Method for the Determination of Vitamin E

(Total Tocopherols) in Serum

Robert G. Martinek*

The use of tripyridyltriazine has been investigated as a chromogenicreagent for the

colorimetric determination of vitamin E in serum. The advantagesof using this re-
agent have beenelucidated. Comparisonis madewith a methodemployingterpyridyl.

A. CONSIDERABLE LITERATURE has developed with respect to the deter-

mination of vitamin E as total tocopherols. Several texts (1-3) pre-
sent both chemical and biologic assay methods. Although the direct
spectrophotometric determination of vitamin E in serum at its absorp-
tion peak of about 295 m is attractive, it is not practical because of
very low sensitivity and the presence of many interfering substances.
Only two of the oxidimetric color reactions for vitamin E appear sen-
sitive enough for analysis of serum. One is oxidation (e.g., with silver
nitrate) to give tocopheryiquinone with an absorption peak at about
265 m; it requires rigid control of conditions for precise results. The
other is the Emmerie and Engel (4) color reaction with ferric chloride
and , ‘-dipyridyl (bipyridyl) to give a red color, a reaction that is
precise and easy to perform, and therefore the approach of choice for
a routine clinical laboratory. Natelson (5) improved the sensitivity of
this technic by substituting terpyridyl (2,2’,2”-tripyridine) for bipyri-
dyl in this color reaction.
Recently Caraway (6) reported a method for the estimation of se-
rum iron and iron-binding capacity employing the very sensitive re-
agent, 2,4,6-tripyridyl-s-triazine (TPTZ). Because of the obvious ad-
vantages of this iron color reagent, it was decided to adapt it to the
oxidimetric color reaction in which ferrous iron, produced by the re-
duction of ferric iron by vitamin E (tocopherols), is used as an index

From the Clinical Chemistry Laboratory, Iowa Methodist Hospital, Des Moines, Iowa.
Received for publication Sept. 19, 1963. Accepted for publication Oct. 8, 1963.
*Present address: Division of Laboratories, Chicago Board of Health, Chicago, Ill.

1078
Vol. 10, No. 12, 1964 DETERMINATION OF VITAMIN E 1079

of the concentration of the vitamin in serum. This adaptation and a

demonstration of its advantages is the subject of this paper.

Glassware
As indicated by Caraway (6), all glassware should be cleaned by
soaking in approximately 6N HC1 followed by thorough rinsing with
distilled or demineralized water.

Reagents
Stock standard -tocopherol, 200 ntg./100 ml. Transfer 500 mg. of
c-tocopherol (General Biochemicals*) to a 250-ml. volumetric flask.
(Racemic rL-tocopheroi may be used.) Dilute to the 250-ml. mark with
absolute ethanol and mix thoroughly. This solution is stable at room
temperature.
Working standard -tocopheroi, 1 mg./100 nil. Dilute 0.5 ml. of
stock standard to 100 ml. with absolute ethanol. This solution is stable
at room temperature.
TPTZ, 0.12% (w/v) Dissolve 0.12 gm. of 2,4,6-tripyridyl-s-tri-
azine in, and dilute to 100 ml. with, n-propanol. This solution is stable
at room temperature. If a precipitate forms, add 0.1 ml. of concen-
trated HC1 per 100 ml. of reagent.
Ferric chloride, 0.12% (w/v) Dissolve 0.12 gm. of FeC13 61120
.

in, and dilute to 100 ml. with, absolute ethanol. This solution is stable
at room temperature.

Procedure
Preparationof Calibration Curve
In four 100-mi. volumetric flasks place 0.23, 0.3, 0.75, and 1.0 ml. of
stock standard -tocopherol, 200 mg./100 ml. Dilute to the 100-mi.
mark in each case with absolute ethanol. These solutions are equiva-
lent to 0.5, I, 1.5, and 2 mg./100 ml. of vitamin E as c-tocopherol. Use
1.0 ml. of each solution in place of the working standard in the routine
procedure. Prepare a graph relating mg./100 ml. of vitamin E (as -

tocopherol) to absorbance. This curve shows sufficient daily variabili-

ty to necessitate using a concurrent standard in the routine procedure.
However, the curve should be drawn to determine the extent of ad-
herence to the Beer-Lambert law with various photoelectric instru-
ments. A typical calibration curve is shown in Fig. 1.

