Variation of erythrocyte glutathione peroxidase activity in Finn sheep

?searchin Veterinary Science 1981, 31. 267-271

'ariation of erythrocyte glutathione peroxidase activity in Finn sheep ,

ATROSHI. S. SANKARI Medicine.

OSTERBERG epartnlent of Anintar rrreeawg. ~grrcurrurarKesearcn Lenrre, Vantaa, Finland

orrege oj verennary Mearcme, HelSlnKI 33, rrn~anc

J.IIIVLJLC gI~tathiok~r peruxiuax t u z i n - r h ) wriiLr1 was analysed on a flock of FilIn sheep (266 sheep) showed variable activity despit e the Samle diet an( selenium intake. Mating experilments sug,gested tha t .. aetermined. .. the GSH-Px activity was genetically The IW GSH-Px animals showed better perf(Irmance a:s lown by a significantly reduced mortal ity rate o f leir offspring. I.:vidence of larger weigh~t gain an(i ool production in the low GSH-Px sheep was btained. The GSH-Px activity slightly decreased wit11 Ce. The erythrocyte GSH-Px activity did not shovv ~uchcorrelation with other genetical markers such a!s le potassium type. GSH type. haemoelobin type 01 :x. It is prolpo&i t h at ~thelow c t i adaptaticon to low !wlenium i~

uvllle a l ~ dhtluralr G I J L I I I U C Y L G > 1143 D I I U W I I L U L U I I L ~ fouraltoms of selenium per enzyme m olecule (Fllohe et al 197:3, Oh et al 1974a, A7tvasthi et a 1 1975). InI every anima I species researched,, including: sheep, a linear .. relationship between the erytnrocyre GSH-Px a ctivitv -.....-I and blood selenium content or selenium intakLe was established (Oh et al 1974b, Allen et al 1975, Thompson et al 1976, Wilson and Judson 19761). The determination of tissue and blood GSH-Px a A+:..:&.. rrlvlly has been widely used to assess the selenium staItus of anima Is. It is notable that in several figures put)lished concerning the correlation of GSH-Px and selenium - - - -can - see that the distribution of the values 1 uric no not represlent a norrr Ition but r;ather a bin~ormal one (I411en et a Vilson antd Judson 1976, Thoml et.al". -sele . pson nium deticiency In sheep has been con nected CJLUI A I H I U N L peroxlaase (LC I . I I.I.Y., t i b ~ with the occurrence of nutritional mu~scular Px) catalyses the reduction of a large variety o degent:ration and poor performance in lambs hydroperoxides using GSH as the reducing agen IFlohe et al 1971). Most o f the acceptor substrates o. (Anderson and Patterson 1976, Paynter et al 19'79). It SH-Px may be cons,idered as undesirable products has also been proposed that selenium defi ciency -king from unspecilTic oxidative processes of free affects the reproduction (Underwood 1977). So far tdical reactions, !such as hydroperoxides of practically no work has been done on the assoc:iation between GSH-Px and other blood parameters ;as well ?saturated lipids, n N A etc ( F l orPr I 7 r u) as the producti on characters of iheep. No other eroxidatio n of the U Imaturated lipids of rnembrane factor:s other tha n the selenium status,has been lmown 3s been c laimed toI result irI an imp;~irment o afff :ct the GSIH-Px activity in she€:p. to ne functio n. This haIS been thc~ g h to iornembra~ . . . . .t bc T- h..e present research was undertaken to inve le immea~atepathogenet~cmecnanlsm In selenium utrition-independent factors determinir~g the ni the tficiency diseases such as nutritional muscula r GSH-IPx activity. The erythrocyte GSH-Px a ctivity :generation (Hoekstra 1975, Combs et al 1975). -. ar~alysedagainst other genetical markers such as GSH-Px was discovered by Mills (195'1) ana wa .,.I.was P ( . -. as .. . K-tvne. .. , haemoelobin type as well 3dbsequently shown to be present in a wide variety o f L"c U'SH-tvne. the pn oduction c :haracteris tics. tissues including red blood cells (Ganther et al 1976)

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Rotruck et a1 (1973) showed that GSH-Px activity ir blood and tissues was denendent on dietarv seleniun take. Subsequently solated frc)m bovine , 267

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ials and m ethods A flock consisting of 266 Finn sheep was analysed.

