Fluorometric determination of tocopherol in sheep plasma

BIOCHEMICAL

MEDICINE

Fluorometric

11,

71-80

(1974)

Determination in Sheep

of Tocopherol Plasma

G. B. STORER Division of Nutritional Research Organization,

Biochemistry, Kintore Received

Commonwealth Avenue, Adelaide, February

Scientific and South Australia,

industrial 5000

14, 1974

A simple, sensitive and precise method is described for the fluorometric determination of tocopherol in sheep plasma. Precautions taken at each step of the procedure to guard against oxidative losses give recoveries of 93-100%. The variation in free tocopherol level with seasonal pasture change and pregnancy ranged from 128 fig tocopherol/lOO ml plasma (SE 6.76) in nonpregnant ewes on dry summer feed to 284 pg tocopherol/lOO ml (SE 10.4) at full term.

The concentration of tocopherol in human plasma has been reported in the range 700-1200 /*g tocopherol/ 100 ml ( l-6), whereas for several animal species including sheep, the usual levels are approximately one fifth of this, viz. NO-250 pg tocopherol/lOO ml (7-11). This places greater demands on the sensitivity of determination for which the fluorometric method is ideal. Many methods for the determination of tocopherol depend on the reduction of Fe”+ and the use of a calorimetric reagent to measure the Fe” produced ( 12-15). Other reductants present may render the determination nonspecific, and allowance has to be made for the presence of carotene at the wavelengths used for spectrophotometry (16-18). Although sensitive and capable of resolution of isomers, gas chromatography requires considerable purification for quantitative work on plasma samples (19, 20). On the other hand the advantages of a fluorometric determination in avoiding difficulties have been pointed out (21-25). Hansen and Warwick published modifications (23, 24) of the original procedure (22)) but the lower limit for determination under their conditions was 60 b>ocopherol/lOO ml which is inadequate for sheep plasma particularly under deficiency conditions (26). This report describes modifications to improve sensitivity and precision. It has been applied to determine variations in plasma tocopherol levels with seasonal change and pregnancy in sheep under field conditions. Copyright All rights

71 @ 1974 by Academic Press, Inc. of reproduction in any form reserved.

72

G. B.

STORER

EXPERIMENTAL

Reagents 1. Ethanol refluxed for 30 min with 1 g KMnO, and 2 g KOH per liter and distilled into a dark glass stoppered bottle. 2. 1% sodium ascorbate pH 4.5, obtained by adjustment of AR ascorbic acid with N NaOH. Stable for one month under refrigeration. 3. Cyclohexane, LR redistilled through a fractionating column into a dark glass container. 4. Hexane, purified and redistilled according to Duggan (22). 5. Twice distilled water from an all glass still. 6. a-Tocopherol standard. dE a-tocopherol (Merck LR) purified by molecular distillation at 190-260°C at l-5 microns pressure. A stock standard was prepared ( 1 mg/ml) by weighing the distilled product and dissolving in freshly distilled ethanol. A working standard of 1 ,J,g/ml was prepared by dilution with ethanol. Standards were stored under refrigeration in the dark. Equipment Kahn shaker; M.S.E. Super-Minor model centrifuge; Aminco-Bowman Model No. 4-8202B spectrofluorometer fitted with an lP28 photomultiplier tube, a Philips CSX 150 xenon lamp, and a S-position rotary turret for 10.5 mm square fused quartz cells; 15 ml standard taper glass stoppered tubes. Procedure All glassware was soaked in “Calgon” inorganic detergent, thoroughly brushed out, and washed repeatedly in tap water, distilled water and finally several times in glass distilled water. Prior to use tubes and stoppers were rinsed twice with small volumes of redistilled ethanol. With all operations done in diffuse light, 0.25 ml of plasma was added to a glass stoppered tube containing 0.75 ml sodium ascorbate and mixed gently. 2 ml of redistilled ethanol were added slowly whilst the contents were agitated on a vortex mixer. The tube was flushed with nitrogen, lightly stoppered, and stood for 5 min to ensure precipitation. The contents were again mixed, 3 ml of cyclohexane added, and the tube flushed with nitrogen as the wetted glass stopper was twisted tightly into place ( 19). The tube was secured horizontally in the Kahn shaker with the stopper firmly clamped to prevent loss. The tube was shaken for 10 min at 500 strokeslmin then centrifuged at 2500 rpm for 5 min. The fluorescence of the cyclohexane extract was measured at 295 nm

