ChemicalBasisof the Sulfo-phospho-vanillin
Reactionfor EstimatingTotal Serum Lipids
Joseph A. Knight, Shauna Anderson, and James M. Rawle
Results with the “sulfo-phospho-vanillin”
reaction, much used for determining total serum
lipids, have been favorably compared with those
for the gravimetric method. We investigated the
basic chemistry of the reaction and determined
the reactivity of this single reagent with various
lipids. Our results suggest that: (a) The reaction
requires
a carbon-carbon
double
bond. (b) Concentrated sulfuric acid reacts with unsaturated
lipids in the initial step to form a carbonium ion.
(c) Phosphoric acid reacts with vanillin to produce
a phosphate ester, with a resulting increase in the
reactivity of the carbonyl group. (d) The carbonium ion reacts with the carbonyl group of phosphovanillin to form a colored compound, which is
stabilized by resonance. (e) Unsaturated compounds with more than one double bond react,
but reaction may vary with steric hindrance. (f)
The method is reasonably precise, but its accuracy
depends primarily on the reference standard used.
The “sulfo-phospho-vanillin”
reaction
for determination
of total serum lipids was first introduced by Chabrol
and Charonnat
(1).
Subsequently,
there have been several
modifications
(2-4)
of the basic procedure.
The method
is
simple, rapid, and reasonably
precise.
The results reportedly
compare favorably
with those obtained by methods based on extraction
and gravimetry (3, 5, 6), and it has been recommended
as an
acceptable
procedure
for routine
use.
Because
the reaction
mechanism
has not been studied,
it
seemed
advisable
to do so to clarify the specificity
of the method.
Chemical Co., St. Louis, Mo. 63118), 0.6 g, was dissolved in 8-10 ml of absolute ethanol before diluting to 100 ml with distilled water. This solution
was mixed with 400 ml of concentrated
H,P04
with constant
stirring.
It was stored in a dark
bottle at room temperature.
All other aromatic
aldehydes
(Eastman
Organic
Chemicals,
Eastman
Kodak
Co.,
ilochester,
N. Y. 14650)
were
treated
exactly as for the phospho-vanillin
reagent.
Concentrated inorganic acids, analytical
grade.
“Dade Total Lipid Standard”
(Dade Division,
American
Hospital
Supply
Corp.,
Miami,
Fla.
33152).
Substrates.
Olive oil, cholesterol,
organic acids
(oleic, linoleic, linolenic, stearic, palmitic),
triolein,
tripalmitin,
or phosphatidyl
choline
(all from
Sigma) were dissolved
(10 g/liter)
in absolute
ethanol, and appropriately
diluted as needed.
Procedure
The procedure
was essentially
as previously
described
(4), with minor
variations.
Concentrated H2SO4, 5 ml, was added to a test tube containing 0.1 ml of serum. The tube was heated for
10 mm in a boiling water bath, cooled, and a 0.4ml aliquot was placed in a clean, dry tube labeled
“unknown.”
A blank contained
0.4 ml of concentrated
H2SO4. To each was added
6 ml of
phospho-vanillin
reagent.
Absorbances
were read
on a Beckman
DBG spectrophotometer
with use
of 10-mm (i.d.) cuvets. The results were the same
when 0.2 ml of the mixture was used and readings
made with 19-mm cuvets.
The final color was
measured at 525 nm (4) after the mixture had set in
the dark for 45 mm.
Materials and Methods
Results and Discussion
Reagents
Phospho-vanillin.
This was prepared
essentially
as previously
described
(3). Vanillin
(Sigma
From the Department
of Pathology,
Hospital, Provo, Utah 84601.
Presented at the 23rd National Meeting
Washington,
Received
August
8-13,
Utah
L.l).S.
of the AACC, Seattle,
1971.
Sept. 2, 1971; accepted
Valley
Nov. 1, 1971.
Need for Carbon-Carbon Double Bond
It has been suggested
(3), but now shown, that
the reaction requires a carbon-carbon
double bond.