‘General Biochemicals, Chagrin Falls, Ohio.

1080 MARTINEI< Clinical Chemistry

Assay
Pipet 1.0 ml. of absolute ethanol’ into each of two glass-stoppered
test tubes (13 X 100 mm.) (screw-cap tubes with Teflon liners in caps
are also satisfactory). Mark one tube “sample” and the other
“blank.” To the sample tube add 1.0 ml. of serum. A(ld the serum

0.40

0
0
Fig. 1. Typical calibration
curve obtained on a Coleman
0
z
Junior spectrophotometer, model
a CD. Reagent blank; 10-mm.
a: round cuvets.
0
(I)
4 0.10

0.5 1.0 .5
VITAMIN E IT000PHEROLS) MG. /100 ML.

slowly, with shaking, to obtain a finely divided protein precipitate. To

tile blank tube add 1.0 ml. of distilled water. To a third glass-stoppered
test tube, marked “standard,” add 1.0 ml. of working standard and 1.0
ml. of distilled water. To each tube add 1.0 ml. of reagent-grade xylene.
Stopper the tubes tightly and shake vigorously (either manually or on
a mechanical shaker) for at least 30 sec. Centrifuge all tubes for at
least 5 miii. at an RCF of 350-450. [Vitamin E is stable in the stop-
pered, centrifuged mixture for at least 24 hr. in the refrigerator (ap-
proximately ) J. Pipet 0.5 ml. of tile xylene (top) layer from each
5#{176}
tube into properly labeled 10-mm. round cuvets. To each cuvet add 0.5
ml. of TITZ solution and mix. Readings are made in a Coleman Junior
spectrophotometer, model 61). Set the instrument to zero absorbance
with the blank at 460 m and measure the absorhance of the sample
only within the next 4 mm. This will he used in the subsequent calcula-
tion to correct for the interference in the reaction from extracted
carotenes. Theii add to each cuvet 0.1 ml. of ferric chloride solution at
definite, timed intervals and mix. Again set the instrument to zero ab-
sorbance with the blank and measure the absorhance of both the sam-
*In all places where absolute ethanol is specified, this reagent was found to be essential for
time highest sensitivity.
Vol. 10, No. 12, 1964 DETERMINATION OF VITAMIN E 1081

pie and the standard at 600 m. The color continues to fade with time
but fading is proportional iii the sample and the standard U to 12 huh.
after adding the ferric chloride solution.

absorbanee of sample Coo n,i - (0.40 X absorhanee of saimiple40 ,,) =

absorhanee of standardoo nibs.

vitamin E (mug. / 100 miii.)

Note: Tile factor, 0.40, above was derived by determining the ab-
sorbaiice of a solution of carotene’ (0.4 mg./100 ml. in xylene) used iii
place of 0.5 ml. of xylemue extract in the routine procedure above. (The
exact concentration of carotene used is not important except insofar
as photometric sensitivity is concerned.)
absorbance600 nun = factor

ahsorbance460 rng

The factor must be determined by the individual laboratory. The char-

acteristics of the reactioii of carotene with TPTZ are shown in Fig. 2.
The significance of this correction for caroteuies in serum is shown iii
Table 1.
Experimental
Comparisonof Methods
In order to validate this modification, the present method was com-
pared with that of Natelson (5). The results are shown in Table 2.
One large series, involving twenty serums analyzed iii duplicate, the
standard deviation, calculated by tile formula:

S.I). = (r1 -

N-i

was 0.03 for both tile Natelson method and for the preseilt method.