DETERMINATION

OF

PLASMA

TOCOPHEROL

73

excitation and 340 nm emission. A reference cr-tocopherol solution was used to check the constancy of instrumental response. A blank was carried through the whole procedure by substitution of glass distilled water for plasma. A standard series was prepared with each batch of determinations by addition of working standard tocopherol to blank mixtures, allowance being made for the volume of ethanol used. RESULTS

AND DISCUSSION

TLe proposed procedure was derived from the following experiments to test each step of the method. Eficiency

of Protek Precipitation

Various volume ratios of ethanol to plasma showed that a 2: 1 mixture gave the best result, with a clear cyclohexane phase after centrifuging, although the aqueous phase was still hazy. However if the pH of the plasma was buffered with sodium ascorbate to the iso-electric point of lipoproteins (pH 5.2-5.4) prior to the addition of ethanol in 2: 1 ratio, the protein precipitate compacted to a hard deposit on centrifuging, leaving both phases clear. This avoided any risk of protein haze being included in the solvent phase. When the volumes of plasma and ascorbate reagent were used as described, the pH of the mixture always lay in the range 5.2-5.5. If 1% ascorbic acid was used without pH adjustment or if pH of this reagent was >5, the precipitate once again remained as a haze after centrifuging. Effect of Sodium Ascorbate and Cyclohexane Duplicate blanks and standards, and quadruplicate pooled plasma samples were determined in three series; (A) using the method as described, (B) substituting water for the ascorbate and (C) as for (A) but using purified hexane as solvent ( Table 1) . A significantly lower resuIt (p < 0.01) occurred in the absence of ascorbate. Higher concentrations of ascorbate were used with no improvement over the 1%reagent. No effect was noted on the fluorescence of standard tocopherol extracted from aqueous ascorbate buffered at pH 4.5 or 5.3. Ascorbate has been proposed as an antioxidant in alkaline hydrolysis of tocopherol esters (6, 25) but the redox potential of ascorbate falls rapidly as neutral pH is approached. Other antioxidants such as butylated hydroxy toluene interfered with fluorometric measurement of tocophero! without prior removal by e.g., TLC. Although there was no significant difference between the means (A) and (C), the fluorescence was about 11%higher with cyclohexane ( 20).

74

G. B.

EFFKCT

OF SODIUM

TABLE

I

ASCORH.ITE

MD CYCYLOHF:XA~-~: ml plasma -

,.g tocopherol/lOO

(With

ascorbtte extracted with cyclodexane)

(Xo

STORER

ascorbze extract)ed with cyclohexane)

(With

c ascorbate, extracted with hexane)

264 264 272

256 244 24s 260

260 368 264 264

Mean = 268 SE = 2.3

Mean = 252 SE = 3.6

Mean = 264 SE = 1.15

272

Extraction

Time

Duplicate blanks, standards and pooled plasma were extracted by the method described for various intervals from 0.5 to 30 min. Blanks and standards gave constant readings after 1 min shaking, but plasma duplicates did not achieve constant values until 10 min. Linearity

and Sensitivity

of Standard

Response

Rectilinear graphs were obtained up to a concentration of 2 pg of tocopherol/3 ml cyclohexane extract, equivalent by the procedure used, to 800 Fg tocopherol/lOO ml plasma. This was measured with the following instrumental settings: monochromator slits 5 mm, cell slits 1 mm, scatter slits 3 mm, meter multiplier 0.01 at a sensitivity of approximately 40. Under these conditions and using reagents purified as described 0.01 pg tocopherol could be detected corresponding to 4 pg tocopherol/lOO ml plasma. Recovery Using pooled plasma of low ( 300 pg/lOO ml) tocopherol levels, 0, 0.2, 0.5, and 1.0 pg amounts of .a-tocopherol were added to duplicate 0.25 ml amounts of plasma prior to deproteinization ( Table 2 ) . Comparison

of Precision

Twelve replicates of well mixed plasma together with duplicate standards and blanks were prepared and determined by the proposed method (A). This series was also determined by the method of Hansen and Warwick (24): except that 0.25 ml plasma and 3 ml of hexane (instead of 5 ml ) were used. with shaking for the published 30 set ( B ) (Table 3).