This requirement
is well substantiated
by our
experiments
(Figure
1). Saturated
fatty
acids
and triglycerides
(stearic acid, palmitic
acid, and
tripalmitin)
as well as glycerol fail to react. All
CLINICAL
CHEMISTRY,
Vol. 18,
No. 3, 1972 199
other compounds
or mixtures shown in Figure 1
have at least one double
bond. The carboxyl
group is evidently
not necessary
for the reaction.
That a double bond is needed is more specifically
shown by the results illustrated
in Figure 2. Here,
the concentration
of various mixtures of oleic acid
(cis-9-octadecenoic
acid) and stearic
acid. (octadecanoic acid) are plotted against absorbance
(A).
Clearly
A increases
only in proportion
to the
amount of the unsaturated
fatty acid (oleic acid).
The reaction
apparently
is specific for unsaturated organic compounds.
Therefore,
one should
expect results to vary according
to the molecular
weight and chemical
structure
of the lipid being
reacted,
which in turn means that serum values
reported
by use of this method depend greatly on
the composition
of the reference
standard
used.
Table 1 illustrates
this point.
The Dade
total
lipid standard
(a mixture of stearic, palmitic,
and
oleic acids) gives the smallest and olive oil (a mixture of triglycerides)
gives the greatest
values.
Pikaar and Nijhof (7) have shown that about 30%
of the total fatty acids in serum, after hydrolysis
of
the various esters, are saturated
and the remaining
70% are unsaturated.
This suggests to us that a
mixture
having a molar percentage
ratio of 70%
oleic acid (the most abundant
naturally
occurring
unsaturated
fatty acid) to 30% palmitic
acid or
stearic acid would best simulate the saturation
of
the lipids normally
present
in serum.
(We estimate, by comparison,
that the Dade standard
contains
about 80% oleic acid.)
This mixture
(70:30, molar) produces
results
intermediate
to
those for the other two standards
(Figure 1 and
Table 1). In addition,
it corresponds
more closely
to cholesterol
(Figure
1), thereby
decreasing
the
error owing to the measurement
of this substance.
This standard
is admittedly
not ideal, because
phospho-lipids
and triglycerides
yield moieties, on
hydrolysis,
that do not react with the reagent.
This is apparent
in that triolein reacts less than
oleic acid. Glycerol, which does not react with the
vanillin
reagent,
contributes
about
10% to the
total molecular
weight.
Nevertheless,
we think
that this 70:30 mixture should give more nearly accurate results than either olive oil or the Dade
standard.
Olive oil would appear to be the least
desirable
of the three. An alternative
approach
would be to use standardized
pooled serum. In
any case, the value obtained
is an estimate,
and
depends on the degree of unsaturation
of the serum
lipids. It would perhaps
be more correct to refer
to an “index of total lipids” when this method is
used.
Role of Sulfuric Acid
It has been inferred (package insert, Dade total
lipid determination)
that a major function
of the
sulfuric acid is to hydrolyze
the lipid esters. The
200 CLINICAL CHEMISTRY, Vol. 18, No. 3, 1972
I
C
N
I
I
mg/lOO ml
Fig. 1. Comparison of absorbances of various lipids after
the sulfo-phospho-vanillin
reaction
E
C
N
0
U
C
0
.0
I.
0
a
.0
4
mg/lOO ml
Fig. 2. Comparison
ratios
of linoleic
phospho-vanillin
of absorbances
given by various
acid to palmitic acid after the sulfo-
reaction
initial reaction
takes place in concentrated
sulfuric acid in which the serum-to-acid
ratio is 1:50.
If hydrolysis
were the only process, less concentrated
acid should
give equivalent
results.
To
examine this idea, we ran the reaction with various
concentrations
of sulfuric acid, as well as with concentrated
hydrochloric
and phosphoric
acids (Table
2). It is apparent
that concentrated
sulfuric acid is
the only one in which an appreciable
reaction takes
place. In addition, pure oleic, linoleic, and linolenic
acids all fail to react appreciably
with phosphovanillin unless previously treated with concentrated
Table 1. Dependence of Total Lipid Values for
Table 2. Absorbances after Phospho-Vanillin
10 Unselected Patients’ Sera on the Reference
Standard Used
Reaction with Olive Oil (10 gJliter) When Various
Concentrations of Sulfuric and
Other Acids Are Used
R.f.r.nce
standards
mg/100 ml
Olsic: palmltic,
70:30
Dad.
