Spectrofluorometric determination of tocopherols

Spectrofluorometric determination of tocopherols

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Spectrofluorometric 116-122 84, Determination (19%) of Tocopherols Daniel E. Duggan From the Labor...

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ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Spectrofluorometric

116-122

84,

Determination

(19%)

of Tocopherols

Daniel E. Duggan From

the Laboratory National

of Chemical Pharmacology, National Heart Institutes of Health, Bethesda, Maryland Received

January

Institute,

19, 1959

INTRODUCTION In the course of a broad survey of the visible and ultraviolet fluorescence and its congeners of biologically important materials (1, 2), a-tocopherol were found to exhibit intensive ultraviolet fluorescence. A more detailed investigation of the fluorescence of tocopherols and related compounds has provided the basis for a simple and extremely sensitive method for the determination of free tocopherols and tocopherol esters in tissues. EXPERIMENTAL Spectrophotojluorometer The Aminco-Bowman spectrophotofluorometer, equipped with violet-sensitive photomultiplier, was used for these studies. This commercial adaptation of the experimental spectrophotofluorometer and operation are described in detail elsewhere (3).

the IP28 instrument whose

ultrais a design

Materials 1. d-ol-Tocopherol and d-ol-tocopheryl acetate were used as obtained from the d-@-Tocopherol and d-&tocopherol were Nutritional Biochemicals Corporation. purchased from Distillation Products Industries. 2. Hexane. Matheson “hexane” is repeatedly washed with concentrated sulfuric acid until the acid layer is essentially colorless, washed with dilute alkali and with water, dried, passed over a column of silica gel of sufficient size to retain the dark yellow band, and distilled. 3. Alcohol. Absolute ethyl alcohol was distilled from potassium hydroxide. 4. LiAZH4 Reagent. Twenty milliliters of cold, freshly distilled ether is shaken with 200-300 mg. of finely powdered LiAIHd and filtered through Pyrex wool.

Procedure To a 2.0.ml. aliquot of plasma are added 2.0 ml. water, 4.0 ml. alcohol, and, after thorough mixing, 8.0 ml. hexane. To a second aliquot is added a small volume of an alcoholic stock solution containing equimolar quantities of a-tocopherol and tocopheryl acetate (0.05 amole constitutes a convenient internal standard for normal plasma). After thorough shaking to insure equilibration of the added material with 116

SPECTROFLUOROMETRIC

DETERMINATION

OF

117

TOCOPHEROLS

the sample, water, alcohol, and hexane are added as above. Thereafter, sample and internal standard are treated identically. The 40-ml. conical centrifuge tubes are securely stoppered, using a minimum quantity of silicone lubricant on the joint, agitated for 10 min. on a mechanical shaker, and lightly centrifuged. The individual components are then determined as follows: A. TocopheroZ. A l.O-ml. aliquot of the clear organic phase is removed and diluted with 3 vol. alcohol, and its fluorescence at 340 rnp upon activation at 295 rnp is measured. B. Tocopheryl Acetate. A 4.0-ml. aliquot of the organic phase is withdrawn and evaporated to dryness under nitrogen. A stream of nitrogen is then drawn through the apparently dry tube for several minutes more to insure removal of the last traces of alcohol. The residue is redissolved in&Iml. hexcooled in ice, and treated with 1.0 ml. of the LiAlH4 reagent. After intermittent shaking for 5 min., 1.0 ml. of cold alcohol is carefully added,’ then 1 vol. of 0.1 N sulfuric acid. The tubes are stoppered and shaken as before; then a small aliquot of the clear organic phase is removed and diluted with 3 vol. alcohol, and its fluorescence measured as above. A standard solution containing an appropriate volume of the tocopherol and tocopheryl acetate alcoholic stock solution diluted to 2.0 ml. with water may be carried through each of the above procedures to serve as checks upon the internal standards. Samples of pure water must be carried through both procedures as blanks.

Calculations Each sample is activated at 295 rnp and its fluorescence at 340 rnp is determined. The fluorescence of sample B will be equivalent to the total of free and esterified tocopherol originally present in the sample, while A will be due to free tocopherol only. For

Free [XA/@A

Tocopherol. -

XA)]

X GM S/2.0) = rmoles free tocopherol/ml.

plasma

(1)

where XA and SA are the fluorescence intensities, corrected for blank, of the untreated tissue sample and internal standard respectively, and PM S is the number of micromoles of free tocopherol added to internal standard. For

Total

[X,/(&

Tocopherol

(to find acetate by difference)

- Xs)] X (PM S/2.0) = pmoles total tocopherols/ml.

