Simultaneous fluorometric determinations of vitamins A and E in human serum and plasma

HIOCHEMlCAL MEDICINE 8, 403414 (1973)

Simultaneous Fluorometric Determinations of Vitamins A

and E in Human Serum and Plasma

J. N. THOMPSON, PAULA ERDODY, AND W. B. MAXWELL

Research Laboratories, Health Protection Branch, Department of National Health and Welfare, Ottawa, Canada

Received February 12, 1973

Simple procedures for the fluorometric determination of vitamin A (1-3) in blood give erroneous answers because of interference from a fluorescent lipid tentatively identified as the carotenoid phytofluene (4). Several methods for eliminating the interference have been investigated (4, 5). In the most rapid method, a correction formula was applied to fluorescence readings at two wavelengths (4).

Hitherto fluorometric determinations of vitamin E have been combined with nonspecific fluorometric determinations of vitamin A (2). De- tails of a method in which the correction formida procedure for vitamin A (4) was combined with a determination of vitamin E are now described. The results of tests of both assays are reported.

EQUIPMENT AND REAGENTS

A Hitachi-Perkin-Elmer MPF3 spectrophotofluorometer was used to measure fluorescence. Glass distilled hexane (Burdick and Jackson Labo- ratories, Inc.) was used as obtained. The specifications of other solvents and reagents have been described (4,6).

FLUOROMETRIC DETERMINATION OF VITAMINS A AND E

Principle. The vitamin A and vitamin E were extracted in hexane from ethanol treated blood. The correction formula for the vitamin A determination has previously been explained in detail (4). Vitamin E was determined on the same hexane extract from its native fluorescence at 295 nm excitation and 320 nm emission. During the vitamin A determination the fluorometer sensitivity was set to a constant value at 365 nm excitation and 480 nm emission using a standard solution of quinine sulphate; the sensitivity at other waveIengths was thus fixed at values which varied from day to day according to the spectral characteristics of the instrument. A factor for the calculation of the vitamin E levels from the fluo-

rescence readings was obtained during calibration of the instrument and 403

Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

404 THOMPSON, ERDODY, AND MAXWELL

_I ,i-i,;;do FIG. 1. Fluorescence of wtocopherol (2 pg/ml) in mixtures of ethanol and hexane.

The fluorescence in a hexane-ethanol phase prepared as in the extraction of blood is arbitrarily set at 100 units. Fluorescence excitation 295 nm, emission 325 mn.

an additional correction factor for subsequent changes in sensitivity was obtained by measuring on each occasion the fluorescence of a standard ethanolic solution of &ocopherol. The vitamin E content of the plasma extract could not be calculated by direct comparison with a hexane solution of tocopherol because the plasma extract contained both hexane and ethanol, and the relative fluorescence of tocopherol in the mixture was between that in hexane and that in ethanol (Fig. 1). It was therefore essential to extract the tocopherol standard with hexane from ethanol and water, as in the analysis of blood (see below), for the initial calibration of the instrument.

Retinol Standard. Oil from a vitamin A acetate capsule (USP vitamin A reference standard) was saponified in a 15 ml centrifuge tube by heat- ing 20 min in a boiling water bath with 1 ml ethanol, 1 ml water, and 0.5 g KOH. The digest was cooled, 1 ml water was added and the mixture was extracted with 5 ml hexane. A portion of the extract was chromatographed on alumina as described previously (4). The fraction containing retinol was evaporated to approximately 0.5 ml and then diluted with hexane until the absorption at 325 nm was near 0.4. A twenty-fold dilution was prepared and its retinol content was calculated from the absorption of the original solution.

lso,?ation of Phytofluene. Difficulties have frequently been encountered when isolating phytofluene by the method described previously (4). In- vestigations subsequently revealed that the cis but not the tram form was readily separated from other carotenoids during chromatography on alumina. Often phytofluene was isomerized in canned juice and ketchup and thus fresh tomatoes were preferred as a source. The extracts were at all times protected from light. Fresh tomatoes (400 g) were homogenized

