Method For The Determination of Vitamin E (Total Tocopherols) in Serum
Method For The Determination of Vitamin E (Total Tocopherols) in Serum
Method For The Determination of Vitamin E (Total Tocopherols) in Serum
Robert G. Martinek*
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
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 -
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.
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.
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
S.I). = (r1 -
N-i
was 0.03 for both tile Natelson method and for the preseilt method.
0.40 -
Ssut SAMPLE
I CARoTENC (90% BETA-, 0% ALPHA),
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
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
‘MeaIl ± S.D. Figures 111 parentheses are auniber of samples studied, in duplicate.
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.
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.