[42]

D E T E R M I N A T I O N OF A L D E H Y D E S

407

[42] D e t e r m i n a t i o n o f A l d e h y d i c L i p i d P e r o x i d a t i o n
Products: Malonaldehyde and 4-Hydroxynonenal
By HERMANN ESTERaAUERand KEVlN H. CHEESEMAN
Introduction

Aldehydes are always produced when lipid hydroperoxides break

down in biological systems,X-3 and it is of interest to identify and measure
these compounds both as an index of the extent of lipid peroxidation and
as an aid to elucidate the role of aldehydes as causative agents in certain
pathological conditions. 2-4 We deal here with current analytical methods
used for the qualitative and quantitative determination of aldehydes in
biological systems, and we pay particular attention to 4-hydroxynonenal
(HNE) and malondialdehyde (MDA).
4-Hydroxynonenal is produced as a major product of the peroxidative
decomposition of to6 polyunsaturated fatty acids (PUFA) and possesses
cytotoxic, hepatotoxic, mutagenic, and genoroxic properties. 2,4,5 Increased levels of HNE were found in plasma and various organs under
conditions of oxidative stress (for review, see Refs. 6 and 7). In addition
to HNE, lipid peroxidation generates many other aldehydes (alkanals, 2alkenals, 2,4-alkadienals, protein- and phospholipid-bound aldehydes)
which may also be of toxicological significance.2,4.6,8
Malondialdehyde is in many instances the most abundant individual
aldehyde resulting from lipid peroxidation, and its determination by
thiobarbituric acid (TBA) is one of the most common assays in lipid
peroxidation studies. In vitro MDA can alter proteins, DNA, RNA, and
many other biomolecules.8 Recently, it has been demonstrated with
t H. Esterbauer, in "Free Radicals, Lipid Peroxidation and Cancer" (D. C. H. McBrien
and T. F. Slater, eds.), p. 101. Academic Press, London, 1982.
2 H. Esterbauer, in "Free Radicals in Liver Injury" (G. Poli, K. H. Cheeseman, M. U.
Dianzani, and T. F. Slater, eds.), p. 29. IRL Press, Oxford, 1985.
3 W. Grosch, in "Autoxidation of Unsaturated Lipids" (H. W. S. Chan, ed.), p. 95. Academic Press, London, New York, 1987.
4 M. Comporti, Lab. Invest. 53, 599 (1985).
5 H. Esterbauer, H. Zollner, and R. J. Schaur, IS1Atlas Sci. Biochem. 1, 311 (1988).
6 H. Esterbauer, H. Zollner, and R. J. Schaur, in "Membrane Lipid Oxidation" (C. VigoPelfrey, ed.), Voi. 1, p. 239. CRC Press, Boca Raton, Florida, 1990.
7 H. Esterbauer and H. Zollner, Free Radical Biol. Med. 7, 197 (1989).
8 E. Schauenstein, H. Esterbauer, and H. Zollner, "Aldehydes in Biological Systems: Their
Natural Occurrence and Biological Activities." Pion Press, London, 1977.

METHODS IN ENZYMOLOGY, VOL. 186

Copyright 1990by Academic Press, Inc.

All rights of reproduction in any form reserved.

408

ASSAY AND REPAIR OF BIOLOGICAL DAMAGE

[42]

monoclonal antibodies that malonaldehyde-altered protein occurs in atheroma of hyperlipidemic rabbits. 9

Standard Determination of Malonaldehyde with Thiobarbituric Acid
In the TBA test reaction one molecule of MDA reacts with two molecules of TBA with the production of a pink pigment having an absorption
maximum at 532-535 nm. The reaction should be performed at pH 2-3 at
90-100 for 10-15 min. Typically, the tissue sample (e.g., a liver microsomal suspension) is mixed with 2 volumes of cold 10% (w/v) trichloroacetic acid (TCA) to precipitate protein. The precipitate is pelleted by
centfifugation, and an aliquot of the supernatant is reacted with an equal
volume of 0.67% (w/v) TBA in a boiling water bath for 10 min. After
cooling the absorbance is read at 532 nm and the concentration of MDA
calculated based on an e value of 153,000. This value is the average of
several slightly differing figures reported in the literature. 1 The crystalline M D A - T B A adduct in water shows an absorption maximum at 532 nm
(e 159,200)) 1
The TBA reagent should be prepared as an aqueous solution and
requires heating to disgolve the TBA solid. A standard curve can be
prepared using malonaldehyde bisdimethyl- or bisdiethylacetal as the
source of MDA. A 10 mM stock solution is prepared by adding 1 mmol of
the acetal to 100 ml of 1% (v/v) sulfuric acid and leaving the mixture at
room temperature for 2 hr to achieve complete hydrolysis. For the preparation of the standard curve the MDA stock is further diluted to about 110/~M and then reacted with TBA as above. The concentration of the
MDA solution can be checked by measuring the UV spectrum. In 1%
H 2 S O 4 the absorption maximum is at 245 nm (e 13,700). 12 In alkaline
solution (10 mM NaaPO4) the maximum is at 267 nm (e 3 1 , 5 0 0 ) . 13
Many factors influence the results obtained with the TBA test, as
discussed previously in this series. 12,14Briefly, conditions and procedures
to be avoided if free MDA is to be measured are as follows: preparation of
the TBA reagent in strong acid solutions, high concentrations of metals,
such as iron, high concentrations of sugars, such as sucrose, and use of
the whole tissue sample in the assay. To ensure that no lipid oxidation
9 M. E. Haberland, D. Fong, and L. Cheng, Science 241, 215 (1988).
10 H. Esterbauer, K. H. Cheeseman, M. U. Dianzani, G. Poli, and T. F. Slater, Biochem. J.
2118, 129 (1982).
11 V. Nair and G. A. Turner, Lipids 19, 804 (1984).
t2 H. Esterbauer, J. Lang, S. Zadravec, and T. F. Slater, this series, Vol. 105, p. 319.
~s T. W. Kwon and B. M. Watts, J. Food Sci. 28, 627 (1963).
14 R. P. Bird and H. H. Draper, this series, Vol. 105, p. 299.