Recovery of Added Vitamin E

Recovery of vitamm E was studied by adding knowiu concentrations
of -tocopherol to serum. Results are shown in Table 3. The excellent
recoveries are consistent with the good precision found.

Spectral Characteristicsof the Reaction Mixture

Absorption spectra of the reaction product were determined on the
extract from a serum sample and from a standard (0.5 mg./100 ml.).
Figure 3 is a graph of the results. Maximum absorption occurred at
600mg.

‘90% beta; 10% alpha-carotene, Mann Research Laboratories, New York 6, N. Y.

1082 MARTINEK Clinical Chemistry

0.40 -

Ssut SAMPLE
I CARoTENC (90% BETA-, 0% ALPHA),

0.35 0.4 us. /100 uL. M-XYLENE

0.30-

0.25 -

0.20-/

0.15

0.10 -

400 425 450 475 500 525 550 575 600 625 650 675 700
WAVE LENGTH, MILLIMICRONS

Fig. 2. Absorption spectra of reaction of carotene with tripyridyltriazimie. Coleman CD

spectrophotometer. Reagent blank; 10-mm. round cuvets.

Table 1. SIONIFLOANCE OF CoRREcTIoN FOIl. INTERFERENCE FROM CAROTENES IN SERUM

Corrected for Not corrected Per rent error

carotenes for carotenes caused by
Test No. (mg./iOO ml.) (,ng./100 ml.) carotenes

1 1.20 1.37 14
2 0.87 0.98 13
3 0.84 0.94 12
4 0.91 1.04 14
Vol. 10, No. 12, 1964 DETERMINATION OF VITAMIN E 1083

Table 2. ASSAY RESULTS WITH Two METHODS FOR SERUM VITAMIN E

Nat etson method (5) Present m ethod

Test No. (mg../100 ml.) (mg./100 ml.) Difference

1 1.21 1.20 0.01

2 0.84 0.87 0.03
3 0.88 0.84 0.04
4 0.91 0.91 0
5 0.99 0.95 0.04
6 0.93 0.98 0.05
MEAN 0.96 0.96

Table 3. RECOVERY OF ADDED VITAMIN E IN SERUM

a-Tocopherol added a-Tocopherol found Per cent

(mg./100 ml.) (mg./100 ml.) recovery

0.0 0.52 ± 0.03 (10)’ -

1.0 1.47 ± 0.04 (7) 97

1.5 1.92 ± 0.06 (6) 95

‘MeaIl ± S.D. Figures 111 parentheses are auniber of samples studied, in duplicate.

Fig. 3. Absorption spectra

of reaction of ferrous iron with
tripyridyltriazine. Coleman CD
spectrophotorneter. Re agent
blank; 10-mm. round cuvets.

WAVE LENGTH, MILLIMICRONS

Stability of Vitamin E in Serum

Vitamin E was found stable in separated serum up to 1 day at room
temperature (approximately 25#{176}),
up to 2 weeks in the refrigerator
(approximately 5#{176}),
and up to 2 months in the freezer (-20#{176}).Dr.
Kirch of the University of Illinois reports about 1 day stability in the
presence of the clot provided no hemolysis has occurred.
1084 MARTINEK Clinical Chemistry

Comparisonof Sensitivityof ChromogenicReagents

Caraway (6) stated that the molar absorptivitv of the irOil complex
of TPTZ was 22,600. This was confirmed. r he comparable molar ab-
sorptivity for terpyridyl was found to he 9,100. Furthermore, Natel-
Son (5) found ter)yridy1 more sensitive than bipyridyl.

Optimal Extraction Time

A serum sanupie was divided into five portiolis and analyzed for
vitamin E by the present method except that extraction with xylene
was conducted for varying periods of time ranging from 13 sec. to
60 nun. The results are shown in Table 4. A 30-sec. extraction with
xyleiie was found sufficient.

pH Control
rilile use of x\-lene extracts of serum and alcoholic solutions of the
reagents obviated the riced for buffering with arnrnonium acetate as
described 1w Caraway (6).

