Fig. 1. Results of a 24-h continuous monitoring during copper oxidation of a LDL pool of conjugate diene formation, measured by spectrophotometry at 234 nm left panel and of ApoB-emitted fluorescence, determined by exciting the LDL solution at 360 nm and measuring
emitted fluorescence at 430 nm right panel. The arrows and letters indicate the aliquots harvested for testing their reactivity with reference sera with known concentrations of oxLDL antibodies Fig. 2 and LDL denaturation by Western blot and agarose gel electrophoresis Fig. 3.
washed four times with 0.05 Tween 20 Sigma in PBS, pH 7.8. The absorbed and unabsorbed aliquots
were spun at 9000 × g in an Eppendorf centrifuge model 5413 for 30 min. The supernatants of the
centrifuged samples, unabsorbed and absorbed, were tested at two final dilutions: 1:2 and 1:4 for purified
antibodies and 1:20 and 1:40 for sera. Then 100 ml of both dilutions were transferred to the wells of the
oxLDL-coated plates and after overnight incubation at 4°C and washing, 150 ml of peroxidase-conjugated rab-
bit anti-human IgG IgG fraction Cappel Organon Teknika, Durham, NC, diluted 1:5000 in PBS-BSA,
was added to each well. After incubation for 1 h at 4°C, the unbound conjugate antibody was washed off, and
0.5 mmoll of 2,2-azino-di-3-ethylbenzthiazoline-6-sul- fonate ABTS, Sigma, St Louis, MO and 3 vv
hydrogen peroxide in a 45-mmoll concentration of citric acid buffer, pH 4.0, was added as substrate. Color
was allowed to develop for 10 min at room tempera- ture, in the dark. The reaction was stopped with 0.1
moll citric acid, pH 2.1, and the absorbance was measured at 414 nm in a VMax enzyme-linked im-
munosorbent assay reader Molecular Devices, Sunny- vale, CA. The optical density values were calculated by
subtracting the OD values measured on oxLDL-ab- sorbed aliquots from those measured on unabsorbed
aliquots at identical dilutions. A human oxLDL anti- body standard was used to calculate the concentration
of oxLDL antibody in serum and isolated antibody preparations as described in an earlier publication [22].
In all EIA studies testing different oxLDL prepara- tions the same oxLDL preparation was used to coat the
wells of the Immulon plates and to absorb free anti- body on antibody-containing samples.
3. Results
The longitudinal changes in the formation of fluores- cent compounds and of conjugated dienes during oxi-
dation of a LDL pool pool 1 for 24 h are presented in Fig. 1. The nine aliquots of this LDL pool labeled ‘a’
to ‘i’ in the figure were obtained at different stages of oxidation and tested for their reactivity with five differ-
ent reference sera Fig. 2. In the example presented in Fig. 1, fluorescence emission at 430 nm reached its
maximum level 6 – 7 h after starting oxidation. We observed maximal reactivity with oxLDL antibodies 4 h
Fig. 2. Study of the reactivity of a series of aliquots of LDL harvested at different times during the oxidation reaction Fig. 1 with five
different reference oxLDL antibodies. The antibody concentration in the five reference sera was 47 mgl serum 1, 45 mgl serum 2, 20
mgl serum 3, 27 mgl serum 4, 204 mgl serum 5. The results are expressed as the difference in OD measured in unabsorbed and
absorbed aliquots of each one of the sera, using identical LDL samples for EIA plate coating and antibody absorption.
Fig. 3. Top panel: Reproduction of a Western blot analysis of the degree of LDL fragmentation and aggregation associated with differ-
ent times of oxidation. Native LDL a and five samples collected at the times shown in Fig. 1 were separated by SDS – PAGE and
exposed to a polyclonal rabbit human ApoB antibody. Samples ‘g’ and ‘i’ had maximal reactivity with reference oxLDL antibodies Fig.