[42]

DETERMINATION OF ALDEHYDES

409

TABLE I
TBA REACTION WITH DIFFERENT COMPOUNDSa
Compound
Malonaldehyde
Alkanals
2-Alkenals

2,4-Alkadienals

4-Hydroxyalkenals

Amino acids preincubated with 0.9 m M Fe

Sugars preincubated with 0.9 m M Fe
Monohydroperoxides from arachidonic acid

Conditions b
A
A,B,C
A without
A with Fe
B
C
A without
A with Fe
B
C
A without
A with Fe
C
D
D
E without
E with Fe

Fe

Fe

Fe

Fe

e value
153,000
0
14-66
30-90
100-200
130-160
48-160
184-280
1100-2600
4500
12-119
38-124
320
50-620
90-2700
3200-8100
12400-34100

a Absorbance at 530-535 nm is expressed per mole of compound.

b A, 5% TCA, 10 rain, 100, in the presence or absence of 3 /zM FeSO4 (Ref. 10);
B, water, 60 rain, 95 (Ref. 15); C, 1 N glacial acetic acid, 120 min, 100 (Ref. 16); D,
glacial acetic acid, 30 min, 100 (Ref. 17); and E, 10% TCA, 30 min, 100, in the
presence or absence of 2.5 m M FeSO4 (Ref. 18).

occurs during the assay, butylated hydroxytoluene (0.01 vol % of a 2%

BHT solution in ethanol) and EDTA (1 mM final concentration) can be
added to the sample prior to TCA precipitation.
Determinations from TBA Test Measurements
It is well documented that the TBA test is not specific for MDA. A
great variety of substances other than MDA under appropriate conditions
also form pink TBA complexes; moreover, MDA or MDA-like substances
can arise during the assay from acid-catalyzed or thermal decomposition
of precursors (other aldehydes, MDA bound to proteins, oxidized lipids,
amino acids, sialic acid) (Table I). 1s-18It would seem, however, that using
15 R. Marcuse and L. Johansson, J. Am. Oil Chem. Soc. 50, 387 (1973).
~6G. Witz, N. J. Lawrie, A. Zaccaria, H. E. Ferran, Jr., and B. D. Goldstein, J. Free
Radicals Biol. Med. 2, 33 (1986).
17 j. M. C. Gutteridge, FEBS Leu. 128, 343 (1981).
18j. Terao and S. Matsushita, Lipids 16, 98 (1981).

410

ASSAY AND REPAIR OF BIOLOGICAL DAMAGE

[42]

the protocol described above for peroxidized tissue samples, e.g., microsomes, there is little artifactual production of MDA or interference with
other TBA-positive substances. This is not merely conjecture but has
been demonstrated in practice. 10,12,19,20In liver microsomal suspensions in
which lipid peroxidation has been stimulated by ADP-iron, CC14, o r
ascorbate-iron, the direct determination of free MDA by the HPLC
method described below gave precisely the same value as did the TBA
test, indicating that in those systems the standard TBA test measures only
free MDA and not MDA-like substances. Also, in oxidized low density
lipoprotein 80% of the TBA-reactive substances (TBARS) were free
MDA. 21
This does not contradict the low specificity of the TBA test but can be
explained as follows. First, in the standard procedure most of the potential MDA precursors, e.g., protein-MDA complexes or oxidized lipids,
are removed by TCA precipitation in the cold prior to the actual assay.
Second, other TBA-positive compounds that could be present in the deproteinized supernatant, such as aldehydes, amino acids, sugars, and
fatty acid hydroperoxides, give only a very weak color in the standard
TBA assay. On a molar basis, the absorption at 530-535 nm produced by
such compounds is several orders of magnitude lower than the absorption
produced by MDA (Table I). The TBA-positive compounds would therefore have to be present in the sample in extremely high concentrations to
interfere significantly with the standard determination of MDA. Suspensions of peroxidized rat liver microsomes (ADP-iron, 30 min) contain,
e.g., 58 nmol free MDA and 95 nmol of the other aldehydes listed in Table
I. 1A rough estimate shows that 99.7% of the absorbance at 535 nm which
would be found in the standard TBA assay results from MDA (e 153,000),
and only 0.3% or less is due to all other aldehydes (assumed average e
300).
The situation may, however, be completely different if the standard
reaction conditions are significantly altered, e.g., heating in the presence
of the complete tissue fraction, prolonged reaction times, the use of other
acids, and supplementation of the reaction mixture with iron. There can
be no doubt that such modified TBA tests are much less specific, and it
seems appropriate to refer in such cases to the measurement of TBApositive substances, TBARS, or simply the TBA value rather than specifying MDA.
Frequently used modifications of the TBA test employ the whole acidt9 H. Esterbauer and T. F. Slater, 1RCS Med. Sci. 9, 749 (1981)
2o j. Lang, P. Heckenast, H. Esterbauer, and T. F. Slater, in "Oxygen Radicals in Chemistry and Biology" (W. Bors, M. Saran, and D. Tait, eds.), p. 351. de Gruyter, Berlin, New
York, 1984.
21 H. Esterbauer, G. Jiirgens, O. Quehenberger, and E. KoUer, J. Lipid Res. 28, 495 (1987).

