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The estimation of α-tocopherol in pasture samplesand a comparison of α-tocopherol levels in samplesfrom areas differing inselenium statusK.R. Millar aa Wallaceville Animal Research Centre ,Department of Agriculture , Private Bag,Wellington , New ZealandPublished online: 24 Jan 2012.

To cite this article: K.R. Millar (1971) The estimation of α-tocopherol in pasturesamples and a comparison of α-tocopherol levels in samples from areas differing inselenium status, New Zealand Journal of Agricultural Research, 14:1, 142-148, DOI:10.1080/00288233.1971.10421310

To link to this article: http://dx.doi.org/10.1080/00288233.1971.10421310

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THE ESTIMATION OF a·TOCOPHEROL IN PASTURESAMPLES AND A COMPARISON OF a·TOCOPHEROLLEVELS IN SAMPLES FROM AREAS DIFFERING IN

SELENIUM STATUS

By K. R. MILLAR*

(Received 4 September 1970)

ABSTRACT

Extraction of pasture samples with acetone immediately aftercollection prevented losses of a-tocopherol for up to 11 days. For theanalysis of a-tocopherol, column chromatography was a preferred alter­native to thin-layer chromatography, and use of the method describedenabled recoveries of 3H-a-tocopherol usually greater than 80%.

No relationship was found between a-tocopherol concentrations andselenium concentrations in pasture samples collected from areas withdiffering selenium status.

INTRODUCTION

Myopathies occurring in New Zealand sheep from selenium-deficientareas appear to be indistinguishable from those reported in the literaturefor animals affected with a presumed vitamin E deficiency (Hartley andDodd 1957). The physiological roles of selenium and a-tocopherol remainobscure, although it is claimed by some workers that both functionmainly, if not solely, as antioxidants, protecting animal tissues againstlipid peroxidation (Tappel 1965). Desai and Scott (1965) suggest thatselenium may enhance vitamin E activity through its effects on tocopherolretention and prevention of tocopherol destruction in blood and cellularconstituents. However, more fundamental roles, particularly for selenium,have been proposed (Schwarz 1962).

Compounds of selenium identified in tissue from animalsdosed with Se75-labelled sodium selenite resemble those foundin some plants. (Peterson and Butler 1962; Virupaksha and Shrift 1965).Thus selenium is associated with proteins, and may also be present asfree seleno-amino acids. If, as the antioxidant theory suggests, seleniumcould have a sparing effect on a-tocopherol in animal tissues, a similareffect might pertain in plants. Moreover, although there is limitedevidence suggesting that, for sheep, a-tocopherol is ineffective againstselenium-responsive unthriftiness, or selenium-responsive infertility

* Wallaceville Animal Research Centre, Department of Agriculture, Private Bag,Wellington, New Zealand.

N.Z. Journal of Agricultural Research (1971), 14: 142-8

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K. R. MILLAR 143

(Hartley 1963), it appears to be at least partially effective in controllingwhite muscle disease (Drake, Grant, and Hartley 1960), and the possi­bility of some major involvement of the vitamin in the selenium meta­bolism of grazing animals is by no means excluded. Hence it is of interestto investigate the a-tocopherol status of pastures on which animalsbecome selenium-deficient.

The most satisfactory method at present available for the extractionof a-tocopherol from plant material appears to be that of Booth (1959),which involves grinding a small sample with acetone, followed by extrac­tion into hexane. Unfortunately, when leaves are damaged, tocopherolsare lost by the action of enzymes released during maceration of thesample before extraction (Booth 1962). Because of this limitation,pasture samples must be extracted immediately.

The present paper' describes a method based on the maceration ofpasture with solvents immediately after collection. This extract can thenbe transported to the laboratory and analysed for a-tocopherol severaldays after the initial collection. Results are presented for the a-tocopherolcontent of samples of pasture (predominantly white clover (Trifoliumrepens) and perennial ryegrass (Lolium perenne» from a known selen­ium-deficient area in New Zealand, an area with a marginal seleniumdeficiency, and an area known to have high pasture-selenium concen­trations.

