Professional Documents
Culture Documents
[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
TABLE I
TBA REACTION WITH DIFFERENT COMPOUNDSa
Malonaldehyde A 153,000
Alkanals A,B,C 0
2-Alkenals A without Fe 14-66
A with Fe 30-90
B 100-200
C 130-160
2,4-Alkadienals A without Fe 48-160
A with Fe 184-280
B 1100-2600
C 4500
4-Hydroxyalkenals A without Fe 12-119
A with Fe 38-124
C 320
Amino acids preincubated with 0.9 m M Fe D 50-620
Sugars preincubated with 0.9 m M Fe D 90-2700
Monohydroperoxides from arachidonic acid E without Fe 3200-8100
E with Fe 12400-34100
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., micro-
somes, 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 poten-
tial 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 de-
proteinized 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 there-
fore have to be present in the sample in extremely high concentrations to
interfere significantly with the standard determination of MDA. Suspen-
sions 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. 1°A 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 TBA-
positive substances, TBARS, or simply the TBA value rather than speci-
fying MDA.
Frequently used modifications of the TBA test employ the whole acid-
t9 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 Chemis-
try 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
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.
:a.
22:6 (-)
20 : 4 (---)
A
09
¢r
<
400 TSARS r]
p-
:a.
i"
II "..,m- , ~_~ TSARS
25 "O
e-
tO
300 // / ...."'.. 20 •
i11 m~ MDA
I Jr',, ,,'/ -",,
".
I..
100 // ~\
"o tf / \~
4 8 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.
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 sam-
pie-(0.1 g) is reacted with dansylhydrazine in hydrochloric acid containing
FeC13. The formed dansylpyrazole is separated by HPLC with fluori-
metric 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 decompo-
sition.
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 TBA-
reactive 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 conve-
nient TBA test.
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 identifi-
cation 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 dinitrophenyl-
hydrazine reagent. Second, it enables certain contaminating carbonyls to
be eliminated; the DNPH forms of formaldehyde, acetone, and acetalde-
hyde are always found at this stage even in the reagent blank. Apparently
these carbonyls are always present in laboratory air and standard solu-
tions. Finally, analysis of the hydrazones in each zone (I, II, and III)
greatly facilitates clear separation of the individual compounds and pro-
vides more confident identification of the peaks in the HPLC chromato-
gram. 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 pro-
grams; however, the resulting chromatograms are complicated, and it is
extremely difficult to make definite peak identifications.
A typical determination of aldehydes produced during lipid peroxida-
tion 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-dini-
trophenylhydrazine 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 Spheri-
FRONT
UV-vis
ZONE III <
| •
alkanals, 2-alkenals
!
i
1
•
2,4-alkadienals, ketones
acetone, | ~ 4" O
HPLC
formaldehyde,~- UV-vis
acetaldehydeJ
ZONE I I
C2~2Zq[) osazones
CS.2223
C.~T/-D 1 <
HPLC
DNPH reagent -O [ 1 <Ov-via
HNE ~O I ] I ZONE I, hydroxyalkenals
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, 2,4-
alkadienals, 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-dihydroxy-
decenal4° and two aldehydes that are probably (based on their mass spec-
tra) 4-hydroxy-4,5-nonadienal and 5-hydroxyoctanal. 4] Zone III contains
propanal, butanal, pentanal, hexanal, nonanal, 2-proper~al, 2-pentenal, 2-
hexenal, 2-heptenal, 2-octenal, 2-nonenal, 2,4-heptadienal, 2,4-deca-
4oA. Benedetti, M. Comporti, R. Fulceri, and H. Esterbauer, Biochim. Biophys. Acta 792,
172 (1984).
41 p. Heckenast, Thesis, University of Graz, Austria, 1983.
418 ASSAY AND REPAIR OF BIOLOGICAL DAMAGE [42]
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 deter-
mined, 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 cyclohexane-
dione 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 pro-
duced 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).
[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 wide-
spread 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,