HUMAN SERUM AND PLASMA VITAMIN A ASD E 405

in a domestic blender with 400 ml methanol. The mixture was filtered with suction through coarse filter paper, the solid residue rinsed twice with 100 ml portions of methanol and the methanol extracts discarded, The red residue was stirred in 400 ml hexane for 30 min and the filtered extract was evaporated and chromatographed on a column of basic alumina (8 cm X 2-cm diameter) weakened with 2X water. The column was eluted with hexane until phytofluene moved ahead of the colored bands: the phytofluene was located by periodic, but very brief, use of a hand ultraviolet lamp. The phytofluene eluted completely with 2% diethyl ether in hexane. The product contained predominantly cis phytofluene. The fluorescence spectra was recorded using narrow excitation sIits (2 nm band pass): the excitation spectrum was well resolved and was similar to the absorption spectrum. Irradiation rapidly isomerized the carotenoid and the fluorescence intensified to almost twice its original value. The comparatively wide excitation slit used in the analysis of blood also re- sulted in isomerization. Therefore the phytofluene was gently irradiated to stabilize its fluorescence before being used in the calibration of the instrument. An aliquot of the purified phytofluene was diluted in hesane unti1 its fluorescence with 8 nm slits could be measured at a convenient sensitivity setting (Table 1).

Quinine Standard. Quinine sulphate dihydrate was dissolved in 0.1 N

H,SO, (0.1 ELM quinine/ml ). TocopheroZ, Sensitivity Control SoZution. A solution of cu-tocopherol

SolntionQ Excitation Emission Sennit,ivity IIe:tding -~ -

Quinine standard 365 480 3 Set, al. 50

330 480 3 Q Phytoflllene standard 365 480 0.3, 1 .O or 3.0 PI

330 4x0 as above P, I!etinol standard 365 450 10 11,

330 480 10 I:,

Reagent blank 365 480 30 HI 330 480 30 B! 295 ml I c

Tocopherol, sensitivity control 295 wo 0. :3 b> Tocopherol standard 295 320 1 I)

a See text.


(100 pg/ml) was prepared in ethanol. The concentration was checked periodically by measuring the absorption at 292 nm (E, a:,! = 72). This solution was diluted to 2 pg/ml for use as a fluorometric sensitivity control.

Tocophrol, Standard. The procedure for the extraction of blood (see below) was followed except that 1 ml ethanol containing 10 pg cu-tocopherol was substituted for pure ethanol and that water was used instead of serum.

Calibration of the MPF-3 SpectrophJoto@oromter. The accurately diluted retinol standard solution (containing ca. 0.1 pg/ml) was prepared on the same day as the instrument was calibrated. In addition to the retinol, quinine, and tocopherol standards, a reagent blank was prepared by following the extraction procedure for blood (see below) using water in place of serum. The slits were set at 8 nm and the measurements listed in Table 1 were recorded.

In the derivation of the formula for the calculation of vitamin A values, R was the fluorescence reading of the retinol standard at 330 nm, S was the concentration of standard retinol solution, B, and B, were the reagent blank fluorescence readings at 365 and 330 nm at the time of calibration. A was the ratio of the fluorescence at 365 nm to that at 330 run for pure retinol at the time of calibration and P was the equivalent ratio for phytofluene. Q was the quinine reading at the time of calibration and g was the quinine reading at 330 nm at the time of analysis when x was the 336 nm reading of the sample and y was the 365 nm reading of the sample (see below).

\‘alues were calculated from the readings listed in Table 1 as follows: R = R,F where F was the measured attenuation between settings 30

and 10 (i.e., usually F = .‘1)

A = RI/R2

P = Pl/P2.

The vitamin A concentration in blood was calculated from the previously derived (4) formula: retinol ( pg/ 100 ml serum or plasma)

= [+&* - R,) - & (y - R,)];YMO. (1)

At the time of calibration of the instrument for the analysis for tocopherol, E was the fluorescence reading of the standard in ethanol, C was the blank and D was the fluorescence reading obtained by passing 10 ,ug tocopherol through the procedure. At the time of analysis the fluorescence of the sample was e and that of the standard in ethanol was t. Then:

HUMAN SERUM AND PLASMA VITAMIN A AND E 407

vitamin E (pg/ml serum) = (2)

Extraction and Analysis of Blood. Serum or plasma (0.2 ml) was diluted with 1 ml water and mixed with 1 ml ethanol in a 15 ml stoppered centrifuge tube. Hexane (5 ml) was added and the mixture was blended thoroughly for 1 minute using a Vortex mixer and then centrifuged for 5 minutes at 1000 rpm. The tubes were kept stoppered and protected from right unti1 the hexane extracts could be transferred to the fluorometer. Slits were set at 8 nm. The quinine standard was inserted first with the coarse sensitivity control at position 3. The fine variable control was adjusted until the quinine read 50 at 365 nm excitation and 480 nm emission and the reading (4) at 330 nm was recorded. The fine control was not altered from then onwards during the analysis, The tocopherol solution (2 pg/ml) was inserted, the coarse control was switched to 0.3, and the fluorescence reading (t) at 295 nm excitation and 320 nm emission was recorded. The hexane extracts of blood were then read first at 480 nm emission with 330 (x) and 365 nm (y) excitation and coarse sensitivity 30 and second at 320 nm emission and 295 urn excitation (e) with the coarse sensitivity at 1. The concentrations of vitamin A and E were then calculated from Eqs. (1) and (2 ). respectively.