RECOMMENDED ANALYTICAL METHOD

Extraction of a-tocopherol from pasture

Samples of pasture (4 g) are homogenised with 50 ml of redistilledacetone and 1 g of purified sand (BDH) for 2 min in a homogeniser(M.S.E.). Fifty millilitres of light petroleum (b.p. 4Q-60°c) is added,and the mixture homogenised for 15 sec. The supernatant is decantedinto a measuring cylinder, and the extraction procedure repeated twicemore.. The volume of the combined supernatants is adjusted to 400 mlwith a mixture of acetone and light petroleum (1: 1), and 1 ml of anethanol solution of 3H-labelled a-tocopherol (DL-a-tocopherol (5­methyl-T) The Radiochemical Centre, Amersham) having a specificactivity of 280 clsl fLg is added to monitor recoveries. This solution canthen be kept at room temperature for several days without appreciableloss of a-tocopherol.

Duplicate 30 ml volumes are removed from the combined super­natants and washed free of acetone with five washings using an equalvolume of water. Vigorous shaking with water should be avoided tolessen the risk of emulsion formation. The light petroleum fraction re­maining is dried with 2 g of anhydrous sodium sulphate and evaporatedto dryness at 40°c under nitrogen.

Column chromatography

The residue is dissolved in a minimum volume of hexane (0.2-0.5ml) and applied to a column of alumina (0.6 g), basic zinc carbonate(0.6 g), and celite 545 (0.3 g), prepared according to the method of

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144 a-tocopherol in pasture samples

Bieri, Pollard, Prange, and Dam (1961). The column is developed witha mixture of benzene and cyclohexane (1: 4 v/ v). The first 3 m1 fromthe column contains p-carotene and is discarded. The next 12 ml, con­taining a-tocopherol, is collected and evaporated to dryness.

Estimation of a-tocopherol

The residue, containing a-tocopherol, is dissolved in 1.6 ml ofethanol, and 0.1 ml is removed for counting to determine the recovery(Millar and Caravaggi 1970). a-tocopherol in the remaining 15 ml isestimated by the method of Tsen (1961) using bathophenanthroline(4,7-diphenyl-l, 1O-phenanthroline).

EXPERIMENTAL AND RESULTS

Examination of plant extracts for a-tocopherol esters

If a-tocopherol is present in plant material in the form of esters,a hydrolysis step would be required to free the a-tocopherol for chroma­tography and estimation.

Samples of clover or ryegrass (0.6 g) were extracted by the methodof Booth (1959). The extracts were concentrated and applied as bandsto thin layers (250 mfL) of silica gel G. The thin-layer chromatogramswere developed in cyclohexane-ether (80: 20) as described by Stahl(1965). Areas of the plate corresponding to marker spots of a-tocopherol

TABLE I--Stability of Acetone-Petroleum Ether Extracts of a-Tocopherol Storedat Different Temperatures

Recovery of a-tocopherolStorage Time (days) 3H-a-tocopherol content of

temperature (%) pasture*(Il-g/g)

0 84.5 59.7

Ambient 2 85.2 58.8

(20 0 e) 4 83.6 60.7

II 75.3 54.0

0 86.2 60.7

2°e 4 83.8 59.1

11 84.5 60.3

0 81.3 57.3

-75°e 4 73.1 58.9

II 65.0 58.6

* Corrected for re~overy

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K. R. MILLAR 145

acetate, a-tocopherol phosphate, and a-tocopherol succinate were re­moved and eluted with 10 m! of acetone followed by 10 ml of ether.The acetone and ether extracts were combined, evaporated to dryness,and the residue hydrolysed with a mixture of ethanol (3 ml), water(3 ml), and 50% wjv potassium hydroxide (0.3 ml) for 15 min at 90°c.The hydrolysis mixtures were cooled in ice and extracted three timeswith 20 ml volumes of ether. The combined ether extracts were concen­trated to 0.2 ml and applied as 1 cm bands to a silica gel G thin-layerplate which was developed with cyclohexane-ether as described above.The areas corresponding to a-tocopherol marker spots were removed andeluted with 10 ml of acetone and 10 ml of ether. The solvents wereremoved at 40°c under nitrogen, and the residue dissolved in 1.5 mlof ethanol for a-tocopherol estimation.

For neither white, clover nor ryegrass was there evidence for thepresence of esters of a-tocopherol in any samples examined.