[42]

DETERMINATIONOF ALDEHYDES

411

ified sample. Typically22.23 1 volume of tissue sample, e.g., 10% (w/v)

homogenate, is mixed with 6 volumes of 1% phosphoric acid and 2 volumes 0.6% aqueous TBA solution and heated for 45-60 min in a boiling
water bath. After cooling, 8 volumes of n-butanol are added and mixed
vigorously. The butanol phase which contains the colored TBA reaction
products is separated by centrifugation and its absorbance measured at
532 nm. The method with phosphoric acid is basically developed to measure MDA and/or TBARS bound to proteins; it seems that under the
assay conditions the binding is, at least in part, reversible. With this
method significantly elevated levels of TBARS were found in various
fresh tissues of rats fed a vitamin E-deficient diet, e.g., 570 nmol/g liver of
vitamin E-deficient rats compared to 76 nmol in controls. 23 In another
procedure, 0.2 ml of 35% TCA and 1 ml of 0.5% TBA are added to 1 ml of
sample, and the mixture is heated at 60 for 90 min, after which dispersed
lipids are extracted with 3 ml CH2C12 and the clear aqueous phase measured at 532 nm. 24--26With this method 0.22-0.40 nmol TBARS/ml were
found in control plasma. A u s t 27 reported a method where 1 ml of sample is
mixed with 2 ml of a TCA-TBA-HCI reagent [15% (w/v) TCA, 0.375%
(w/v) TBA, 0.25 N HCI], the complete mixture is heated on a boiling
water bath for 15 min, and after centrifugation, the absorbance is measured at 535 nm.
Numerous other variations have been introduced, but heating the
complete assay sample with TBA in acidic solution is common to all.
These methods can be subject to various sources of errors if only the
absorption at 530-535 nm is measured. For example, yellow compounds
with maxima at 450-490 nm and significant absorption at 530-535 nm are
often formed and would lead to an overestimation of TBARS. It is therefore strongly recommended that the spectrum in the 430-600 nm range be
recorded to prove the existence of the 532 nm maximum typical for the
MDA-TBA complex and to correct for possible background absorption
by interpolation. The exact amount of the MDA-TBA complex in the
reaction mixture can also be determined by HPLC. 14,28 The neutralized
reaction mixture or a butanol extract is separated on an ODS column with
methanol-water (85 : 15) and detected at 530-535 nm. In a TCA extract of
fresh pork liver 30 nmol TBARS was found by the spectrometric method,
36% of which was MDA as determined by HPLC. 28
22 M. Uchiyama and M. Mihara, Anal. Biochem. 86, 271 (1978).
23 M. Mihara, M. Uchiyama, and K. Fukuzawa, Biochem. Med. 23, 302 (1980).
24 K.-L. Fong, P. B. McCay, and J. L. Poyer, J. Biol. Chem. 248, 7792 (1973).
25 D. M. Lee, Biochem. Biophys. Res. Commun. 95, 1663 (1980).
26 F. Bernheim, M. L. Bernheim, and K. M. Wilber, J. Biol. Chem. 174, 257 (1948).
27 S. D. Aust, this series, Vol. 52, p. 302.
28 R. P. Bird, S. S. O. Hung, M. Hadley, and H. H. Draper, Anal. Biochem. 128, 240 (1983).

412

ASSAY AND REPAIR OF BIOLOGICAL DAMAGE

[42]