COMPARISON OF THE PROPOSED METHOD WITH OTHER PROCEDURES

Procedure 1: Estimation of Vitamin A by Column C1womutograph.y. Extracts of human serum were analyzed by the formula method and also by chromatography on alumina. Each sample of serum (1 ml) was mixed with ethanol (1 ml), homogenized with hexane (5 ml), aud centrifuged. A 0.5 ml portion of the supernatant was diluted to 2 ml and the fluorescence measured for the calculation of the retinol content by the correction formula. A 4 ml portion of supernatant was evaporated and then chromatographed on alumina as described previously (4) except that hexane was used instead of petroleum ether. The phytofluene content was measured fluorometrically in the hexane fraction and the retinol was measured fluorometrically in the Fjo% diethyl ether in hexane fraction.

Procedure 2: Calorimetric Determination of Vitamin E. Serum ( 1 ml) was mixed with ethanol (1 ml), h omogenized with hexane ( 5 ml), and centrifuged. One portion (1 ml) of the hexane extract was diluted to 5 ml for the fluorometric determination of vitamin E. The solvent used to dilute the extract was prepared by homogenizing hexane (56 ml) with water ( 10 ml ) and ethanol ( 10 ml). The remaining extract of blood

408 THOMPSON, JBDODY, AND MAXWELL

(4 ml) was evaporated and taken up in ethanol. Vitamin E was estimated calorimetrically by adding ethanolic ferric chloride and 4,7- diphenyl-l,IO-phenanthroline (bathophenanthrohne) using orthophos- phoric acid to arrest the reaction after 20 seconds (7).

Procedure 3: Chromatographic Determination of Vitamin E. Serum ( 1 ml) was mixed with ethanol ( 1 ml), homogenized with hexane (5 ml), and centrifuged. One portion ( 1 ml) was used for a direct fluorometric determination of vitamin E as described above. Another portion (4 ml) was evaporated to less than 0.2 ml but not to dryness, dissolved in hexane and chromatographed with the aid of a pump on a 50 cm column of hydroxyalkoxypropyl Sephadex (HAPS) using hexane, ben- zene, and ethyl acetate (67:30:3) as the eluting solvent (6). The portion of the eluatc containing a-tocopherol was made up to 5 ml with the solvent mixture and the tocopherol content was calculated from the fluorescence with 340 nm emission, 290 nm excitation, and 8 nm slits. Other peaks were collected and the fluorescence was measured and expressed as equivalents of a-tocopherol (Table 2).

TABLE 2 SP;PAISATI~N OF FLUORESCENT LIPIDV IN HUMAN BLOOD RY

HAPS CHROMATOGRAPHV

Direct HAPS frnctionsd e&ma-

Sampleb tjionr Total 01 Fraction 1 Fraction 3 j3f-Y --__---.

1(256) 32.0 33.8 19.0 6.6 5 0 3 2 2(294) 28.0 29.6 18.4 4. 0 5.0 2.2 3(414) 31.7 36.8 14.7 6.2 9 3 6.6 4(192) 19,s 19.2 12.0 4.x 15 0.9 5(213) 18.S 22.1 11.4 2 ‘2 6.3 2.2 S(200) 15 n 17,s 9.7 3 6 :i 6 0 !J 7(161) 11.5 12.4 S.6 ., “.M 6) 1.7 0 8(16Y) 11.0 11.8 7.3 2.2 I.6 0. 7 9(134) 11 .:: 11.9 7.0 :;. 0 1.5 0. 4 lO(143) s.s 11.0 6.2 1 .9 2.5 0.4 ll(149) 10.0 10.4 52 2 4 2 h 0 12(123) 5.8 0.7 4.7 0,s 1.2 :z 0 13(94) 7.5 6.7 4. 0 O.!) 1.4 0.4 14(96~ Cl.5 s !) 3.6 :; 1 1.7 0 15(70) 5.5 4.5 1 7 0 6 1.S 0 4

- a &pressed as the fluoromet,rically equivalent amount of cu-tocopherol (pg/ml serum)

measured at 290 nm excitation and 340 nm emissiorl. h Cholesterol levels (mg/lOO ml) in parenthesis. c By recsommended flllorometric method. d See text. and (6).


