality,low glucosinolate flour (1, 2). Therefore,in order that the glucosinolates be rem ovedcom pletely from flours prepared from glu-c osin ola te -ric h se ed s, m ore e xte nsiv e tre atment with hydrogen peroxide than described herein would be necessary. A t the

same time, the present treatment had amarked effect in oxidizing m ethionine tomethionine sulfone, a form of this aminoacid unavailable to the grow ing rats (20).4The decrease in nutritional value of theflours caused by chem ical detoxificationcould not be reversed completely by methionine supplementation. In the case ofthe RF HoO o preparation, its high contentof glucosinolates was probably one of theinhibiting factors in the restoration of foodconsumption and growth to normal. Am arked effect was noted by adding methionine to diets formulated with the WWRFH2O2 sample, which contained very lowle ve ls o f g lu co sin ola te s. H ow ev er, a lth ou ghgrow th and food consumption increased,these param eters w ere still som ewhat lowin the supplemented groups comparedwith those of the WWRF-fed groups. Perhaps a sufficient intake of food was inhibited by the m ethionine sulfone contentTABLE 8E xp erim en t 2 : p la sm a s ulfur a mino a cid co nte nts o f ex pe rim en tal g ro up s

Dietary treatment Amino acidaAddedm ethionin eP rote in sourceM ethion ineM ethionine sulfoxideM ethionineulfoneHalfcystineCysteiccidTaurine%

pano les / 100l0.00.150.30CaseinRFH2O2WWRFWWRFH2O2CaseinRFH2O2WWRFWWRFH,O,CaseinRFH2O2WWRFWWRF

H2O26.814.254.192.679.504.785.974.1111.708.008.198.448.651.084.8416.740.957.939.9719.191.298.075.33.

15.199.1814.058.2412.672.563.175.715.955.233.107.

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32 4 AND ERSON , LI, JO NES AND BEN DERof the protein, which led to high levels ofthis am ino acid in the WWRF H^O 2 g roups.In a subsequent study,4 it was observedthat very small additions of methioninesulfone to the diets decreased food intakeb y w ea nlin g ra ts .There is considerable controversy surrounding the accuracy of m ethionine sulf-oxide determ ination in acid or base hy-drolysates of protein ( 21 ) and the separation of m ethionine, m ethionine sulfone,and m ethionine sulfoxide in plasm a has notbeen reported. However, the plasma reflected an increased concentration of methionine sulfone or sulfoxide, which wasproportionate to that found in the H^Oo-treated protein sugg esting that the m ethodsof analysis used in this study were correct.Plasma cysteic acid levels were not as directly related to the cysteic acid level ofthe protein. For exam ple, cysteic acid w ashighest in the WWRF H2O , protein, butplasma levels were highest in the rats fedthe RF H2O2 sample. Furthermore, theplasm a levels of cysteic acid changed littlein response to increased consumption ofprotein co ntaining cysteic acid as occ urredwhen methionine additions were made.Probably cysteic acid levels of the plasm adid not correlate with that consumed because cysteic acid m ay be easily oxidizedfurther to taurine. In contrast, m ethioninesulfone and sulfoxide would need to be reduced prior to oxidation in the metabolicpathways, and this is not a favorable reaction, at least for the sulfone, which appears to be prim arily excreted either beforeor after acetylation (22 ).It is concluded from this study that hydrogen peroxide, a strong oxidizing agent,may be utilized as a means of destroyingrap eseed glu cosinolate s. H ow ever, the p roduction of methionine sulfone, an aminoacid that cannot be used in mammalian systems, and possibly cysteic acid, from meth io nin e a nd c vste in e, re sp ec tiv ely , re du ce sconsiderably the nutritional value of therapese ed protein. T herefore, before hydrogen peroxide treatment can be recommended as a means of destroying thioglu-cosides in rapeseed preparations, morework m ust be done to develop an effectivemethod that is not accom panied by the form ation of the oxidized sulfur am ino acids,in p artic ula r m eth io nin e su lfo ne .