Acid
A,
520
540
640
H2S04:H20(3:1)
510
740
530
H2S04:H20(1:3)
485
500
630
930
595
820
575
890
780
460
405
1110
H2S04:H20
0.00
0.00
0.00
0.00
Table 3. Absorbances after Sulfo-Phospho-
835
570
500
1380
1160
(1:1)
HCI, concd.
H3P04, concd.
1060
715
600
705
480
420
675
0.47
0.19
H2S04, concd.
Olive oil
Vanillin Reaction with Oleic Acid (10 g/liter) at
Various Temperatures”
T.mp.,”C
A,525nm
100
25
Similar results with olive oil, linoleic, and linolenic acids.
example, concentrated
sulfuric acid reacts little or
not at all, and diluted sulfuric acid (one vol of acid
plus one vol of water) produces a cloudy brown-red
color. Nitric acid oxidizes the vanillin,
producing
variable
dark-brown
solutions.
Hydrochloric
acid,
surprisingly,
did produce
a brilliant
(although
somewhat variable) purple color.
The vanillin reagent is prepared
in water, but
this is mixed with concentrated
phosphoric
acid
before use. Color development
is minimal (and the
color rapidly fades) if no acid is used. Phosphoric
acid undoubtedly
reacts with the hydroxyl
group
to produce an aromatic phosphate
ester. The phosphate group is presumed
to change the electron
distribution
of the vanillin molecule such that the
reactivity
of the carbonyl
group is enhanced
and
the final product of the reaction stabilized.
If concentrated
hydrochloric,
nitric, or sulfuric acids are
substituted,
the final color varies, and in each case
is not suitable for quantitative
measurement.
For
NH
#{149}
-#{233}.#{234}-
0.26
0.08
50
Role of Phosphoric Acid
H8$O,
0.53
0.44
75
sulfuric
acid. Hence,
although
hydrolysis
presumably
takes place, the lipid must react specifically with sulfuric acid. This information
is most
consistent
with the theory of carbonium
ion formation (8). Here, an unsaturated
double bond (weak
base) accepts a proton from sulfuric acid (strong
acid) to form a highly reactive carbonium
ion. The
ion is efficiently formed at 100#{176}C
[lower temperatures lead to considerably
less reaction (Table 3)].
On cooling, the ion so formed is stable for at least
several hours.
I.
525 nm
Proposed Reaction Sequence
On the basis of the information
at hand, we believe that the reaction takes place in three steps
(Figure 3): (a) Unsaturated
compounds
react with
sulfuric acid to produce a carbonium
ion, (b) vanillin reacts with phosphoric
acid to produce an aromatic phosphate
ester, and (c) the carbonium
ion
reacts with the activated
carbonyl group of phospho-vanillin
to produce a charged colored complex
that is stabilized
by resonance
and absorbs maximally at about 525 nm (4). Reportedly,
alcohols
NH
4-6-
(Ne)
H
H
2
Q
OCH,
#{149}
N,P04
Fig.
N,
3.0
0CM,
+
N
3.
quence
vanillin
c.0
H
.-0-.
-e
N
-
Proposed
for the
reaction
reaction
se-
sulfo-phospho-
I
sic.
CLINICAL CHEMISTRY, Vol. 18, No. 3, 1972 201
Table 4. Absorbances of the Reaction Products of Oleic, Linoleic, and Linolenic Acids (10 g/liter)
with Phospho-Vanillin and Other Phospho-Aromatic Aldehydes”
p-Hyd roxybonzald.hyd.
(500 nm)
o.M.thoxy.
benzaldehyd.
(520 nm)b
p-Methoxybenzald.hyd.
(495 nm)
Oleic
0.53
0.37
0.58
0.29
Lirioleic
0.43
0.25
0.53
0.28
0.78
Linolenic
0.39
0.17
0.32
0.18
0.49
“Absorbance
b
o-Hyd roxyb.nzaldehydo
(490 nm)
Vanhi in
(525 nm)
Acid
wavelengths
Initial absorbance.
indicated
in parentheses.