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S. Sankari, S. Osterberg, M. Sandhe'"'

The a1~ i m a l incl s uded ewes and rams between o ne and six yezu s old ancI their thre:e-month-(,Id lambs. c:.,,,< -1 r r tF Lvl ~ r r rvr A r nulul rn cr *A ,r r u. . . Iu y juEulal :..-..I..zrlt v u u w.or a3 c uncture into Vac tubes (B lson) contiaining K I m anticoal

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bis-(2-nitr,o benzoic acid) in 1(10 ml of tl w(as added (Ellman 11959). Aft 4I I V C lrluruica ."buffer, * I... auauluarlCc -I.-- -I.--,... ...a a -' A * ? -irrc waa,. -,.--..--. IIrcaLurcu LL ~ I 1114 L 3SH stanclards contiaining 8, LI,2, 1, 0.:5 and 0 ml 3SH in 1 nnM Na,EI: )TA were 1processed in parallel I

-".",nlrnatron OJ CYAH-Y: ,

On1: hundreci pl of packed erythrocytes was haemcAysed in 900 pl of distilled \uater and stored frozer1 Ior one week until.. analysed (Table 1). The ana lysis was done by the method used by Giinzller et al (197'4) but slightly modified to suit the Gilford ISystem 35a3and ovineerythroc'ytes. The assay system c:ouples the GSH-Px reaction (tertiary butyl hydrope:roxide ana GSH) with the glutathione reductase system. In this coupled assay the glutathione is continuously regenerated by glutathione reductase and NADPH. --.. . of oxidation of NADPH was recorded at 340 The rate nm ac~d37°C. The: effort of detecting any gene1tic influen done Iby groupirig the shee:p accordi,ng to their D* v ", :tivity. The peroxidase activity of the erythr ocytes was classifiec1 high if it was more than lo0Ocikat per litre or low i f i t was le!5s than 1000 pkat per litre. These two grc,ups of s,beep were then .- -.~. - - - - -.. ..compi for other blood paramerers and production charac

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Uetermrnatron oJ fhe haenloglobln concentratron The determination was done by the cyanmethaemoglobin method. Twenty pI of whole blood 1was pipette:d into 5 m l of modif ied Drabkiin's solutic 2ind the A.540 measu red (Van Kampen iand Zijlst 11961). ~eternlmatronoJthehaenloglob~ntYPe

The blood cells were washed four times wltn 5 ml ot 0 . g Der cent sodium chloride and 100 of the packr :ells were Ideep froze:n to haennolyse the cells. Zor blectropho~resis on sta.rch slab ge-1swas used to separa he haemo;globins according tc their electrophoret -".-...". 1Qfm r . ... (Braend and. ktrpmnv . 1 lin types 1vere classi fied accor Ire suggest:ed by Har ris and Wa

beterntmotron tration

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erythrocyte potasslum concen-

~fGSH (acrid soluble thiols) Deteri One hundred pI of washed, packed erythrocytes was . . . .. Freshly collected blood (1.5 ml) was centrifuged haemolysed in 9.9 ml of distilled water. TI using an Eppendorf centrifuge (12,000 rpm for two 'Ontent was determined by flan minutes). The plasma was removed by suction and 5 0 0 photometry at 766 nm (Unicam SP-90 with flan p. nf .,. the packed cells [ransferred to another,. ttthe photometry attachment). The K - t ~ ~ ethe sheep w' contai ning 5 0 0 pl of 10 per cent sulphosalicyli c acid, :lassified h~ighif the K concent ration excc:eded 19 n mixed and cooled on ice for one hour. The 1)rotein )er litre, arid low if ttie concent ration was less than : precip itate was spun to the bottom of the tube by the ng per litrc.. r--,... =lJlJc'~ d o r fcentrifuge and 200 ul of the super I I a L a I l l trans6erred into a tube containing 8 ml of I0 . 3 M I: 'roduction characteristics phosp hate buffer pH 6.13. An am ount of sodium hydro xide was added !50 as to neutrali: ie the The blood parameters measured were compared I * . - - - . .. .. SUlphC)salicylic acid (78 pl o f I N soalum hydrox~deper the litter size born, litter size alive, weight at 12 hour hree days and weeklly weighin gs, weight of the ea )er cent :iulphosalic:ylic acid) 5 0 0 fJ .nd wool p roduction hundr lman reag ent consislting of 1OC I

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TABLE 1: Determination of GSH-hl Reagent

Volume

-PackecI cells 100 pl distillelj water 900 PI

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Final concentration

Men

1's solution Drabki~

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P h ~ s p.=+a t . " .h,,ffa. ~ 0.25 M , pH 7.0 with 2.5 m M EDTA NADPti GSH GSSG-reductase El Tertiary butyl hydroperoxide

