A Colorimetric Method for Determination of
Total Serum Lipids Based on the
Sulfo-phospho-vanillin Reaction
CHRISTOPHER
S. FRINGS,
PH.D., AND RALPH
T.
DUNN,
M T (ASCP)
Medical Laboratory Associates, 1025 South 18th Street, Birmingham,
Alabama 35205
Abstract. Frings, Christopher S., and Dunn, Ralph T.: A colorimetric method
for determination of total serum lipids based on the sulfo-phospho-vanillin
reaction. Am. J. Clin. Path. 53: 89-91, 1970. A simple, rapid, and precise
colorimetric method for total serum lipids based on the sulfo-phospho-vanillin
reaction is described. The method uses 0.10 ml. of serum and the resulting
chromogen follows Beer's law at 540 m^. up to lipid concentrations of 1,000
mg. per 100 ml. The colorimetric method gives good agreement with a gravimetric-extraction method and has a coefficient of variation of 3.5%. The simplicity, speed, and reliability of the proposed method makes it suitable for
large-scale analyses.
WIDELY-ACCEPTED METHODS for measurement of total serum lipids by extraction and subsequent gravimetric analysis 4 ' 7 are tedious and time-consuming. A
simple, rapid, reliable method for the assay of serum lipids which has the analytical
integrity of the gravimetric method is
needed. Several basic approaches to this
problem have been made.
One approach is to perform cholesterol,
phospholipid, and triglyceride assays, and
then calculate the total lipid concentration
from the values of the lipid fractions.2 This
approach has the disadvantage of being
very time-consuming because three independent chemical determinations are required, and in addition it is necessary to
accept the often-incorrect assumption that
73% of total cholesterol represents cholesterol esters.
The phenol turbidity method of Kundel
and associates 5 for assay of total lipids is
rapid, but was shown by Cheek and Wease 2
to compare unfavorably with the gravimetric method.
A method based upon the oxidation of
an alcohol-ether extract of serum by a
K 2 Cr 2 0 7 -H 2 S0 4 reagent and subsequent
determination of the reduced chromium
ion has been described by Bragdon. 1 This
method is time-consuming because the solvent must be evaporated to dryness at 60 C.
In addition, determinations of cholesterol
and phosphatides are necessary so that the
corresponding oxidation factors may be applied.
Drevon and Schmit 8 reported on the
color reaction given by lipids with vanillin
in a medium of sulfuric acid and phosphoric acid. Postma and Stroes ° mentioned
a sulfo-phospho-vanillin method for serum
lipids, but the details were not described.
In this paper we describe an improved
colorimetric method, based on the sulfo-
THE
Received June 23, 1969; accepted for publication
August. 10. I'OO'.l.
Presented in part at the 21st National Meeting of
the American Association of Clinical Chemists, August, 1969, in Denver, Colorado.
89
90
Vol. 53
FRINGS AND DUNN
cold water bath for about 5 min. Transfer
TABLE 1. Comparison of Colorimetric and Gravimetric
Total Lipid Methods
a 0.10-ml. aliquot of the mixture into a
Total Lipids
Colorimetric Gravimetric
Sample
Method
Method
(mg. per 100 ml.)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
MEAN
552
360
665
640
569
569
570
415
665
650
565
665
1378
1300
595
526
1442
1395
641
659
561
720
680
573
669
714
583
721
764
612
707
721
phospho-vanillin reaction, for the determination of total serum lipids.
Materials and Methods
Reagents
1. Concentrated sulfuric acid.
2. Vanillin, 0.6% (w/v).
3. Phospho-vanillin reagent. Place 200
ml. of 0.6% vanillin solution in a 2-liter
Erlenmeyer flask. Add 800 ml. of concentrated phosphoric acid, with constant stirring. Store in a brown bottle at room temperature. This solution is stable for at least
6 weeks.
4. Standards. Working standards of 200,
400, 600, and 800 mg. per 100 ml. are prepared by diluting a stock standard (1.000
Gm. olive oil per 100 ml. absolute ethanol)
with the appropriate amount of absolute
alcohol. These standards are stable for at
least a month at 4 C.