Stability of a-tocopherol in acetone-petroleum ether extracts

A 4 g sample of pasture i(predominantly white clover and ryegrass)was extracted with acetone-petroleum ether by the recommended method.To the combined extract was added 15.5 /Lg of 3H-a-tocophero1 (specificactivity 280 cjsj /Lg). This solution was divided into three parts, one ofwhich was stored at room temperature, another at 2°c, and the third at-75°c. At successive times 30 ml aliquots from each solution werewashed with water, chromatographed on alumina-basic zinc carbonate­celite 545 columns, and estimated for a-tocopherol. The recovery of3H-a-tocopherol and the corrected a-tocopherol content of the originalpasture (expressed as /Lgjg wet wt) are given in Table 1. .

In the sample stored at -75°c the acetone-petroleum ether mixtureseparated into two phases, which probably explains the lower recovery,after equivalent times, of 3H-a-tocopherol in this sample.

Thin-layer chromatography as an alternative to column chromatographyAny system chosen for the chromatography of pasture extracts must

be capable of separating a-tocopherol free of interfering materials,particularly the coloured chlorophylls and carotenoids. Dilly and Crane(1963) and Roughan (1967) describe thin-layer chromatographicmethods for the estimation of a-tocopherol in plant material. Despite thefact that the thin-layer method is slower than the column method, it wasinvestigated as an alternative.

The residue from 30 ml of an acetone-petroleum ether extract ofpasture was dissolved in chloroform (0.2 ml) and applied as a 10 cmband to a silica gel G plate (250 m,a layer). After development in ben­zene the plate was sprayed with a mixture of equal parts of batho­phenanthroline (6 X 1O-3M in ethanol) and ferric chloride (1 X 1O-3Min ethanol). In all samples examined two pink bands were apparent, oneat Rf 0.53 corresponding to a-tocopherol marker spots and the other ofalmost equal intensity at Rf 0.49. This other band was suspected to beubichromenol, but this was not confirmed. If the thin-layer plates wereallowed to age for one or two days before application of the sample, thepink bands were found to almost overlap, making it impossible to

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146 a-tocopherol in pasture samples

TABLE 2-a-Tocopherol and Selenium Levels in Pasture from Whangaehu

Paddock No.

1

2

3

4

5

6

7

8

a-tocopherolp.gjg wet wt

30.4

33.0

34.8

34.3

40.3

31.2

37.0

39.8

SelenuimJLgjg dry wt

0.600

0.275

0.041

0.210

0.125

0.023

0.058

0.270

remove the a-tocopherol area free of interfering material. The recoveryof a-tocopherol from the plate was 89-91 %.

Examination of the a-tocopherol fraction from a column by thisthin-layer method showed only one band (corresponding to a-tocopherolmarker spots) reactive to the colorimetric reagents.

a-tocopherol content of pasture samples from areas of different seleniumstatus

Three areas based on earlier surveys of pasture selenium levels(Grant, pers. comm.) were chosen for examination.

1. Rerewhakaaitu (Rotorua)-an area of known animal responsesto selenium;

2. Wallaceville-an area of marginal selenium deficiency;3. Whangaehu (Wanganui)-an area having variable concen­

trations of selenium in the pasture.

Paddocks in all areas were sampled in a zigzag pattern, approxi­mately 1 g of pasture being collected every 20 paces. All samples were

TABLE 3-a-Tocopherol and Selenium Levels in Pasture for Each Area

a-tocopherol

ISelenium

No. of(p.gjg wet wt) (JLg(g dry wt)

Locality samplesmeans and I I means and I

std. deviations range std. deviations range

Rerewhakaaitu 8 34.8 ± 4.5 29.7-42.2 0.012 ± .002 0.009-.015

Wallaceville 6 36.5 ± 3.1 31.1-41.6 O.oI7 ± .005 0.009-.024

Whangaehu 8 35.1 ± 3.4 30.4-40.3 0.208 ± . .178 0.023-.600

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K. R. MILLAR 147

collected in April. For each paddock the collected pasture was thor­oughly mixed, and duplicate 4 g samples were removed and immediatelyhomogenised with acetone-petroleum ether. After the addition of 3H-a­tocopherol the solutions were kept in darkened containers at ambienttemperature for 2 days during transportation to the laboratory, wherethey were stored at 2°c.

Thirty ml aliquots were chromatographed and estimated for a­tocopherol between 4 and 6 days after collection. The recovery of 3H-a­tocopherol was calculated, and this figure was used to correct the meas­ured a-tocopherol values.

The selenium content of all pasture samples was determined ondried material by the method of Grant (1963).

Because of the wide range of pasture selenium concentrations foundin the Whangaehu samples, the results for this area are given in detail inTable 2.