1.25ml

0.20 m M

0.25 rnl

0.33 Ulml 0.85

4.0mM

'-Px activic TABLE 2: The association between haemoglobin. glutathione z Finn sheep (means t SE) GSH High

Low

igh

(rnmolllitre)

-GSH low GSH high Potassium low

1;

Potassium high

1:

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ey 1970) w,as lnce (Harv~ Least sq uare analy: ~ s e dto ass;ess the re1 between GiSH-Px lev,el --., marrters, numoer . ." .- L--inu "nenerlcal or larnos Durn arld . .. --weaned, ewe bodyvveight an(i wool weight, Ian'b weight at clifferent a13es. Chi scp a r e anal!pis was al!$0 lsed to examine diffe'rences am1ong the GS;H-Px type:S' 3

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The erytnrocyte u s t i - r x acrlvlty me; Im individuals of the flock kept on the same :fr lives showed a wide range of activities (rlg I ) . I r'1s variation does not come from analytical errc": coefficient of variation between replicate analyses was 6.2 per cent. The association between the Hb typ'ep GSH type , potassiurn type and Hb concentration as related wit h the GSkI-Px activity is show n in Table 2. 1 266 sheep

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LLE", l'GJ Ix1hen vv I the sheep were divided into two cat---:-accol*ding to their erythrocyte GSH-Px activity ter o r less than 1000 pkat per litre) and the:groups thus formed compared for their production charac[erlstics (Table 3), the only significant differe nce was seen in the production of living offspring. Lambs from low GSH-Px sheep gave birth to 3 . 24 living offsl: . . . ring compared with the average of 2.59 for the hlgh peroxidase group. There were also somle other prod uction characters in favour o f t he low per,oxidase groulp: greater bodyweight at three and six Hleeks as well i3s better wool production. Th e age of the sheep did have some bearin g on its GSH -Px activity (Fig 2). The enzyme activ,ity was somewhat higher in the lambs compared wit.h adult ewes.. Also the activity slightly declined from 01 ne to six .. years of age. TIie age dist~ ribution orf the high iand low .._I..

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TABL,E 3: The relationship bet1ween erythrcx y t e GSH-Px activity and PI,oduction characteristics in Finn shee!P

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Glutathiorle peroxidas FIG I: Dicrribution of erythrocyte GSH-Px activity in a flock of ;!66 Finn chccp. Thc GSH-Pt activity is expressed as Pkat per line of packed eryth~

Litter size born Litter size alive Weight at 12 hours (kg) Weight at 3 days (kg) ight at 1 week (kg) ight at 2 weeks (kg) ight at 3 weeks (kg) ight at 4 weeks (kg) ,-5t at 5 week? IL-' ~tat 6 week! Ewe vveight (kg) Wool weight Ikg)

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NS Not s~gnif~cant Significant at P = 0'01

Sheep wit) low GSH-P activity (means)

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4.185 5,637 7.279 8.857 +n.r n c

ep with GSH-PX :tivity leans)

F. Atroshi, S. Sankari, S. Usterberg, M. Sanclhnlnr

13y a single pair of au tosomal alleles, the g:ene for hi1 3SH-PX b eing domi nant to th at for low GSH-Px. . .. . . --. appears tnat tne lnnerltance or tne erytnrocyte clslygenic in I IPx activity in Finn stleep is pol

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Discussion

- . . It has been claimed that a low W H - r x actlvity is ; indicator of poor selenium status and represents a ri of nutritional muscular degeneration particularly f rapidly growing young animals (Anderson at Patterson 1977, Whanger et al 1978). The prese results are contrary to the concept of using GSH-I purely as an indicator of selenium intake and :riterion for animal performance. Ma1 mvestigaticons have snown a linear relationsh between thle seleniumI status antj theeryth rocyte GS1 Px activit] of anim;als raised on differc:nt seleniu intakes.

u 1 I n sheep.

Effect of agc

. .. . L GSH-P x actlvllv IS exuresseu as UKar per llrre paCKeU eryrnrurytes. Means +SE are indicated. Th e line fo y=143 6-44x, whet.e y = GSH-P:x activity and ..-:--I I-x dlllllldlb wab u3n-n-.

