.J. COMP.PATH.

1978.VoL.88.

181

GLUTATHIONE ERYTHROCYTES SHEEP AND ITS

PEROXIDASE AND MUSCLE RELATIONSHIP

ACTIVITY IN OF CATTLE AND TO SELENIUAI

BY

P. H.

ANDERSON, MAFF.

SYLVIA Central

Veterinqv

BERRETT Laborntoyy.

~Vew

and D. S. P. Haw.

Wqbridge,

Surrq,

PATTERSON l’.K.

INTRODUCTION

A number of diseases in farm animals (empirically described as selenium responsive) are treated or prevented by selenium administration (Andrews, Hartley and Grant, 1968; Muth, 1970). Four enzyme-catalyzed reactions, 2 in bacteria and 2 in mammals, have been shown to involve a selenium-containing protein (see review by Stadtman, 1974). One of these reactions is catalyzed b) glutathione peroxidase (GSHPx; E.C. 1.11.1.9), and its activity in blood and tissuesof the rat has been shown to be dependent upon the selenium content of’ the diet (Rotruck, Pope, Ganther, Swanson, Hafeman and Hoekstra, 1973; Reddy and Tappel, 1974; Chow and Tappel, 1974; Hafeman, Sunde and Hoekstra, 1974; Smith, Tappel and Chow, 1974). Furthermore, it has bcru proposed (Rotruck et al., 1973) that many of the nutritional effects of selenium could be explained by the action of GSHPx. More specifically erythrocyte GSHPx of both cattle (Flohe, Giinzler and Schock, 1973) and sheep !Oh, Ganther and Hoekstra, 1974) has been shown to contain 4 atoms of selenium per molecule of enzyme. Following these discoveries, there has been considerable interest in the relationship between erythrocyte GSHPx activity and blood selenium concentrations as selenium/vitamin E deficiency diseases are still prevalent in farm animals. Allen, Parr, Anderson, Berrett, Bradley and Patterson (1975) and Boyd (1975) described the correlation between erythrocyte GSHPs activity and blood selenium concentration in cattle, and Wilson and Judson (1976) and Thompson, McMurray and Blanchflowcr (1976) made sin&u observations in cattle and sheep. This paper describes an investigation into the correlation between erythroc\,tc GSHPx activity and blood selenium concentration in cattle and a less extensive study in sheep. The effects of selenium deprivation and treatment on erythroq-tc. and muscle GSHPx activities are also described. MATERIALS

Animals

and blood sampling. Table

AND

METHODS

1 shows the types of animals

used for the various

experiments. All blood sampleswere taken by jugular venepuncture into heparinized “Vacutainer” (Becton-Dickinson) tubes. Feeding regimen. To study the effect of a low selenium intake on the erythrocyte and muscle GSHPx activities of yearling cattle, a diet was prepared from kibbled barley?

182

P.

H.

ANDERSON,

S. BERRETT

AND

D.

S. P.

PATTERSON

urea, hay and a mineral/vitamin supplement. The hay and barley had been obtained from areas in Britain which had been shown previously to produce forage of low selenium concentration. Analyses of this diet showed that the total selenium content was about 0.01 pg selenium per g feed. TABLE 1 A DESCRIPTION

Description

OF THE

ANIMALS USED AND THEIR INDICATED IN MATERIALS

hitmbers animals

of experiment

DIETS IX THE AND METHODS

SERIES

OF EXPERIMENTS

of Description

of animals

-___ The correlation between erythrocyte glutathione peroxidase activity and whole blood selenium

The effect of selenium intake on erythrocyte and muscle glutathione peroxidase activity and whole blood selenium

143 Cattle

hlostly Friesian or Friesian cross, between 64 and 18 months old, located on 15 farms in England or Wales. Diet essentially locally-grown herbage.

48 Cattle

Field cases submitted from Veterinary Investigation Centres in England and Wales. Breed and age not known.

25 Sheep

Cross-bred bridge, hay.

18 Sheep

Field cases from animals in England and Wales. Breed and age unknown.

15 Cattle

Friesian or Friesian cross yearlings on a diet containing about 0.01 pg per g selenium. Five were injected subcutaneously every 2 weeks with 2.5 mg selenium as sodium selenite and 750 mg vitamin E on 5 occasions.