Other modifications of the TBA assay were designed to analyze lipid

hydroperoxides. As can be seen from Table I, including iron in the TBA
reaction mixture significantly increases the yield of pink products from
various substances. A particularly high absorption is obtained with monohydroperoxides from arachidonic acid, and this is the basis of a TBA test
for microdetermination of lipid peroxides. The recommended procedure 18,29is briefly as follows. One milliliter of methanol solution containing the oxidized lipid is mixed with 2 ml of 20% TCA containing 20/~mol
ferrous sulfate and 1 ml 0.67% TBA. The mixture is heated in a boiling
water bath for 30 min, and after cooling 2 ml CHCI3 is added (to extract
turbid material). The mixture is centrifuged, and the absorbance of the
clear supernatant is measured. In the case of methyl arachidonate monohydroperoxide isomers (MeHPETE), a linear relationship is found between the absorption at 532 nm and the concentration of MeHPETE.
Although each isomer gave a different response, the lowest amount of
MDA was found from the 8-OOH isomer (0.081 mol MDA/mol
MeHPETE); the highest amount gave the 5-OOH and 15-OOH isomers
(0.22 mol MDA/mol MeHPETE). To prevent additional lipid oxidation
during the color development, 0.01 vol% of 2% BHT in ethanol can be
added to the TBA reagent just prior to use.
Another TBA test to measure preferentially lipid peroxides was developed by Yagi for the analysis of serum or blood. 3,31 Here, the proteins
and lipids are first precipitated with phosphotungstic acid. The sediment
(equivalent to 20/.d serum) is then suspended in 4 ml water, 0.5 ml glacial
acetic acid, and 0.5 ml 0.33% aqueous TBA solution. The mixture is
heated for 60 min at 95 and after cooling extracted with 5 ml n-butanol.
The concentration of the T B A - M D A complex in the butanol extract is
determined fluorimetrically at 553 nm with excitation at 515 nm. The
amount of TBARS (assumed to be lipid peroxides) found by this test in
normal subjects was in the range of 1.8-3.9 nmol/ml serum. This is about
10 times the amount found with another modified TBA test 25 and clearly
shows that different procedures yield very different results. Direct comparison of data reported by different investigators is often not possible.
In conclusion, what is measured by the TBA assay is strongly influenced by the reaction conditions. In assays where the whole sample is
heated in an acidic TBA solution, the resulting absorption at 530-535 nm
(or fluorescence at 553 nm) can come from all preexisting MDA, proteinbound MDA, and lipid peroxides, as well as any other substances that
give rise to MDA or TBARS in the hot acid. Tests using only the TCA29 T. Asakawa and S. Matsushita, Lipids 15, 137 (1980).
3o K. Yagi, this series, Vol. 105, p. 328.
31 K. Yagi, in "Lipid Peroxides in Biology and Medicine" (K. Yagi, ed.), p. 223. Academic
Press, London, New York, 1982.

[42]

DETERMINATION OF ALDEHYDES

413

soluble fraction of the sample are more specific for free MDA. Here again,
however, interference can be caused by other TCA-soluble compounds,
in particular, if free MDA is low, such as in fresh tissue samples. In any
case, additional analyses should be performed to elucidate the nature and
the source of the pink color. Such analyses include the demonstration of
the TBA-MDA complex by HPLC 14,28 and the direct detection of free
MDA as described below.
Direct Determination of Malondialdehyde by HPLC
An HPLC method for the determination of free MDA in biological
samples has been described at length previously in this series, 12 and we
only outline it here. The principle of the method is that an aqueous sample
containing MDA at pH 6.5-8.0 is separated by HPLC using an aminophase column with acetonitrile-30 mM Tris buffer, pH 7.4 (1 : 9, v/v), as
the mobile phase. The effluent is monitored at 267 nm, the absorption
maximum of the enolate anion form of free MDA (e 31,500 at this pH and
wavelength). The system is calibrated and the sample MDA peak identified by comparison with a solution of MDA. Using an injection volume of
20/zl, the smallest concentration of MDA in the original solution that can
be quantified this way is 0.25/zM. To protect the column it is best to
deproteinize the sample, and we find that addition of an equal volume of
acetonitrile followed by centrifugation is satisfactory. A typical estimation would be as follows. An aliquot of sample (e.g., liver microsomal
suspension) is mixed with an equal volume of acetonitrile and the protein
precipitate pelleted at 3000 g for 10 min. A sample (20/zl) of the supernatant is injected into the HPLC and separated on an S-5 Spherisorb-NH2
column (Phase Separations Ltd.) at I ml/min.
With this method it w a s s h o w n 1A2,19,2 that what is measured with the
standard TBA test (use of TCA supernatant) in peroxidized microsomes
or mitochondria is exclusively free MDA. In addition, this method was
u s e d 6 to compare the amount of free MDA and TBARS formed during
oxidation of various PUFA (Fig. 1). At the stage when the PUFA were
completely oxidized, the yields of free MDA on a molar basis were 0.5%
for linoleic acid, 4.5% for linolenic acid, 4.9% for y-linolenic acid, 4.7%
for arachidonic acid, and 7.6% for docosahexaenoic acid. The corresponding yields of TBARS were slightly higher, namely, 0.55, 4.9, 5.1,
6. l, and 8.6%. The very low yield of MDA from linoleic acid agrees with
the proposal 32 that MDA is only formed from PUFAs with three or more
double bonds.
We have also used this method in a system completely unrelated to
32 W. A. Pryor, J. P. Stanley, and E. Blair,

Lipids 11, 370 (1976).

414

ASSAY AND REPAIR OF BIOLOGICAL DAMAGE

:a.

22:6 (-)
20 : 4 (---)

09
r
<
r]
p-

:a.

400

TSARS

i"

II

300 //

25
"..,m- ,

~_~

i11

m~

,,'/

"O
etO

TSARS

/ ...."'..

I Jr',,

[42]

20

MDA

-",,

".
I..