2. The positions of molecular weight standards included in the run are indicated on the left margin of the figure. Bottom panel: Repro-
duction of an agarose gel electrophoretogram of the samples sepa- rated by Western blot. The samples were applied at origin O.
conjugated diene peak nor the OD reading of that peak correlated with the maximal fluorescence readings. Af-
ter the early 234-nm peak is reached, the OD values continued to slowly increase. This secondary increase in
OD is not due to continuing formation of lipid perox- ides but rather to the decomposition of lipid peroxides
to aldehydes and other products, which also absorb light at 234 nm. The decomposition of lipid hydroper-
oxides is associated with fragmentation of apolipo- protein B and aggregation of the fragments. A Western
blot study of LDL aliquots collected at different times of LDL oxidation showed that aliquots with maximal
antibody reactivity aliquots ‘e’ and ‘g’, Figs. 1 and 2 showed virtually complete disappearance of the 512-
kDa ApoB fraction, replaced by small molecular weight fractions and larger molecular weight aggregates Fig.
3. On agarose gel electrophoresis, aliquots ‘e’ and ‘g’ showed a marked increase in electronegativity, relative
to native LDL and aliquots collected at earlier times.
Based on these results and the results obtained with one other LDL pool and two LDL preparations iso-
lated from different individuals, we decided to monitor the reaction with fluorescence emission data due to the
fact that the peak of fluorescence was closer in time than the early conjugated diene peak to optimal oxida-
tion and to stop the reaction 4 – 6 h after the peak fluorescence value was reached. Spectrophotometric
and fluorescence data obtained with 13 LDL pools oxidized and monitored under the outlined conditions
showed that the average time needed to reach maximal fluorescence values was 10 9 4 h, with a maximum of
19 h.
The wide range of time required to reach maximal modification was even more dramatically illustrated
with an LDL pool that showed increasing fluorescence values for over 24 h of oxidation Table 1. In that
particular study monitoring was done at various time periods and the highest values of fluorescence were
recorded in aliquots harvested after 48 h of oxidation, which also showed maximum reactivity with reference
oxLDL antibodies. Both parameters remained stable when oxidation of LDL was continued for an addi-
tional 24 h after peak fluorescence was reached.
The experiments described above suggest that once the maximum levels of fluorescence and antibody reac-
later. The antibody reactivity of oxLDL aliquots har- vested at later times up to 24 h of oxidation remained
unchanged Fig. 2. Monitoring optical density values at 234 nm showed a very early peak, approximately 2 h
after starting the reaction, preceding the stabilization of fluorescence values by 5 h Fig. 1. The time needed
to reach the conjugated diene early peak correlates well with the time needed to reach maximal fluorescence
emission, but neither the time needed to reach the
Table 1 Longitudinal follow-up of fluorescence and reactivity with reference oxLDL antibodies of LDL harvested at 2, 10, 12, 24 and 48 h of oxidation
a
2 10
Time of oxidation h 12
48 24
72 1.800
1.680 0.440
Fluorescence 360–430 nm 3.930
3.801 2.600
0.106 0.253
0.242 Reactivity with purified antibody
0.350 0.696
0.695 0.024
0.065 0.067
0.158 0.342
0.361 Reactivity with reference serum
a
Antibody reactivity values are expressed as optical density values calculated by subtracting the OD measured at 414 nm on oxLDL absorbed-aliquots from the OD measured on unabsorbed aliquots of our reference oxLDL antibody and of a reference serum containing 201 mgl
of oxLDL antibody. The EIA wells were coated with LDL samples collected during oxidation at the indicated times.
Table 2 Reactivity of oxLDL aliquots of an LDL pool harvested at 3, 17, 36,
and 47 h, after the fluorescence reached the maximum level with purified oxLDL antibody, and two reference sera containing 47 mgl
of oxLDL antibody serum 1 and 204 mgl of oxLDL antibody serum 5
a
17 h Antibody
36 h 3 h
47 h 0.576
Purified Ab 0.774
0.755 0.789
0.424 0.460
0.208 0.466
Serum 1 0.608
Serum 5 0.543
0.478 0.551
a
Reactivity is expressed as optical density values calculated by subtracting the OD values obtained on oxLDL-absorbed aliquots
from those measured on unabsorbed aliquots. Final dilutions were 120 for serum and 12 for isolate.