Procedure
Pipet 2.0 ml. of concentrated H 2 S0 4 into
a tube containing 0.10 ml. of serum, and
mix well. Heat the tube for 10 rain, in a
boiling water bath. Cool the tube in a
tube labeled unknown. To an additional
tube labeled blank, add 0.10 ml. of concentrated H 2 S0 4 . Add 5.0 ml. of phosphovanillin reagent to each tube and mix well.
Incubate all tubes for 15 min. at 37 C.
Cool the tubes for 5 min. at room temperature. Measure the absorbance of unknown at 540 m^. against the blank within
an additional 5 min. The color is stable for
at least 10 min.
The serum lipid concentration is calculated from absorbance values of standards
taken through the entire procedure, as described above.
Results and Discussion
A plot of absorbance vs. lipid concentration is linear to 1,000 mg. per 100 ml. at
540 lrifi. Beer's law is not followed at lipid
concentrations greater than 1,000 mg. per
100 ml., even though a sample containing
1,000 mg. of lipid per 100 ml. results in
an absorbance of only 0.46 at 540 m^. in
19-mm. cuvettes. This finding is probably
due to the solubility characteristics of vanillin. Serum samples which have total
lipid concentrations greater than 1,000 mg.
per 100 ml. should be diluted with 0.9%
(w/v) NaCl and reassayed.
To evaluate the reliability of the colorimetric method, we compared it with the
gravimetric-extraction method of Jacobs
and Henry. 4 The data in Table 1 show
that the colorimetric method and the gravimetric-extraction method compare favorably. These results were selected at random and are typical of comparisons of
more than 60 serum samples. The colorimetric method exhibits a coefficient of variation of 3.5%. In our laboratory the gravimetric method of Jacobs and Henry has a
coefficient of variation of 9.6%.
Olive oil was determined to be a suitable
standard. Our evidence is based upon the
January 1970
COLORIMETRIC DETERMINATION OF LIPIDS
findings obtained from the comparison of
the colorimetric method, using olive oil as
the standard, with the gravimetric method
(Table 1). Olive oil, triolein, oleic acid,
linoleic acid, linolenic acid and cholesterol
react quantitatively in the method. Either
olive oil, which in our opinion is a suitable standard, or serum which has been assayed previously by a gravimetric-extraction
method can be used.
The exact chemistry involved in this
method, just as in many methods in lipid
chemistry, is not completely understood.
It appears that a carbon-to-carbon double
bond is necessary for the reaction to proceed as described above. In human serum,
there are essentially no interfering substances since naturally-occurring compounds with carbon-carbon double bonds
other than lipids (fatty acids, cholesterol,
cholesterol esters, triglycerides and phospholipids) are found in very small amounts.
Secobarbital was found to react in the procedure, presumably because of the CELCH
=CH« group, but is not considered an
interfering compound because the levels of
91
secobarbital necessary to cause color formation are incompatible with life.
The small volume of serum required
(0.1 ml.), in addition to the simplicity,
speed, and reliability of the proposed
method, make it suitable for large-scale
analyses as well as for analyzing pediatric
samples.
References
1. Bragdon, J. H.: Colorimetric determination of
blood lipides. J. Biol. Chem., 190: 513-517,
1951.
2. Cheek, C. S., and Wease, D. F.: A summation
tcchnic for serum total lipids. Clin. Chem., 15:
102-107, 1969.
3. Drevon, B., and Schmit, J. M.: La reaction sulfophosplio-vanillique dans l'etude des lipides seriques. Bull. Trav. Soc. Pharm. (Lyon), 8: 173178, 1964.
4. Jacobs, S. L., and Henry, R. J.: Studies on the
gravimetric determination of serum lipids.
Clin. Chim. Acta, 7: 270-276, 1962.
5. Kunkel, H. C , Alliens, E. H., and Eiscnmenger,
W. J.: Application of tuibidimetric methods
for estimation of gamma globulin and total
lipid to the study of patients with liver disease. Gastroenterology, 11: 499-507, 1948.
6. Postma, T., and Stroes, J. A. P.: Lipid screening
in clinical chemistry. Clin. Chim. Acta, 22:
569-578. 1968.
7. Sperry, W. M.: Gravimetric determination of
total lipids in blood serum or plasma. In Seligson, D. (ed.): Standard Methods of Clinical
Chemistry. Vol. 4. New York, Academic Press,
Inc., 1963, pp. 173-182.