"o

100

tf

//

~\

\~

12

incubation time, hours

FiG. 1. Free malonaldehyde (MDA) and MDA-like substances (TBARS) formed during
autoxidation of arachidonic acid (20 : 4) and docosahexaenoic acid (22 : 6). The fatty acids
(0.1 mg/ml) were incubated in Tris buffer, pH 7.4, with ascorbate-iron (10 raM-0.4 raM) at
37. Consumption of the fatty acids was measured by GC, free MDA by HPLC, and TBARS
by the standard TBA assay as described in the text.

lipid peroxidation: the production of MDA from cleoxyribose degradation

by OH. attack. 33 Further, a modification of this method has been reported
in which the proportion of acetonitrile is increased to 80% in order to
achieve better separation from interfering substances when measuring
plasma. 34
Two other HPLC methods for measuring free MDA directly have been
published. One method 35 uses an ODS column with acetonitrile-water
(14 : 86), 50 mM myristyltrimethylammonium bromide, 1 mM phosphate
buffer, pH 6.8, as mobile phase at 1 ml/min, with detection at 267 nm. The
basis of the separation is ion-pairing chromatography. A reasonably good
equivalence between this direct determination and the TBA test was
found when measuring peroxidized microsomes. The other method 36 uses
a size-exclusion column (Spherisorb TSK G 1000 PW, Phase Separation
33 K. H. Cheeseman, A. Beavis, and H. Esterbauer, Biochem. J. 252, 649 (1988).
34 C. Largilliere and S. B. Melancon, Anal. Biochem. 170, 123 (1988).
35 A. W. Bull and J. Marnett, Anal. Biochem. 149, 284 (1985).
36 A. S. Csallany, M. D. Guan, J. D. Manwaring, and P. B. Addis, Anal. Biochem. 142, 277
(1984).

[42]

DETERMINATION OF ALDEHYDES

415

Ltd.) with 0.1 M phosphate buffer, pH 8.0, and detection at 267 nm. A
poor equivalence was found by this method when measuring MDA in beef
or pork muscle or rat liver, e.g., 43 nmol (TBA) versus 11 nmol (HPLC)
per 1 g of rat liver.
Several other chromatographic methods for the detection of MDA
were reported. In one procedure, 37 developed for vegetable oil, the sampie-(0.1 g) is reacted with dansylhydrazine in hydrochloric acid containing
FeC13. The formed dansylpyrazole is separated by HPLC with fluorimetric detection. In another method, 38 developed for investigation of the
formation of MDA from lipid peroxidation products, the oxidized lipid
(20-25 mg) is treated for 18 hr at ambient temperature with 1 ml of 5%
anhydrous HC1 in methanol and I ml trimethyl orthoformate. The amount
of MDA-tetramethylacetal formed is determined by gas chromatography.
Both methods certainly do not measure free MDA but rather the amount
of MDA that can be formed from precursors by acid-catalyzed decomposition.
Although in the systems we have studied the TBA test is demonstrated
as measuring free M D A , 12'19'20'33 this will not be true in all systems. If the
investigator is concerned in knowing whether MDA is the only TBAreactive product in the test system, then the measurement should be
validated with a direct measurement of free MDA by HPLC. If the two
determinations are equivalent, the investigator can use the more convenient TBA test.
Determination of Aldehydes via Dinitrophenylhydrazone Formation

The methods most frequently used for determination of aldehydes in

biological tissues are based on treatment of the sample with 2,4-dinitrophenylhydrazine. Aldehydes react with dinitrophenylhydrazine to
form the corresponding dinitrophenylhydrazone (DNPH) derivatives. In
contrast to most free aldehydes the hydrazone derivatives are stable and
not volatile, greatly facilitating the subsequent workup procedure. Moreover the D N P H derivatives have a strong yellow color (hmax 360-380 nm,
e 25000-28000 M -1 c m - l ) which is of great help in detecting the compounds on TLC plates or by HPLC.
An outline of the procedure we routinely use is as follows. The sample
is mixed with dinitrophenylhydrazine reagent and allowed to react. The
DNPH derivatives are extracted into an organic solvent, concentrated,
and preseparated by TLC to yield DNPH classes of different polarity
(here termed zones I, II, and III from their positions on the TLC plate).
37T. Hirayama, N. Yamada, M. Nohara, and S. Fukui, J. Sci. FoodAgric. 35, 338 (1984).
E. N. Frankei and W. E. Neff, Biochim. Biophys. Acta 754, 264 (1983).

416

ASSAY AND REPAIR OF BIOLOGICAL DAMAGE

[42]