Color fades rapidly.
with more than two carbon atoms also react (4),
because they are readily dehydrated
by concentrated sulfuric acid to unsaturated
compounds
(8),
which subsequently
react as just described.
Compounds with Multiple Unsaturated
The intensities
are compared
with
vanillin
(Table 4).
case as the number
the para isomers
highly colored and
because the hydroxy
separated
from
the
of the resulting
carbonyl
group.
colors
On
the other hand, because the reacting
carbonyl
is
close to the hydroxy
and methoxy
groups,
the
ortho isomers not only react less but in the case of
Q-methoxybenzaldehyde,
the reaction
product
is
highly unstable,
and the color fades rapidly.
Because the electronic structures
of each pair of aidehydes are similar,
the differences
are considered
primarily
to result from steric hindrance.
202 CLINICAL CHEMISTRY, Vol. 18, No. 3,
1972
Oleic
.50
Linolonic
Linoleic
C
those obtained
with phosphoAbsorbance
decreases
in each
of double bonds increase, and
produce
compounds
that are
stable, as would be expected,
and methoxy
groups are well
reacting
.60
E
Bonds
We initially assumed that when compounds
with
more than one double bond, were reacted,
they
would give a more intense color than would those
with a single double bond. Using oleic, linoleic, and
linolenic acids, we found the opposite Lo be the case
(Figure 4). The reasons for this are at least twofold: (a) only a single carbonium
ion is formed per
molecule,
because
multiple
ones would not be
stable; and (b) steric hindrance
evidently
becomes
an important
factor in the case of the multiplyunsaturated
compounds.
For example,
oleic acid
(9-octadecenoic
acid) is the cis isomer. Linoleic
acid (9,12-octadecadienoic
acid) is cis,cis, and
linolenic
acid (9,12,15-octadecatrienoic
acid) is
cis,cis,cis.
These molecules are curled increasingly
tighter into a ring, thereby making it more difficult
for the relatively
large and bulky phospho-vanillin
molecule to react at the proper site. Evidence supporting
this hypothesis
was elicited not only by
studying
molecular
models, but also by reacting
the three acids with phosphoric
acid-treated
o- and
p-hydroxybenzaldehyde
and o- and p-methoxybenzaldehyde.
0.90
i0
N
.40
a
V
U
C
a
.0
0
C
.0
4
.30
.20
.10
500
mg/lOO
ml
Fig. 4. Comparison of absorbances of oleic, linoleic, and
linolenic acids after the sulfo-phospho-vanillin
reaction
References
1. Chabrol,
l’etudes
E., and Charonnat,
des lipides:
L’oleidemie.
R., Une nouvele
reaction
pour
Presse Med. 45, 1713 (1937).
2. Drevon,
B., and Schmit, J. M., La reaction sulfo-phosphovanillique
dans l’etude des lipides seriques.
Bull. Tray. Soc.
Pharm. Lyon8, 173 (1964).
3. Frings, C. S., and Dunn, R. T., A colorimetric
method for
determination
of total serum lipids based on the sulfo-phosphovanihin reaction. Amer. J. Clin. Pathol. 53, 89 (1970).
4. Zoeliner, N., and Kirsch, K., Uber die quantitative
Bestimmung von Lipoiden (Mikromethode)
mittels der vielen naturlischen Lipoiden (alien bekannten
Plasmalipoiden)
gemeinsamen
sulfophosphovanillin
Reaction. Z. Gesamte Exp. Med. 135, 545
(1962).
5. Postma, T., and Stroes, J. A. P., Lipid screening in clinical
chemistry. Clin. Chim. Ada 22, 569 (1968).
6. Sperry, W. M., Gravimetric
determination
of total lipids in
blood serum or plasma. Stand. Methods Ciin. Chem. 4, 173 (1963).
7. Pikaar, N. A., and Nijhof, J., Microdetermination
of the fatty
acids in blood serum. Biochem. J. 70, 52 (1958).
8. Morrison,
R. T., and
Boyd,
ed., Allyn & Bacon, Inc., Boston,
R. N., In Organic Chemistry,
Mass., 1968, pp 161-162.
2nd