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effect of age can be neglected when comparing these g r o wIS of animals for their perform lance. Alslo when e were ex;amined ewes . . . of high and low GSH-Px typ . .. wtth~r 1 age grouping (one to sir: years), slmllar differ ence in per was seen vvithin each pair of g r o wIS.. . . Tat)le 4 snows Dreeaing aata lor m e a, Matir~g between low GSH-PX sheep resulted in low GSH-Px in the erythrocytes of their offspring as meas1x e d at three months of age. On the other hand matin g between high GSH-Px types resulted in both .nign . .s 3es b f offs pring. The,se results s hat the GISH-Px levels are cor the h! TABLE 4: Breeding data of GSH-PX types tn mnn snec

Sire numbe r

GSH-I'x type of P: Irents Sir1e Dam

Number G SH-Px type of lambs High Low of lambs

The pt.esent study suggests two additior variations: one of genetic origin and the other ag change in the GSH-Px activity. The effc dependent 'of . .... different selenium intakes was overcome in t present study by analysing sheep from a single flock with uniform diet. It would appear that the gene tic heterogeneity in the GSH-Px activity (Fig 1) is 1somewhat ~ masked by the age-dependent effect I- IGSH-Px (Fig 2). It :ieems reas onable to assume th 1the groups would ha ve been m ore neatly separated Ithe animals were all of the sawle age. The lam~ b swere not analysed until they were thr'ee months olci a s i t h a s t Ieen showrI that fetal erythrocyl:es tin variab le amount s of GSHI-Px activiity may cont: . . (Ganther 1976). [ f animals on different selenium i ntakes wf :re compared to establish the genetic diffe rence in t he erythrocyte GSH-px activity, one shoul d rememt~ e r that the selenium is incorporated into the a- w v .-c slrc- o f GSH-PX dluring erythrocyte maturation and b e c a ~ the average life span of an erythrocyte is some three four mont hs, the erythrocyte GSH-Px activity reflec the mean selenium supply during the last months. As the animals \:vere classified into high or low GSH-Px types, it was pos!;ible to compare the production characte,r with the phenotype. A striking lgrl l l l y of offspring in the hi-'result was the higher l l t u r l aI:... GSH-Px group. Some idea of better performance in the low GSH-Px animals may be seen in Table 4. T'he low GSH-Px animals had greater bodyweight a nd yielded more wool than the high GSH-Px anim although statistically significant-limits were r reached. Similarly lambs from the low GSH-Px she:ep were heavier at three and six weeks old. T his difference is striking because one wouldl anticipata-r alower milk yield gain per lam b in cases when a e we drops a high number of lambs I t can be noted that the G S n - r n a u r r r t y wab riot higher in the low GSH type of sheep but the contrary #

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8 13

2085

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activity > 1000pkat per litre w a s classified tiigh IH) GSt4-Px . .. . ... . . GSt1-tJx actlvlty < 1000pkat per litre was classlttea I ow IL) ~berofdams

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on in shee, de was evident. This means that in ti detoxification mechanism, the low GSH substra.-, t P level is not compensated with a higher enzyme activity. Genetic variation in the GSH-Px activity may have given an opportunity for selection among sheep population. The low peroxidase activity may represent an adaptation to low selenium intake. Plant a11d :ereals grown in Finland are known to be extreme:Iv poor in selenium and selenium deficiency diseas es en such as nutritional muscular degeneration have bec.. :ommon in Finland (Oksanen and Sandholm 1970). Further research is needed to establish if the low GSHpx really represents an adaptation to low selenium ntake. It would be important to pinpoint tt-a lib 3iochemical difference, if-alternative "on seleniu m iependent pathways of peroxide detoxification occur n the low GSH-Px sheep. Such information would 1be mportant in the search for .the pc,sslolllty 1O r selecting animals resistant to seleniu m 0

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Received for public

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COMESS, G.F.. NC .. -.

Pro1:emurgs M,1

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E L L M A N , ~ . L .(1959)Archrveso~BiochenlislryandBiop 70 FLOH Ciba Founda ium, June 695-1 ZLER, W., JIUNG, G. & SCHAICH. FLOH "-.. ^:.^"I...:C, rile ..,-.L..-:-qecrrrrtrrr J J L,,IJY-lOY "UP pr arym 3 L r r r a r r r r y t j u r phy~iologis~hr FLOH 8, L.. GUNZLER, W.A K, H.H. (19'73) FEBS Lett,e n 3 2 , 132-1 34 GANT'HER, H.E. , HAFEMPiN. D.G., LAWRENCI3. R.A., .- . - -.
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ober 6 , 191

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