The individual classes are recovered and separated by HPLC for identification of their constituent individual aldehydes. The importance of the
preliminary separation by TLC should be stressed as it performs several
important functions. First, it enables the removal of excess dinitrophenylhydrazine reagent. Second, it enables certain contaminating carbonyls to
be eliminated; the DNPH forms of formaldehyde, acetone, and acetaldehyde are always found at this stage even in the reagent blank. Apparently
these carbonyls are always present in laboratory air and standard solutions. Finally, analysis of the hydrazones in each zone (I, II, and III)
greatly facilitates clear separation of the individual compounds and provides more confident identification of the peaks in the HPLC chromatogram. For example, zone III can only contain alkanals, 2-alkenals, and
2,4-alkadienals and cannot contain the more polar 4-hydroxyalkenals that
are restricted to zone I. It is possible to apply all of the DNPH derivatives
directly in HPLC without preliminary TLC, e.g., by using gradient programs; however, the resulting chromatograms are complicated, and it is
extremely difficult to make definite peak identifications.
A typical determination of aldehydes produced during lipid peroxidation in liver microsomes, 1,39hepatocytes, 39 or low density lipoproteins, 21
as examples for other biological samples, is as follows. To 1 ml of the
sample, e.g., microsomes at I mg protein/ml, add 0.1 ml of 1% EDTA,
10/zl of 2% BHT, and 0.5 ml freshly prepared DNPH reagent (2,4-dinitrophenylhydrazine recrystallized from butanol dissolved in 1 N HCI at a
concentration of 0.35 mg/ml). Mix vigorously and keep in the dark for 2 hr
at ambient temperature and then for 1 hr at 4. The reaction mixture is
extracted with CH2C12 (2 times 5 ml each); phase separation can be
achieved by centrifugation. The pooled extract is left in a freezer for at
least 2 hr and then rapidly filtered through a folded filter to remove ice
crystals. The extract is brought to dryness on a rotary evaporator (-<35)
and redissolved in a minimum volume of CH2C12 (about 0.1-0.5 ml) for
application to the TLC plate (silica gel 60 precoated, 20 x 20 cm, Merck,
Darmstadt, FRG). The extract is applied across the plate as a band 3-5
cm long; DNPH standards (see Fig. 2) are also applied as a separate spot.
The plate is developed first in CH2C12 (5 cm) and then in benzene (about 15
cm). In Fig. 2, nominal zones I, II, and III are indicated on the developed
plate.
The zones are scraped off the TLC plate and eluted with methanol (2
times, 1 ml each). The methanol extracts are dried in a small conical vial
with nitrogen, and the residue is finally redissolved in 0.1 ml methanol.
Samples (20/zl) are separated by HPLC on an ODS column (5/zm Spheri39 G. Poli, M. U. Dianzani, K. H. Cheeseman, T. F. Slater, J. Lang, and H. Esterbauer,
Biochem. J. 227, 629 (1985).

[42]

DETERMINATION OF ALDEHYDES

417

FRONT
|

UV-vis

ZONE III
<
alkanals, 2-alkenals
2,4-alkadienals, ketones

HPLC

| ~ 4" O

acetone,

formaldehyde,~acetaldehydeJ

UV-vis
ZONE I I

DNPH reagent

-O

C2~2Zq[) osazones
CS.2223
C.~T/-D
1
<
[
1

HNE

~O

START

HPLC

<Ov-via

I ZONE I, hydroxyalkenals

~ polar aldehydes~

HPLC
UV-vis

standards

sample

FIG. 2. Scheme showing the determination of aldehydes by the DNPH method.

sorb ODS, 4.6 x 250 mm) with methanol-water (31:9, v/v) at 1.0 ml/min
and detected at a wavelength between 365 and 378 nm. Peak assignment
and quantification are made with reference to chromatograms of standard
hydrazones. Additionally, the peak material can be collected to determine
the assigned structure by mass spectroscopy. Alkanals, 2-alkenals, 2,4alkadienals, or ketones are commercially available (e.g., Aldrich, Merck).
We prepare the corresponding hydrazones as follows. The compound
(about l0 mmol) is dissolved in a small volume of ethanol and added to
82 ml of DNPH reagent (2.4 g 2,4-dinitrophenylhydrazine in 100 ml 30%
HCIO4). The precipitate is filtered, washed acid-free with water, and
recrystallized from ethanol or ethanol-water mixtures. Various syntheses
for 4-hydroxyalkenals are described (for review, see Refs. 5 and 6), and
their hydrazones can be prepared as above.
The compounds identified in zone I include 4-hydroxynonenal and
4-hydroxyhexenal. In addition, this zone can contain 4,5-dihydroxydecenal4 and two aldehydes that are probably (based on their mass spectra) 4-hydroxy-4,5-nonadienal and 5-hydroxyoctanal. 4] Zone III contains
propanal, butanal, pentanal, hexanal, nonanal, 2-proper~al, 2-pentenal, 2hexenal, 2-heptenal, 2-octenal, 2-nonenal, 2,4-heptadienal, 2,4-deca4oA. Benedetti, M. Comporti, R. Fulceri, and H. Esterbauer, Biochim. Biophys. Acta 792,
172 (1984).
41 p. Heckenast, Thesis, University of Graz, Austria, 1983.

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ASSAY AND REPAIR OF BIOLOGICAL DAMAGE

[42]