plasma

(2)

where Xs and Ss are the respective fluorescence intensities of LiAlHa-treated tissue samples and internal standard, andrM S is the total number of micromoles of tocopherol and tocopheryl acetate added to the internal standard. The values of the LiAlHa-treated and -untreated aliquots of the external standards should be equal, respectively, to the denominators of (2) and (1) above, if the original external standard was equal to the added internal standard. These external standards tend to give slightly higher results, however, owing to the absorption of either activating or fluorescence wavelengths by the tissue sample. Where a significant difference between results based upon internal and external standards exists, the former is to be preferred. The internal standard, furthermore, may be made to agree 1 A vigorous evolution of gas should occur at this point. Otherwise, an insufficient quantity of LiAlHh and probable incomplete cleavage of tocopherol esters are indicated.

DUGGAN

118 with the absolute transmission over

or external the fluorescent

standard by correcting light path as described

for calculated elsewhere (1).

percentage

RESIJLTS Qualitative

Aspects

Q-, p-, and &Tocopherols, a-tocopheryl acetate, a-tocopheryl acid succinate, and c+tocopherylquinone were each qualitatively surveyed under various conditions of activation and in various organic and aqueous media. Only the free tocopherols were found to exhibit measurable fluorescence. Qualitatively, the activation and fluorescence spectra of the three compounds (Fig. 1) are identical, any possible differences in maximum nctivation wavelengths corresponding to minor differences in their respective absorption maxima being undiscernible with the degree of resolution chosen for these studies (H6 in. defining slits). Quantitative Aspects Solutions of 1.0 pg./ml. of (Y-, /?-, and &tocopherols in absolute ethanol showed respective fluorescence intensities of 38.5, 44, and 45.5 at 1% of

200

400

600

mP and fluorescence spectra of d-a-tocopherol. Solid curve: pure d-a-tocopherol, in ethanol, 1 pg./ml; broken curve: d-a-tocopheryl acetate, following LiAlHa reduction, in ethanol, concentration corresponding to 1 pg./ml. acetate. Fluorescence spectra (peak at 340 mj~) obtained upon activation at 295 rnp; activation spectra (peakat 295 mp) obtainedwith fluorescencemonochromatorset for 349 w.

FIG. 1. Activation

SPECTROFLUOROMETRIC

DETERMINATION

TABLE -

Effect

of

Tocopherol concentration

Increasing

Tocopherol

upon

Fluorescence

IntensitP DeviationC

rdml.

o Product b Obtained c Figures

119

TOCOPHEROLS

I Concentration

Fluorescence Observed

0 (blank) 0.010 0.050 0.10 0.50 1.0 5.0 10.0 20.0 40.0 60.0

OF

0.002 0.014 0.076 0.14 0.77 1.5 7.6 14 28 51 74

I

Theorya

%

0.015 0.075 0.15 0.75 1.5 7.5 15 30 60 90

C-6)

(+I) C-6) (+2)

(+y, (-5)

C-6) - 15% -18%

of meter reading (1 - 100) X sensitivity (1 - 0.001). from straight-line plot of log Z vs. log concn. in parentheses indicate within experimental error.

maximum available sensitivity when activated at 295 rnp. The ratio of these intensities corresponds almost exactly with that of their respective Evalues at 295 rnp. For each compound, the fluorescence vs. concentration relationship was found to be linear over a wide range of concentrations, extending through three orders of magnitude from the lowest detectable concentration of approximately 0.01 pg./ml. through about 10 pg./ml. An essentially linear relationship still exists as high as 40 pg./ml. (Table I). Recovery of cu-tocopherol from water, plasma, and homogenates of various tissues as determined by either procedure A or B (Experimental section) and of a-tocopheryl acetate as determined by procedure B were excellent. The results of a typical plasma recovery experiment are summarized in Table II. The plasma tocopherol levels of 12 normal human subjects were determined by both the spectrofluorometric procedure and the modified Emmerie-Engel method (5), triplicate determinations being made on each sample. The mean value for the former method was 10.8 f 1.9 pg./ml. with an average reproducibility in the triplicate values for each sample of 2.5 %; the respective values by the latter method were 11.3 f 1.9 pg./ml. and 3.5%. The average difference between values for individual samples as determined by both methods was 5 %, the Emmerie-Engel procedure giving the higher result in almost every case. Only negligible differences between values of normal plasma treated by

DUGGAN

120

TABLE Recovery

Water

No.

_n&z. 1 2 3 4 5 6 7 8 9 10 11

-

5 5 5 5 5 0 0 0 0 0 0

T

of Tocopherol

II and

Tocopheryl

T

Plasma ml. 0 0 0 0 0 5 5 5 5 5 5

Acetate Fluorescence

Proc. A Irg.