The mean results for the concentration of selenium and a-toco­pherol in pasture samples for each of the three areas are given in Table3.

CONCLUSIONS

The procedure described enables a-tocopherol analyses to be doneon plant material collected from areas remote from the laboratory. Noappreciable loss of a-tocopherol could be detected in the acetone/petrol­eum ether extracts of pasture even after storage at ambient temperaturefor several days. Other workers report rapid breakdown of a-tocopherolonce plant material has been macerated. Roughan (1967), for example,reported a loss of 20% of a-tocopherol in a petroleum ether solution in2 days, even when stored at 2-4°c. From the results presented in thispaper it appears that the presence of acetone prevents rapid destructionof a-tocopherol, possibly by inhibiting the action of certain enzymes.The column chromatographic method of Bieri et aT. (1961) is more rapidthan thin-layer methods, and is less liable to interference from othercompounds that give a colour with ferric chloride and bathophenanthrol­ine (Millar and Caravaggi 1970).

The levels of a-tocopherol found in pasture samples containingwidely differing amounts of selenium were subject to little variation, andit seems unlikely that, within the range of concentrations examined,selenium exerts. any effect on a-tocopherol levels in plant material.

Acknowledgments

Dr A. B. Grant and Mr A. D. Sheppard for selenium analyses; J. Craig andG. Bond for technical assistance.

REFERENCES

BIERI, J. G.; POLLARD, C. J.; PRANGE, I.; DAM, H. 1961: The deter­mination of a-tocopherol in animal tissues by column chromato­graphy. Acta Chemica Scandinavica 15: 783-90.

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148 a-tocopherol in pasture samples

BOOTH, V. H. 1959: The extraction of pigments from plant material.Analyst 84: 464--5.

1962: The effect of autolysis on lipids of green leaves.Biochemical Journal 84: 85.

DESAI, I. D.; ScoTT, M. L. 1965: Mode of action of selenium in relationto biological activity of tocopherols. Archives of Biochemistry andBiophysics 110: 309-15.

DILLY, R. A.; CRANE, F. L. 1%3: A specific assay of tocopherols inplant tissue. Analytical Biochemistry 5: 531-41.

DRAKE, c.; GRANT, A. B.; HARTLEY, W. J. 1960: Selenium and animalhealth. Part 1. The effect of alpha-tocopherol and selenium in thecontrol of field outbreaks of white muscle disease in lambs.N.Z. Veterinary Journal 8: 4-6.

GRANT, A. B. 1963:, Determination of selenium in biological material.N.Z. Journal of Science 6: 577-88.

HARTLEY, W. J. 1963: Selenium and ewe fertility. Proceedings of theN.Z. Society of Animal Production 23: 20-7.

HARTLEY, W. J.; DoDD, D. C. 1957: Muscular dystrophy in New Zealandlivestock. N.Z. Veterinary Journal 5: 61~6.

MILLAR, K. R.; CARAVAGGI, C. 1970: Determination of a-tocopherol inserum. N.Z. Journal of Science 13: 329-36.

PETERSON, P. J.; BUTLER, C. W. 1962: The uptake and assimilation ofselenite by higher plants. Australian Journal of BiologicalScience 15: 126-46.

ROUGHAN, P. G. 1967: A simple and rapid method for the quantitativedetermination of a-tocopherol (vitamin E) in leaves. AnalyticalBiochemistry 19: 461--7.

SCHWARZ, K. 1962: Vitamin E, trace elements and sulfhydryl groups inrespiratory decline. (An approach to the mode of action oftocopherols and related compounds.) Vitamins and Hormones20: 463-84.

STAHL, E. (Editor) 1965: "Thin-layer chromatography. A laboratoryhandbook." Academic Press Inc., New York and London. 230-1.

TAPPEL, A. L. 1965: Free-radical lipid peroxidation damage and itsinhibition by vitamin E and selenium. Proceedings of AmericanSocieties for Experimental Biology 24: 73-8.

TSEN, C. C. 1961: An improved spectrophotometric method for thedetermination of tocopherols using 4,7-diphenyl-l, 10-phenan­throline. Analytical Chemistry 33: 849-51.

VIRUPAKSHA, T. K.; SHRIFT, A. 1965: Biochemical differences betweenselenium accumulator and non-accumulatol' Astragalus species.Biochimica et Biophysica Acta 107: 69-80.

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