dienal, butanone, 2-pentanone, 3-pentanone, and 2-octanone. Long-chain

aldehydes such as hexadecanal and octadecanal are also present in zone
III. They are unrelated to lipid peroxidation but result from plasmalogens.
Zone II contains mostly osazones, none of which have been identified.
In multiple analyses of the same sample the reproducibility of the
overall procedure is in the range of +--10-15%. In the literature 1,21,39,42-51
the amount of individual aldehyde present in the analyzed sample is usually reported as the figure obtained in the analysis described above, i.e.,
without considering recovery loss. This seems sufficient for many in vitro
studies where only the qualitative aldehyde pattern and the relative
changes compared to a control sample are needed. For determining the
absolute amount, however, recovery losses must be taken into account.
The recovery of aldehydes from biological samples is rather variable .6
The addition of 4-hydroxynonenal to fresh liver microsomes yields a recovery figure of 40 -+ 6%; for hexanal and 2-heptenal the figures were 29 +
4.2 and 72 +- 8%, respectively. Based on these recovery figures we find in
peroxidized rat liver microsomes (ADP-iron, 30 min) the following
amounts of aldehydes per milligram of microsomal protein: 4-hydroxynonenal, 20 nmol; hexanal; 40 nmol; propanal, 24 nmol; 2-propenal, 1.6
nmol; and 2-octenal, 2.0 nmol.
The DNPH method has broad applicability and is reasonably selective
and sensitive. The detection limit for a single aldehyde is about 1 pmol per
20/xl of injected sample.
The methanol solutions of zones I, II, and III remaining after HPLC
42 p. Winkler, W. Lindner, H. Esterbauer, E. Schauenstein, R. J. Schaur, and G. A. Khoschsorur, Biochim. Biophys. Acta 796, 232 (1984).
43 W. E. Turner, R. H. Hill, W. H. Hannon, J. T. Bernert, E. M. Kilbourne, and D. D.
Bayse, Arch. Environ. Contarn. Toxicol. 14, 261 (1985).
G. Poli, U. Ramenghi, O. David, F. Biasi, G. Cecchini, R. Carini, E. Chiarpotto, and M.
U. Dianzani, in "Free Radicals, Cell Damage and Disease" (C. Rice-Evans, ed.), p. 187.
Richilieu Press, London, 1986.
42 G. Poli, G. Cecchini, F. Biasi, E. Chiarpotto, R. A. Canuto, M. E. Biocca, G. Muzio, H.
Esterbauer, and M. U. Dianzani, Biochim. Biophys. Acta 883, 207 (1986).
46 H. Esterbauer, A. Benedetti, J. Lang, R. Fulceri, G, Fauler, and M. Comporti, Biochim.
Biophys. Acta 876, 154 (1986).
47 M. U. Dianzani, G. Poll, R. A, Canuto, M. A. Rossi, M. E. Biocca, F. Biasi, G. Cecchini,
G. Muzio, M. Ferro, and H. Esterbauer, Toxicol. Pathol. 14, 404 (1986).
A. Benedetti, A. Pompella, R. Fulceri, A. Romani, and M. Comporti, Biochirn. Biophys.
Acta 876, 658 (1986).
49 M. Curzio, H. Esterbauer, G. Poli, F. Biasi, G. Cecchini, C. Di Mauro, N. Cappello, and
M. U. Dianzani, Int. J. Tissue React. 9, 295 (1987).
50 G. D. Buffinton, N. H. Hunt, W. B. Cowden, and I. A. Clark, Biochern. J. 249, 63 (1988).
Sl A. Pompella, A. Romani, R. Fulceri, A. Benedetti, and M. Comporti, Biochim. Biophys.
Acta 961, 293 (1988).

[42]

DETERMINATION OF ALDEHYDES

419

separation may be used to determine the total aldehyde concentration in

each of the fractions. 39,44,45,47For that determination the samples are diluted about 20-fold with methanol, and the UV-VIS spectrum is recorded
in the range of 200-600 nm. The concentration is calculated from the
absorption maximum using an average value of 27,000 for zones I and III
(hmaxbetween 360 and 375 nm) and 44,000 for zone II (hmaxaround 400430 nm). 1
An alternative HPLC method has been described for the detection of
aldehydic lipid peroxidation products in plasma and liver homogenates. 52-54 This method is based on the reaction of aldehydes with 1,3cyclohexanedione to yield fluorescent dehydroacridine derivatives that
can be separated by HPLC. The sample (0.5 ml) is mixed with an equal
volume of methanol and centrifuged. The clear supernatant (0.5 ml) is
reacted with 1 ml of 1,3-cyclohexanedione (CHD) reagent at 60 for 1 hr.
The CHD reagent is prepared by dissolving ammonium sulfate (10 g),
glacial acetic acid (5 ml), and CHD (0.25 g) in 95 ml water. The CHD
reaction mixture (1 ml) is poured onto a Sep-Pak C18 cartridge for clean up
and eluted with 2 ml methanol. This methanol solution is separated by
HPLC on a 5-/xm ODS column (6 x 100 nm) with fluorescence detection
at 445 nm and excitation at 380 nm (flow rate 1 ml/min). The mobile
phases for elution are the following: 0-18 min, MeOH-H20, 3:7; 1832 min, tetrahydrofuran (THF)-H20, 26 : 74; 32-42 min, THF-H20, 4 : 6;
and 42-50 min, THF. The peaks are assigned using a reference chromatogram; peak quantification is based on the use of 5-hydroxypentanal as
internal standard.
The method seems to be rather sensitive and allows detection of about
100 fmol per 100 /zl injected aldehydes. The disadvantage is that the
chromatogram is rather complex and shows very large peaks resulting
from the reagents. With the CHD method various aldehydes including 4hydroxynonenal in plasma and liver of rats were detected (about 1 nmol/
ml plasma or I nmol/g liver); rats fed a vitamin E-deficient diet or treated
with CC14 had significantly increased aldehyde levels.
Direct Determination of 4-Hydroxynonenal by HPLC or GC-MS
The importance of HNE as a cytotoxic lipid peroxidation product has
led to the development of two independent analyses specifically for this
5z K. Yoshino, T. Matsuura, M. Sano, S.-I. Saito, and I. Tomita, Chem. Pharm. Bull. 34,
1694 (1986).
53 K. Yoshino, M. Sano, M. Fujita, and I. Tomita, Chem. Pharm. Bull. 34, 5184 (1986).
54 I. Tomita, K. Yoshino, and M. Sano, in "Clinical and Nutritional Aspects of Vitamin E "
(O. Hayashi and M. Mino, eds.), p. 277. Elsevier, Amsterdam, 1987.