RESULTS

Validity of Vitamin A Estimation. Hexane extracts, rather than the original serum samples, were analysed in comparisons of the methods; this approach was always adopted as it eliminated the irrelevant but unavoidable differences produced when sampling and extracting serum. Comparison of the vitamin A determinations by the formula with those done after chromatography revealed excellent agreement. The ratio of the answers was 1.06 ( kO.07 SD) and the correlation coefficient was 0.97 for 41 samples. The vitamin A levels were also calculated without correction which is essentially the procedure utilized by others ( 13); comparison with the values obtained after chromatography revealed excesses of 20-200X which correlated (ratio = 1.15 + 0.30; coefficient = 0.82) with the amounts of phytofluene isolated by chromatography.

Comparison of Fluorometric and Calorimetric Determinations of Vitu- min E. The determinations by the fluorometric method correlated (coefficient = 0.96) with those by calorimetry. The mean ratio of the answers (calorimetric to fluorometric k SD) was 1.09 + 0.15 for the 40 samples.

Comparison of Fluorometric and Chromutographic Determinations of

Vitamin E. Preliminary experiments indicated that artifacts were being formed during the chromatographic determination of tocopherol in blood. HAPS chromatography of blood extracts, as described previously (6), separated ultraviolet fluorescing lipids into four fractions (Fig. 2) :

-l- Ah- -3- 2 22 0 50

byta gamma

60 30

FIG. 2. Fluorescence at 295 nm excitation, 335 nm emission in eluate from HAPS column chromatography (6) of a blood extract. Abscissa calibrated in minutes.


fast moving components (termed Fraction 1); a-tocopherol; a third peak (termed Fraction 3) ; and finally, a fraction containing p- and y-tocopherols. Tests with solutions of pure tocopherol in place of blood extracts revealed that often less than 80% of the tocopherol was recovered during analysis and simultaneously fluorescence appeared in Fraction 1.

As in previous experiments (6) tocopherol remained intact during passage through the column. The destructive step was traced to the final stages of the evaporation of the hexane extract prior to chromatography. The loss was negligible when water was omitted from the original extraction mixture or when the hexane extract was incompletely evaporated; the results of a pertinent experiment are listed in Table 3. The relevance of these findings to the analysis of blood was not obvious. When n-tocopherol was added to blood it was completely recovered and there was no increase in the amount of fluorescence in Fraction 1. Nevertheless, as a precaution in the analysis of blood, extracts were not evaporated to dryness before chromatography. The results for 15 blood samples are listed in order according to the a-tocopherol content in Table 2. Only five of the blood samples (numbers 6-9 and 11) had been selected at random; all had been analysed previously and samples l-5 were selected because they were unusually high in tocopherol and samples 10 and 12-15 because they were unusually low. The total amount of fluorescent lipid eluted from the column corresponded closely with the direct determination, but a-tocopherol accounted for only 60% of this

TABLE 3 RECOVERY OF TOCOPHEROL DURING ESTRACTION .IND HAPS CHROM.~TOGKAPHI'

Procedurea

a-Tocopherol Fluorescenceh recovered in Fraction 1

(m) ipgi

A. Extract evaporated to dryness before chroma- 7.7 2 5 tography 8.3 0.9

8.X 1.3 B. Extract evaporated to 0.1 ml before chroma- 9.4 0

tography 9.8 0 9.4 0

C. Water absent, solution evaporated t,o dryness, 9 8 0 before chromatography 9.7 0

9.8 0

a In each test 10 pg a-tocopherol in 1 ml ethanol was homogenized with 5 ml hexane and, in A and B, with 1 ml water. The hexane phase, after being treated as indicated, was chromatographed on HAPS.

* Expressed as the fluoromet.rically equivalent amount of a-tjocopherol.

HUMAN SSRUM AND PLASMA VITAMIN A AND E 411

measurement. Fraction 1, Fraction 3, and p plus y tocopherols both individually and in sum tended to be higher in the samples rich in tocopherol.