DETERMINATION

OF

PLASMA

RMXVERY

OF TOCOPHEROL

TABLE

2 ml Total

Added

level

pooled

PL.ISM.I

FROM

pg tocopherol/lOO

Low

determined

level

pooled

“;. MecLover>

92 16s 2x4 485

plasma

0 High

73

TOCOPHEHOL

96 !I!)

X’S

I

X0 i 200 400

plasma

!El

4108 516 700

100 !M !lY

Interferences A number of possible sources of interference with the fluorescence measured at 295 nm excitation and 340 nm emission were examined. 1. Anhydrous ethanol prepared by azeotrophic distillation with benzene may retain traces of it, which could be recognized by spectral curves. This contamination with benzene contributed considerably to the TABLE: COMPARISON

3

OF PRECISION

12 replicates of pooled pg tocopherol/lOO

plasma ml

A (Proposed

method)

B ref.

(Method

272 264 264 272 272 272 264 276 264 272 264 272

244 260 260 280 264 “60 260 2x0 ‘24” 272 242 260

Mean = 260 SE = 1.31

Mean = 260 SE = 3.76

The difference between SE’s is highly significant

means is significant, (p < 0.01).

(p < 0.05)

and

the

(24))

difference

betweet)

76

2.

3.

4.

5.

G. B.

STORER

fluorescence of blanks, and cannot be removed by distillation. 95% ethanol derived from industrial fermentation processes was satisfactory. At 295 nm excitation the peak of the Raman emission spectrum for cyclohexane was found at 325 nm, which was also the peak of emission for tu-tocopherol. Therefore the emission was measured at 340 nm which diminshed the signal from tocopherol only slightly, but with the slit arrangements specified, reduced the blank emission due to this factor to about 10%of that at 325 nm. Solutions of p-carotene or Vitamin A palmitate in cyclohexane up to concentrations equivalent to I2 pg carotene or 220 pg Vitamin A/100 ml plasma showed no effect on the fluorescence of blank or standard tocopherol. Preservatives in various brands of heparin showed peaks of fluorescence at 280 nm excitation and 315 nm emission, but were undetectable at the wavelengths of the method at the concentration finally present in the cyclohexane extract. Ethanolic extracts of polythene containers or closures gave broad peaks of fluorescence at 280 nm and 305 nm excitation and 350 nm emission due probably to plasticisers such as Ionox 330 (28). Glass containers for reagents and samplesmust be used throughout.

Specificity

To verify that tocopherol is the species being measured by the proposed method, a 2 ml aliquot of the cyclohexane extract of the blanks, standards and plasmas of the series prepared for the recovery experiment was evaporated to dryness in a tube under nitrogen in diffuse light. The inside of the tube was washed down with minimal amounts of hexane, evaporated to small volume, and the residue and two subsequent washings with hexane applied as a spot to a thin layer of purified acid alumina, ruled into channels. The solvent was evaporated by a gentle stream of carbon dioxide, the whole plate being maintained meanwhile under a CO, blanket. The chromatogram was developed in a solvent saturated tank with 93: 7 cyclohexane-ethyl acetate and the tocopherol spots rapidly located by Fe&bathophenanthroline spray of bilateral marker spots of pure (v- and y-tocopherol. The alumina of each channel in the position of a-tocopherol was scraped under CO, blanket into a glass stoppered centrifuge tube and extracted with 0.5%sodium acetate in ethanol. After centrifuging, the a-tocopherol in the ethanolic extract was measured at 295 nm excitation and 340 nm emission in the fluorometer. The

DETERMINATION

OF

PLASMA

TABLE

I_ 11

TOCOPHEROL

4

SPECIFICITY OF MEASURC:MENT Determination (tocopherol, pg/lOO Direct,

Sample Low level pooled plasma Low level pooled plasma tocopherol/lOO ml High level pooled plasma High level pooled plasma tocopherol/lOO ml

+ 200 pg

+ 200 pg

TLC

P&c

ml)

Tocopherol applied to plat,e

50 Kecov-

ery of direct, determination

92 2x4

40 21”