420

ASSAY AND REPAIR OF BIOLOGICAL DAMAGE

[42]

compound) 5,56 Free HNE can easily be detected by HPLC with an UV

detector at 220-223 nm owing to its high molar absorptivity (hmax222 nm,
e 13100, in methanol). A typical analysis of HNE in liver microsomal
suspensions is as follows. To 20 ml of microsomal suspension (1 mg
protein/ml), 20 /.d BHT (10 mg/ml ethanol) is added as well as 200/xl
desferrioxamine (10 mg/ml water) to prevent further oxidation during
sample workup. The suspension is extracted with CH2C12 (2 times 20 ml
each), acetate buffer (2 ml, 0.1 M, pH 3.0) is added to the pooled extract,
and the CH2C12 is removed on a rotary evaporator (-<20). The residual
buffer solution containing HNE is quantitatively applied to a disposable
C18 solid-phase column (Bond-Elut, C18, 3 cm 3 size; Analytichem International, Harbor City, CA) that has been preconditioned with 3 ml methanol
and equilibrated with water. The Bond-Elut column is first eluted with 2
ml hexane to remove unwanted nonpolar materials; HNE is then eluted
with 2 ml methanol-water (8:2) into a 2-ml volumetric flask. Residual
hexane in the eluate is removed by nitrogen gassing, and the volume is
brought to 2 ml with water.
A volume (20/zl) of the cleaned sample is separated by HPLC on an S5 Spherisorb ODS column (4.5 x 250 mm) with acetonitrile-water (4 : 6)
or methanol-water (6.5 : 3.5) as mobile phase at 1 ml/min, with detection
at 220 nm. Peak identification and quantification are done with reference
chromatograms of standard solutions of HNE. The lowest amount detectable by this method is about 2 pmol per 20-/zl injection. The precision is
good and shows a coefficient of variation between 1.0 and 3.2%. With
microsomes the recovery as estimated with 14C-labeled HNE is 73%. The
HNE value found by this method in peroxidized microsomes (ADP-iron,
30 min) is 4.6 - 0.67 nmol/mg protein.
ff the sample contains higher concentrations of HNE (>2 nmol/ml) the
method can be simplified) 7 In such cases the sample is mixed with an
equal volume of acetonitrile-acetic acid (97 : 3), which precipitates most
of the protein and extracts HNE simultaneously. After centrifugation the
clear supernatant is separated by HPLC as described above.
For GC-MS 56 the HNE is first converted under mild conditions to its
pentafluorobenzyl oxime and then silylated. The GC-MS analysis is performed in the negative chemical ion mode with specific ion monitoring.
Recently this method has been used with deuterated HNE as an internal
standard for the quantification of HNE in platelets, monoeytes, and
55 j. Lang, C. Celotto, and H. Esterbauer, Anal. Biochem. 150, 369 (1985).
56 F. J. G. M. Van Kuijk, D. W. Thomas, R. J. Stephens, and E. A. Dratz, Biochem.
Biophys. Res. Commun. 139, 144 (1986).
57 H. Esterbauer, H. Zollner, and J. Lang, Biochem. J. 228, 363 (1985).

[43]

MALONDIALDEHYDE
DETERMINATION

421

plasma. 58 The details of this G C - M S method are described elsewhere in

this volume [40, 41].
Concluding Remarks
The choice of which method for aldehyde analysis should be used
depends on the particular interest of the investigator. Is an overall picture
of the complete spectrum of aldehydes required, or is there an interest in a
specific compound such as MDA or H N E ? ff only MDA is to be determined, the classic TBA test remains a useful method, providing it has
been validated by an HPLC measurement for the particular system under
study. If only H N E is to be determined, the method of choice is direct
HPLC or GC-MS. The latter method is more sensitive, but the resources
required are more expensive.
If the whole spectrum of aldehydes must be measured, then the DNPH
method described is probably more reliable than the current cyclohexanedione method, which separates all aldehydes in one run. As the number of
aldehydes present in peroxidized biological samples may exceed 30 and
their relative proportions vary greatly, complex chromatograms are produced and definite peak identification is difficult. The DNPH method is
less sensitive but gives more confidence in peak identification.
Acknowledgments
The authors' work has been supported by the Association for International Cancer
Research (U.K.) and by the Austrian Science Foundation (to H.E., Project P6176B).
58M. L. Selley, M. R. Bartlett, J. A. McGuiness, A. J. Hapel, and N. G. Ardlie, J. Chromatogr. 488, 329 (1989).

[43] M a l o n d i a l d e h y d e D e t e r m i n a t i o n as I n d e x o f
Lipid Peroxidation
By H. H. DRAPER a n d M. HADLEY

Introduction
The determination of malondialdehyde (MDA) has attracted widespread interest because it appears to offer a facile means of assessing lipid
peroxidation in biological materials. However, the validity of MDA as an
index of lipid peroxidation has been clouded by controversy regarding its
formation as an artifact of analysis and as a product of enzyme reactions,
METHODS IN ENZYMOLOGY, VOL. 186

Copyright 1990 by Academic Press, Inc.

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