The peak of Fraction 3 was encountered in samples of human bIood of diverse sources but it was not seen in fresh animal blood or elsewhere. The excitation maximum was at 283 nm and the emission was at 307 nm. A sample of the fluorescent component was prepared from 1 liter human serum by chromatography on a large column of HAPS first in 90% methanol and second in 5% diethyl ether in hexane. Mass spectroscopy revealed a possible molecular weight of 368 and the substance was tentatively identified as either 2,2’-methylenebis [ 6-tert-butyl-4-phenol] or some other closely related rubber antioxidant. A substance with iden- tical mass. absorption, and fluorescence spectra was extracted by hexane from the rubber stoppers of the vials (Vacutainers) in which blood samples were invariably stored. When human blood was collected in glass stoppered tubes the Fraction 3 peak was absent but when aliquots of the same serum were placed overnight in Vacutainers the peak appeared. Although the antioxidant was detected by the Aminco Bowman instrument during chromatography, it had only a small effect on the fluorescence measurements in the Hitachi-Perkin-Elmer instrument at the chosen settings. A comparison of tocopherol estimations on serum stored in Vacutainers and in glass stoppered tubes is shown in Table 4 for various wavelength settings. The results indicate that the use of 295 nm excitation avoids interference from the antioxidant.

TABLE 4 EFFECT OF WAVELENGTH SETTINGS ON APPARENT TOCOPHEROL CONTENT OF

BLOOD SERUM STORKD IN V.XUTMNF;RS

Wavelength (nm)a Apparent t,ocopherol content (rg/ml)b . --. _--- -- -.

Excitation Emission Glass stopper Rubber stopper ______

285 310 12.2 15.6 290 320 10.7 11.9 290 325 10.9 11.5 295 320 10.9 10.9 295 325 10.3 10.8

n Fluorometric determinations were performed at, the designated wavelength settings with 8 nm slits.

* Pooled sample of serum was stored either in a glass st,oppered tllbe or in a rubber st~oppered Vacutainer.


DISCUSSION

Although the ffuorometric method for determining vitamin A in blood is superior to the alternative calorimetric procedures for a variety of reasons, a unique advantage of using fluorometry is the possibility of simultaneously determining vitamin E (2, 8) at the trivial cost of an extra reading. The combined procedures have proved to be simple and rapid in use once the instrument is calibrated. The most troublesome steps, as judged by reproducibility, were the fluorescence and absorption measurements on the retinol standard, that is, the determination of A, R, and S. The difficulties arose because of the instability of retinol and the care needed during its preparation. Similar problems would be encountered presumably in all methods for vitamin A and thus their existence is not a specific disadvantage of the fluorometric method; however, preparation of several retinol standards is recommended for the calibration procedure to assure reliable measurements.

The vitamin A determination used in the present method has been described earlier (4) when several tests of its validity were published. The comparisons with column chromatography reported now not only confirmed previous experiments in proving that the formula provided accurate answers for vitamin A but also verified with each blood sample that the correction corresponded to the concentration of phytofluene. Furthermore, the phytofluene correction was shown to be of such a magnitude that its omission during anaIysis would have made the results meaningless. Thus in addition to the statistics already quoted, there was no correlation between the levels of phytofluene and vitamin A (coefficient = 0.13). The corrections for phytoffuene ranged from the equivalent of 5.8 to 54.3 pg vitamin A/100 ml which clearly were unacceptable as errors. The mean correction ( t SD) was 28.9 + 13.1 pug which cor- responds closely to the error 34.9 -+ 24.2 pg reported by others (5).

Although Hansen and Warwick ( 2, 9) recommended a fluorometric method for vitamin E, they provided no direct evidence of its validity other than that the answers were of the expected order of magnitude; even then they obtained unusual values for serum tocopheryl acetate which is not usually found in blood. Earlier Duggan (8) described a comparison of fluorometric and calorimetric methods on 12 human blood samples; however, he used a relatively concentrated extract of serum and he considered it necessary to incorporate a correction for quenching of the fluorescence.