LITERATURE CITED1. Tape, N . W ., Sabry, Z. I. & Eapen, K. E.( 1970 ) Production of rapeseecf flour forhuman consumption. Can. Inst. Food Tech-no l. J . 3 , 7 8-8 1.2. Anderson, G . H. & Sabry, Z. I. (1970) Nutritional quality of rapeseed protein. In: Proceedings of the International Conference onthe Science, Technology and Marketing ofRapeseed and Rapeseed Products, St. Adele,Quebec, September 20-23, 1970, pp. 539-554, R apeseed A ssociation of C anada, O ttaw a.3. Staron, T. ( 1 970 ) A method of biologicallydetoxifying rapeseed m eal. In: Proceedings ofthe International Conference on the Science,Technology and M arketing of Rapeseed andRapeseed Products, St. Adele, Quebec, September 20-23, 1970, pp. 321, Rapeseed Asso ciation o f C an ada , O tta wa.4. Bell, J. M ., Youngs, C. G. & Sallans, H . B.T reatm ent of rapeseed m eal. C anadian Patent839653, April 21, 1970, Canadian PatentsOf fice , Ot tawa .5. Rutkowski, A . (1970) Effect of processingon the chem ical com position of rapeseed m eal.In: Proceedings of the International Conference on the Science, Technology and M arketing of rapeseed and rapeseed products. St.Adele, Quebec, September 20-23, 1970, pp.496, R apeseed A ssociation of C anada, O ttaw a.6. Eapen, K. E., Tape, N . W . & Sims, R. P. A .( 1968 ) New process for the production ofbetter quality rapeseed oil and meal. II. Det ox if ic at io n a nd c le hu ll in g o f r ap es ee ds f ea sibility study. J. Amer. Oil Chem . Soc. 46,52-55.7. Ballester, D ., Rodrigo, R ., Nakouzi, J.,Chichester, C . D., Yanez, E . & M onckeberg,F. (1970) Rapeseed meal. III. A simplem ethod of detoxification. J. Sci. F ood A gr. 21,143-144.8. Ballester, D ., Rodriguez, B., Rojas, M .,Brunser, O ., Reid, A ., Yanez, E. & Monckeberg, F . (1973) Rapeseed meal. IV . Continuous w ater extraction and short-term feeding studies in rats w ith the detoxified product.J. Sci. Food A gr. 24, 127-138.9. Kozlowska, H ., Sosulski, F. W . & Youngs, C .G. ( 1972 ) Extraction of glucosinolatesfrom rapeseeds. C an. Inst. Food S ci. Technol.J . 5 , 1 49 -15 4.10. Hergert, G . B . (1973) A laboratory classifier for separation of ground products byterm inal velocity. Can. Inst. Food Sci. Technol. J. 6, 298-299.11. Campbell, J. A . (1963) Methodology ofProtein Evaluation. Am erican University ofBeirut, Beirut, Lebanon, publication no. 21.12. Musten, B . M ., Peace, D . & Anderson, G . H.(1974) Food intake regulation in the wean

ling rat: self-selection of protein and energy.J. N utr. 1 04 , 5 63 -57 2.13. Blackburn, S . (1968) Amino Acid Determination: Methods and Techniques, p. 13,M arcel D ekker, Inc., N ew Y ork.14. Spencer, R . L. & Wold, F. (1969) A newconvenient method for estimation of total

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QUALITY OF HYDROGEN PEROXIDE-TREATED RAPESEED FLOUR 325cystine-cysteine in proteins. A nal. B iochem .32, 185 -190 .15. Kohler, G . O. & Palter, R . (1967) Studieson methods for amino acid analysis of wheatproducts. Cereal Chem . 44, 512-520.16. Ma, T. S. & Zuazaga, G . (1942) M icro-Kjeldahl determ ination of nitrogen. A newindicator and an im proved rapid method. Ind.E ng. Chem ., A nal. E d. 14, 280.17. A ssociation of O fficial A gricultural C hem ists.(1965) Official M ethods of Analysis, ed. 10.A sso ciatio n of O ffic ia l A gric ultu ra l C hem ists,W ash in gto n, D .C .18. Youngs, C . G. & Wetter, L . R. (1967) M i-crodeterm ination of the m ajor individual iso-

t hi oc ya na te s a nd o xa zo lid in eth io ne in r ap es ee d.J. Amer. Oil Chem . Soc. 44, 551-554.19. Steele, R . G. D. & Torrie, J. H . (1960)Principles and Procedures of Statistics. M cG raw -H ill B ook C o. Inc., N ew Y ork.20. Njaa, L. R . (1962) Utilization of methio-nine sulphoxide and methionine sulphone bythe young rat. Brit. J. Nutr. 16, 571-578.21. Lipton, S. H . & Bodwell, C . E. (1973)Oxidation of amino acids by dimethyl sul-foxide. J. Agr. Food Chem. 21, 235-237.22. Smith, R . C. ( 1 972 ) Acetylation of methionine sulfoxide and methionine sulfone by therat. B iochim . B iophys. A cta 2S 1, 304-309.