Use of Anticoagulants
Potassium oxalate, animollium ileparin, sodium citrate, or the di-
sodium salt of etilvleuuedlamihletetraacetic acid may be used as anti-
coagulant with no change iii values stemniuig front analysis of tile re-
sulting plasma.

Effect of Hemolysisand Icterus

Serum hemoglobin up to 0.4% and serum bihirubin up to 22 ung./100
nil, were found to have iio effect Ofl the results of vitamin E assays with
the l)letiehlt niethod.

Table 4. OPTIMAL EXTRACTION TI.rE WITh XYLENE

Extract ion time

it!, .rylc,, c Vita,,, i,, #{163}
Jon ,,,t
‘Pent No. (Iii in.) (mO/i 00 niL)

1 1/4 0.97
2 #{189} 1.21
2 1.17
4 15 1.20
5 60 1.21
Vol. 10, No. 12, 964 DETERMINATION OF VITAMIN E 1085

Normal Values
Twenty fasting serum sauuiples \cn obtanued from hospital person-
nel haviuig 0 Signs of illness. Both sexes were about equally repre-
Seilted in this group and ranged in age from about 19 to 39 years. The
vitamin E determniatioris were perfornledi on these serums vitlnn 1 hi’.
after collection of tile blood at room temperature (approximately
23#{176}).
Tile 93% limits for serumui vitanuin E (total tocoplierols) 0.83-
1.34 mg./100 ml. No statistically significant differeiice between niale
and female levels was found.

Discussion
It is important that the blank, sample, and standard be treated ill
the same manner. Although the final color will fade with time, fading
was fouiud to be proportional in the sample and standard up to 12 mm.
after adding tile ferric chloride solution, providing, of course, they
were treated in identical fashion.
This method could easily be adapted to a micro technic by appro-
priately scaling down the amounts of reagents and by the employment
of a narrow band width spectropilotometer with ultramicro attach-
ments.
The present method does not deterniine esters of vitamin E. how-
ever, Natelsomi (5) feels that the unesterified vitamin is a measure of
vitamin E deficiency.
The serum for the analysis of vitamin E should be protected from
light and undue agitation. Vitamin E darkens on exposure to light
and is slowly oxidized by atmospheric oxygen.
In addition to 2,4,6-tripyridyl-s-triazine, triphenyltetrazolium chlo-
ride (2,3,5-triphenyl-2H-tetrazolium chloride) has also been accorded
the abbreviation “TPTZ” (7). This should be borne in mnu(l by the
laboratory worker to avoid possible substitution of tile latter chemical.
2,4,6-Tripyridyl-s-triazine (TPTZ) has been shown to constitute a
very satisfactory chromogenic reagent in the colorimetric method for
determination of vitamin E. It possesses several distinct advantages
over currently used reagents. These include greater sensitivity, more
uniform composition, and lower cost.
1086 MARTINEK Clinical Chemh+ry

References
1. Gyorgy, P., Vitamin Methods, Vol. 2. Academic Press, New York, 1951, p. 382.
2. Association of Vitamin Chemists, Inc., Methods of Vitamin Assay (ed. 2). Interecience,
New York, 1951, p. 272.
3. Hawk, P. B., Oser, B. L., and Summerson, W. H., Practical Physiological Chemistry (ed.
13). Blakiston, New York, 1954, p. 1272.
4. Emmerie, A., and Engel, C., Eec. Tray. Chim. 58, 283 (1939).
5. Natelson, S., MicrotechniqRes of Clinical Chemistry (ed. 2). 0. 0 Thomas, Springfield, Ill.,
1961, p. 455.
6. Caraway, W. T., Clin. Chem. 9, 188 (1963).
7. The Merck Index (ed. 7). Merck & Co., Rahway, N. J., 1960, p. 1071.