0. 15 0.47

44 7.5

32X 516

240 4.56

o.r(2 1.29

7:; xx

tocopherol content was calculated in reference to standards taken through the whole procedure at the same time (Table 4). With the amounts of extract subjected to TLC, there was no detectable material in the y-tocopherol position either by spray reagent or by eluting and measuring fluorescence ( 3). Estimation of plasma tocopherol at low levels by TLC suffers from losses due to retention near the origin of tocopherol by lipids occurring together in the extract, the magnitude depending on the amount of tocopherol actually present and the ratio of tocopherol to plasma lipid (3,27). Thus the recoveries shown in Table 4 show improvement as the amomlt of tocopherol present increases. Similar results have been obtained using re-chromatographed %-a-tocopherol, recovery again rising with the amounts of ,ar-tocopherol present. Thus it appeared that the direct extraction and fluorometric measurement as described was specific for tocopherol. It did not estimate esterified tocopherol, but this could be done readily by prior treatment with LiAlH, to determine total tocopherol, with esterified tocopherol derived by difference (22). However previous reports showed little or no tocopherol ester to be present in those plasmas tested (22, 23). APPLICATION

OF

METHOD

Thirty-one maiden ewes were selected from a flock grazing improved. predominantly grass pastures in a Mediterranean climate. Blood was sampled from the jugular vein on seven occasions during a period of 13 md. The sample was discharged into an ice cold, heparinized tube. and without delay, it was centrifuged, plasma separated and stored at -20°C

78

G. B.

STORER

TABLE

Season

Pasture

Spring (Oct.) Summer (Dec.) Summer (Feb.) Autumn (Mar.) Winter (June) Wint.er (Aug.) Lat,e Spring (Nov.) Ijifferences

means

Mean tocupherol (pg; 100 ml)

No. of ewes

lllid-growth Mat.ure (in seed) Dry feed I)ry feed New growth Lush growth Mature

between

5

are highly

31 31 31 81 31 (3 mo pregnant) 29 (full term) 30 (post,-weaning) significant

173 s 201.1 167.7 128.7 212.3 2x4. :3 241 .:I

SE 6.76 6.76 6.76 6.76 6.76 10.4 7.x

Cp < 0.001).

before analysis. The ewes were mated to give a lambing time in late August (Table 5). Male and female lambs from this group of ewes, and others, were sampled approximately 2 and 6 wk after birth ( Table 6). Reported normal ranges of tocopherol in sheep plasma have been obtained under experimental pen conditions or have derived values from very few animals (9, 10). The same group of flock animals was used throughout this survey so that biological variation arising from taking a random sample on each occasion from the same flock has been eliminated, as indicated by the constant standard error under nonpregnant conditions. Thus the mean level prior to pregnancy reflected the quality and intake of feed on a seasonal basis, and rose to a peak on mature summer pasture (29, 30). The effect of pregnancy in raising tocopherol levels has been investigated in women using a large sample (31, 32). Although little information is available in sheep, there was an indication of a rise at term (33), but present results show a rise of 50% over non-pregnant values on similar pasture. In line with human data (32), no sex differences were noted in the lambs. TABLE PLASMA

Age (wk)

No. of samples

2 6 IXfferences

TOCOPHEKOL

Male Plasma tocopherol (a/100 ml)

27 28

254.3 244.3 of means

between

SE 14.2 9.4

6 OF LAMBS

No. of samples

Female Plasma tocopherol k/100 nIlI

SO 32

sex and age are not signifivan

339.0 246.2 t.

SE 8l.b 11.3

DETERMINATION

OF

PLASMA

TOCOPHEROL

79

ACKNOWLEDGMENTS The author wishes to thank Mr. W. B. Hall of CSIRO Division of Mathematical Statistics for advice and help with statistical treatments and Mr. A. Mutter and Mr. R. Watson of Glenthorne Field Station for blood samples.