adults located fed locally-grown

at Weybarley

and

Muscle specimens. Thirteen of the 15 yearling cattle on a low selenium intake with or without selenium injections (Table 1) were killed by stunning with a captive bolt followed by exsanguination. A small piece of each of 10 muscles, namely the proximal end of the biceps femoris, the semimembranosus, the middle gluteal, the supraspinatus, the infraspinatus, the triceps brachii, the ulnzris lateralis, the crura of the diaphragm, the masseter and the heart were taken, blotted and placed on ice in a polythene bag for transport to the laboratory. Homogenates of muscle in water were prepared within 2 h of removal from the carcase. Biochemical analyses. (a) Glutathione peroxidase. Blood samples were prepared for the assay of GSHPx activity by the addition of an equal volume of deionized water to whole blood. After centrifugation at 1000 g for 15 min the supernatant was removed for enzyme assay. Dilutions in deionized water, usually 1:6, gave a suitable rate of reaction, The assay was either carried out the same day or the lysate was stored at -20 “C until assayed. The packed cell volume of the whole blood was measured in a microhaematocrit centrifuge, and as the GSHPx activities of cattle and sheep sera are negligible, the activity of the whole blood lysate was expressed in units per ml of erthyroctyes. To determine the GSHPx activity of muscle, a 1 g piece of muscle was cut up with scissors, homogenized in 9 ml of deionized water with a “Silversc In”.. homogenizer/ _ emulsifier and centrifuged at 1000 g for 15 min to remove cell debris. Aliquots of the

GLUTATHIONE

PEROXIDASE

AND

183

SELENIUhf

supernatant were used for the assay of enzyme activity, which was expressed in units per g of wet muscle. Glutathione peroxidase activity in the blood lysate or muscle homogenate \vas assayed by the method of Paglia and Valentine (1967). Modifications described h) Allen, Parr, Anderson, Berrett, Bradley and Patterson (1975) were incorporated but further changes were also made, Cumene hydraperoxide replaced hydrogen peroxide as the substrate; a 10 mmol solution was prepared by the addition of 0.1 ml cumcm hydroperoxide (Koch-Light) to 50 ml ethanol, and this provided a final concentration of 0.33 mmol of substrate in the reaction mixture. Assays were carried out on a Vitatron “Automatic Kinetic Enzyme System (AKES)” analyser. ;I unit of activit) in equivalent tn 1 pmol of GSH oxidized per min at 30 “C:. (b,) Selenium. Concentrations of selenium in whole blood (and a few feed samplrs I were determined by the fluorimetric methodofWatkinson (1966) as modified by W. H. Parr (personal communication). The principal modification was in the digestion of samples, in which a mixture of concentrated acids (nitric, 5 ml; perchlorlr, 2 ml; sulphuric, 2 ml) were used to digest 1 ml of blood completely in about 30 min. Selenium values were expressed as pg Se per ml of whole blood. RESULTS

Er_l;thl-oqyte Glutathione Peroxidase Activity and I/Vhole Blood Selenium Chnceutraiion The correlations between erythrocyte GSHPx activity and whole blood selenium concentration in cattle and sheep are shown in Figs 1 and 2 rcspcctively, the slopes of the regression lines for the 2 species being significantly different- (P < 0.001).

Whole

I’ig.

blood

selenium

i . The correlation between erythrocyte glutathione in 191 cattle. The regression line isy = 296+3-7.257.

pg

per

peroxidase activity (~1 ----I)

ml and 95 per

whole blood cent confidence

seleniuul limits.

Figure 3(a), (b) sh ows the changes in erythrocyte GSHPx activity and wholt: blood selenium in a group of 10 calves on a diet with a selenium content of about 0.01 pg per g and another group of calves on the same diet receiving 2.5 mg of selenium by subcutaneous injection every 2 weeks. The increases of both mean selenium concentration and GSHPx activity in the in.jectcd calves were significant at the P < 0.05 and P < 0.001 levels respectively. The drcrcnsc. in mean GSHPx activity in untreated calves was also statistically significant (P -; 0.001).

P. H. ANDERSON,

G

r

S. BERRETT

0.05

AND

D. S. P. PATTERSON

0.15

0 IO

Whole

”

w

blood

selenium

Fig. 2. The correlation between erythrocyte glutathione in 43 sheep. The regression line isy = 572.7x-

O-20

pg

per

ml

peroxidase activity and whole blood selenium 15.18. (O---O) 95 per cent confidence limits.

Muscle Glutathione Peroxidase Activities

Table 2 shows the geometric means of the GSHPx activities of 10 muscles and the erythrocytes of 9 calves on a low selenium intake for 10 weeks and TABLE 2 THE

GLUTATHIONE

PEROXIDASE ACTIVITY SELENIUM-INJECTED

Level of signiJicance of dzyerence between uninjected and injected

Glutathione peroxidase activity* (geometric mean) (ininjected

Semimembranosus Biceps femoris Middle gluteal Supraspinatus Infraspinatus Triceps brachii Ulnaris lateralis Masseter Diaphragm Heart Erythrocytes * Glutathione

peroxidase

AND

4

Ratio of geometric mean injected :uninjected

Injected

9 169 16.4 102 224 29.4 187 899 359 1390

757 1880 1080 1300 1770 1120 1230 6420 3080 8010

5.4

26.9

activity

OF 10 MUSCLES IN 9 UNINJECTED YEARLING CATTLE

is expressed

P P P P P P P P P P

i < = -: < < < < i,