The tests of the vitamin E method in the present experiments were not as easily appraised as were those of the vitamin A determination. There was good agreement with the calorimetric method although, as found by Duggan (S), the ffuorometric determinations tended to be lower. As


most published standards for serum tocopherol have been based on calorimetry the fluorometric method would be acceptable for clinical and survey projects. However, HAPS chromatography provided evidence that both the calorimetric and fluorometric methods substantially over- estimated the true e-tocopherol content of serum. The error in the colori- metric method was not investigated in our tests although its existence has been noted previously ( 10, 11). The values for a-tocopherol after HAPS chromatography (see Table 2) were approximately 60% of the simple fluorometric and calorimetric estimations and they appear to be lower than what is accepted as normal by some ( 12, 13) and higher than what is considered accurate by others ( 11). The fluorescence measured in the simple procedure was completely accounted for during chromatography: the error was attributed to the presence of Fraction 1, Fraction 3 and non-tu-tocopherols. Of these interferences, only Fraction 1 could conceivably have been produced as an artifact from tu-tocopherol. One component of Fraction 3 was identified as a contaminant from the con- tainers. However this fraction must also have contained other substances.

The quantities of all of the fractions were mutually related to a degree because of their individual dependence on serum cholesterol and prob- ably lipoprotein levels (14). As an illustration of this relationship, cholesterol levels, which had been determined previously as part of another project, have been included in Table 2.

SUMMARY

A simple rapid fluorometric method for simultaneously determining vitamin A and vitamin E in human blood has been described. Interference from phytofluene in the vitamin A assay was eliminated by use of a correction formula. Analysis of 41 samples by this method and by alumina column chromatography gave similar answers for vitamin A (ratio 1.06 + 0.07 SD; correlation coefficient 0.97) and for phytofluene (ratio 1.15 rt 0.3 SD; correlation coefficient 0.82). In the analysis of 40 samples the Emmerie-Engel reaction gave similar values for vitamin E (ratio 1.07 + 0.15 SD; correlation coefficient 0.96). However chromatography of blood extracts on HAPS revealed that a-tocopherol accounted for only 60% of the fluorescence measurement; other fluorescent lipids which contributed included ,& and y-tocopherols and at least two unidentified substances. When samples of blood were stored in Vacutainers an antioxidant from the rubber stoppers was invariably detected during HAPS chromatography. This substance, although fluorescent, had a variable but small effect on the fluorometric determination of vitamin E depending upon the settings of the instrument.


ACKNOWLEDGMENTS

We thank Dr. 0. Pelletier for data on cholesterol levels and the results of preliminary analysis of the serum samples and Mr. Walter Miles for his assistance in the identification of the fluorescent component of Fraction 3.

REFERENCES

1. KAHAN, J., in “Methods in Enzymology” (D. B. McCormick and L. D. Wright, Eds. ), Vol. XVIII, part C, p. 574. Academic Press, New York, 1971.

2. HANSEN, J. G., AND WARWICK, W. J., Tech. Bull. Regist. Med. Techn. 39, 70 (1969).

3. DRUJAN, B. D., in “Methods in Enzymology” (D. B. McCormick and L. D. Wright, Eds.), Vol. XVIII, part C, p. 565. Academic Press, New York, 1971.

4. THOMPSON, J. N., ERIXDY, P., BRIEN, R., AND MURRAY, T. K., Biochem. Med. 5, 67 (1971).

5. GARRY, P. J., POLLACK, J. D., AND OWEN, G. M., Clin. Chem. 16, 766 (1970). 6. THOMPSON, J. N., ERDODY, P., AND MAXWELL, W. B., Anal. Biochem. 50, 267

1972. 7. TSEN, C. C., Anal. Chem. 33, 849 ( 1961). 8. DUGGAN, D. E., Arch. Biochem. Biophys. 68, 1 ( 1957). 9. HANSEN, L. G., AND WARWICK, W. G., Tech. Bull. Regist. Med. Techn. 36, 131

(1966). 10. DAYTON, S., HASHIMOTO, S., ROSENBLUM, D., AND PEARCE, M. L., J. Lab. Clin.

Med. 65, 739 ( 1965). 11. HERTING, D. C., AND DRURY, E., Amer. .l. Clin. Nutr. 17, 351 (1965). 12. BIERI, J. G., TEETS, L., BALAVADY, B., AND ANDREWS, E. L., PTOC. Sot. Exp. Biol.

Med. 117, 131 (1964). 13. UNTJZREKER, W. J., KATER, R. M. H., AND DENTON, M. D., Proc. Sot. Exp. Biol.

Med. 134, 1148 (1970). 14. DAVIS, T., KELLEHER, J., AND LOSOWSKY, M. S., Clin. Chim. Acta 24, 431 (1969).

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Simultaneous fluorometric determinations of vitamins A and E in human serum and plasma