REFERENCES 1. QUAIFE, M. L., AND HARRIS, P. L., 1. Biol. Chem. 156, 499 (1944). 2. HARRIS, P. L., HARDENBROOK, E. G., DEAN, F. P., &SACK, E. R., AND JENSES, J. L., Proc. Sot. Exp. Biol. Med. 107, 381 ( 1961). 3. BIERI, J. G., AND PRIVAL, E. L., Proc. SOC. Exp. Biol. Med. 120, 554 ( 1965 ). 4. HASHIM, S. A., AND SCHUTTRINGER, G. R., Amer. J. Clin. Nub. 19, 137 (1966). 5. DESAI, I. D., Cunud. J. Physiol. Pharm. 46,819 ( 1968). 6. RINDI, G., Int. Z. Vitaminforsch. 28, 225 ( 1958 ). 7. SQUIBB, R. L., GUZMAN, M., AQUIRRE, F., AND SCRIMSHAW, N. S., Am. J. Vet. Res. 14, 484 ( 1953). 8. ROUSSEAU, J. E., DICKS, MARTHA W., TEICHMAN, R., HELMBOLDT, C. F., BACON, E. L., PROUTY, R. M., DOLGE, K. L., EATON, H. D., JUNGHERR, E. L., AND BEALL, G., J. Animal Sci. 16,612 (1952). 9. CARAVAGGI, C., Comp. Biochem. Physiol. 30,585 ( 1969). 10. BYFIELD, R. F., FALK, R. H., AND BARRETT, J. D., J. Chromatog. 36, 54 ( 1968). 11. MILLAR, K. R., Private communication. 12. STROHECKER, R., AND HENNING, H. M.. “‘Vitamin Assay,’ Tested Methods,” p. 283308, Verlag Chemie, Weinheim ( 1965). 13. EIIMERIE, A., AND ENGEL, CH., Rec. Tram. Chim. Pay-Bas 5’7, 1351 (1938). 14. TSEX, C. C., Anal. Chem. 33,849 ( 1961). 15. MARTINEK, R. G., Clin. Chem. 10, 1078 (1964). 16. EDWIN, E. E., DIPLOCK, A. T., BUNYAN, J., AND GREEN, J., Biochem. J. 75, 450 (1960). 17. MILLAR, K. R., AND CARAVAGGI, C., N. Z. I. Sci. 13,329 ( 1970). 18. QUAIFE, M. L., SCRIMSHAW, N. S., AND LOWRY, 0. H., J. Biol. Chem. 180, 1229 (1949). 19. BIERI, J. G., “Chromatography of Tocopherols” in “Lipid Chromatographic Analysis” (Guido V. Marinetti, ed.), p. 459, Vol. 2. Marcel Dekker, New York (1969). 20. THOMPSON, J. N., ERDODY, P., AND MAXWELL, W. B., Anal. Biochem. 50, 267 (1972). 21. UDENFRIEND, S., in “Fluorescence Assay in Biology and Medicine,” Vol. II. Academic Press, New York ( 1969). 22. DUGGAN, D. E., Arch. Biochem. Biophys. 84, 116 (1959). 23. HANSEN, L. G., AND WARWICK, W. J., Am. J. C&. Path. 46, 133 (1966). 24. HANSEN, L. G., AND WARWICK, W. J., Am. J. Clin. Path. 51, 538 (1969). 25. BUNNELL, R. H., “Vitamin E Assay by Chemical Methods” in “The Vitamins” ( P. Gyorgy and W. N. Pearson, eds.), p. 261, Vol. 6. Academic Press, New York (1967). 26. HID~OGLOU, M., JENKINS, K. J., AND CORNER, A. H., Can. J. Anim. Sci. 52, 511 (1972). 27. HORWITT, M. K., HARVEY, C. C., AND HAR~ION, E. M., “Lipids, a-Tocopherol and Erythrocyte Hemolysis” in “Vitamins and Hormones” (R. S. Harris, I. G.

80

28. 29. 30. 31. 32. 33.

G. B. STORER

Wool, and J. A. Lorraine, eds.), p. 487, Vol. 26. Academic Press, New York (1968). KIRKBRIGHT, G. F., NARAYANASWAMY, R., AND WEST, T. S., Anal. Chim. Acta 52, 237 ( 1970 ) . BLAXTER, K. L., AND BROWN, F., Nutrit. Abstr. Reu. 22, 1 (1952). BOOTH, V. H., J. Sci. Food Agric. 15, 342 ( 1964). KRAMER, M., Int. Z. fur Vitaminforsch. 26, 58 (1955). STRAUMFJIORD, J. V., AND QUAIFE, M. L., Proc. Sot. Erp. Biol. Med. 61, 369 (1946). WHITING, F., WILLMAN, J. P., AND LOOSLI, J. K., J. Anim. Sci. 8, 234 (1949).