Pi?.

50 0 100 0 100 0 50 0 100 0 100

0 50 0 100 100 0 0 50 0 loo 100

20

0 40

0 40 22 41 21

(97)d

1 (98) 22 64 (104)

Readingb Proc.

18 18 37 36 74 23 41 40 61 58 94

Be

(100) (97) (102) (98) (98)

(1 Quantity given is equivalent quantity of free tocopherol (pg. tocopheryl acetate added X 430.7/472.7). b Fluorescence readings corrected for blank; for Procedures A and B, 2 and 3, respectively. c Values for Procedure B are a constant percentage lower than for A due to difference in the over-all dilution factors involved. d Figures in parentheses indicate per cent recovery based upon sum of standards in water and found plasma levels.

spectrofluorometric procedures A and B were observed, indicating the presence of little or no esterified tocopherols. DISCUSSION

As compared to the chemical methods extensively used for the estimation of tocopherols in biological materials, the present method affords several marked advantages and suffers from one relative disadvantage. The latter relates to the fact that the molar fluorescence intensities of the CY-,@-, and &tocopherols vary in a similar manner as the values of their respective molar extinction coefficients, thus posing a problem where precise determinations of mixtures of these compounds are to be made.2 Furthermore, the concomitant minor differences in absorption maxima of the pure compounds have, for all practical purposes, no analogy in either activating or fluorescence wavelength maxima, owing probably to the relatively poor 2 A similar difficulty is encountered in the phosphomolybdic acid method, the a-, p-, and &tocopherols showing respective color values of 100,60, and 43 (6); the irondipyridyl method, on the other hand, results in essentially equal values for the three compounds (4).

SPECTROFLUOROMETRIC

DETERMINATION

OF TOCOPHEROLS

121

resolution of the spectrofluorometer, thus rendering a differential analysis on this basis impossible. On the other hand, the spectrofluorometric method offers several distinct advantages in terms of sensitivity, specificity, and the relative mildness of conditions employed. Based upon a fluorescence intensity of ten times the lowest achievable blank value as an arbitrary lower limit for reproducibility, concentrations of 0.005 pg./ml. can be precisely estimated.3 Less stringent purification of solvents will result in a severalfold decrease in ultimate sensitivity as defined on this basis, but will still allow accurate determination of concentrations of tocopherol at least one order of magnitude lower than those assessableto the most delicate calorimetric methods. Vitamin A and other fat-soluble reducing substances which constitute the major source of interference in the commonly used iron-dipyridyl and phosphomolybdic acid methods (6, 7) show no fluorescence similar to that of the tocopherols, thus obviating the necessity of hydrogenation or chromatographic separation. Larger quantities of Vitamin A will interfere, however, by adsorption of tocopherol fluorescence in the 320-330-rnp region, but this difficulty is readily circumvented through correction obtainable by either internal standards or absorption data as previously described (1). An extensive survey of the fluorescence of normal constitutents of mammalian tissuesindicated that the phenolic estrogensare the only compounds which might conceivably interfere under the conditions of extraction and fluorescence measurement employed in this method. SUMMARY

A convenient and sensitive method for the differential determination of free and esterified tocopherols utilizing ultraviolet spectrofluorometry is presented. The introduction of a stoichiometric excess of ethereal LiAlHd into hydrocarbon solutions of tocopherol esters was found to result in a quantitative cleavage to the free alcohols. The method has been applied to the determination of tocopherols in plasma and found to give results in substantial agreement with those obtainable by existing chemical methods. REFERENCES 1. DUOOAN, them.

D. E., BOWMAN, Biophys.

R. L., BRODIE, 68, 1 (1957).

* For most practical the data in Tables under Materials.

purposes, this order

I and II were

obtained

B. B., AND UDENFRIEND, of sensitivity is superfluous. employing solvents prepared

S., Arch.

Bio-

Accordingly, as described

122 2.

DUGGAN

S., DUGGAN, D. E., VASTA, B. M., AND BRODIE, B. B., J. Pharmacol. Therap. 120, 26 (1957). BOWMAN, B. L., CAULFIELD, P. A., ANr) UDENFRIEND, S., Science 123, 32 (1955). EMMERIE, A., AND ENGEL, C., Rec. tram. chim. 67, 1361 (1938). QUAIFE, M. L., AND HARRIS, P. L., J. Biol. Chem. 166, 499 (1944). NAIR, P. P., AND MAGAR, N. G., Indian J. Med. Research 43,557 (1954). ROSENKRANTZ, H., J. Biol. Chem. 224, 165 (1957). UDENFRIEND,

Exptl.

3. 4. 5. 6. 7.