aliquots. All aliquots were dialyzed against PBS- EDTA, filtered, and stored at 4°C, protected from light
and under a nitrogen-rich atmosphere. One aliquot of each of the four pools was oxidized shortly after blood
collection, as indicated. Stored aliquots from two of the pools were oxidized 5 and 5.5 weeks after blood collec-
tion and the other two aliquots were oxidized 9 weeks after blood collection. There was an apparent inverse
correlation between pre-oxidation storage time and the time elapsed before maximal LDL oxidation was
reached Table 3. On the other hand, prolonged pre- oxidation storage appeared to result in a lower degree
of oxidation, as reflected by lower fluorescence and conjugated dienes values Table 3. While all the freshly
oxidized LDL preparations reached fluorescence values in excess of 1.1, only one of the stored LDL pools pool
6 reached a level slightly above 1.0 1.02.
We also tested the reactivity of the eight different oxLDL aliquots with reference antibodies Table 4.
The OD values expressed as the difference in the readings obtained with unabsorbed and absorbed
aliquots varied between 0.46 and 0.52 when tested with reference serum 1 and between 0.76 and 0.85 when
tested with reference serum 5. For all aliquots oxidized after more than 4 weeks of storage time the OD values
were lower than those measured with aliquots oxidized after a shorter storage time.
Finally, we investigated the reproducibility of anti- body assays when samples were tested against different
oxLDL preparations prepared according to what ap- peared to be optimal conditions, i.e. the oxidation
reaction was stopped 4 – 6 h after maximal fluorescence was reached, and the fluorescence value exceeded 1.1
OD U. We used two quality control sera, one contain- ing a low antibody concentration 47 mgl and the
other containing a high antibody concentration 204 mgl, in a series of 13 EIA runs in which four different
tivity are reached, those parameters may remain stable for a considerable period of time. To better assess this
point we carried out an additional experiment in which oxidation of a fourth LDL pool was continued for as
long as 47 h after the maximum level of fluorescence was reached. Values of fluorescence and conjugated
dienes were monitored continuously until 20 h of oxida- tion. After that, fluorescence and conjugated dienes
values were determined simultaneously in aliquots col- lected at specific times. The maximum level of fluores-
cence F
430
= 1.9 was reached after 9 h of oxidation
and it remained stable for a period of 47 h. The reactivity with oxLDL antibodies showed an increase
between the aliquots collected at 3 and 17 h after the peak of fluorescence was reached the 3-h aliquot was
collected too early to reflect maximal reactivity, and remained stable at the maximum level in the LDL
samples harvested 36 and 47 h after the maximum level of fluorescence was reached Table 2.
To estimate the effect of storage of native LDL on the kinetics of oxidation we prepared four LDL pools
pools 5 – 8 and divided each one of them into two
Table 3 Study of the effect of LDL storage prior to oxidation on the levels of fluorescence and conjugated dienes measured after oxidation
a
Conjugated dienes Fluorescence
LDL pool no. Peak fluorescence value 360–430
Time to reach the maximal level of OD 234 nm after 12 h of oxidation
fluorescence h nm
Fresh LDL
b
Fresh LDL Stored LDL
Stored LDL
c
Fresh LDL Stored LDL
1.89 0.75
1.47 1.12
10 13
5 1.20
13 1.39
10 1.12
1.02 6
13 12
1.22 7
0.90 1.44
1.01 1.40
0.71 1.15
8 13
1.01 8
a
One half of four LDL pools was oxidized within 4 weeks of isolation of the different LDL fractions included in the pool fresh LDL and the other half was oxidized after 5–9 weeks of storage stored LDL. The time of oxidation needed to reach the maximum level of fluorescence
and the corresponding values for fluorescence, as well as the levels of conjugated dienes after 12 h of oxidation are presented in the table.
b
Oxidized within 2 weeks of blood collection.
c
Pool 5 was oxidized 5 weeks after blood collection; pool 6 was oxidized 5.5 weeks after blood collection; pools 7 and 8 were oxidized 9 weeks after blood collection.
Table 4 Study of the effect of LDL storage prior to oxidation on the reactivity with reference oxLDL antibodies
a
LDL pool no. Reactivity with reference serum 5
Reactivity with reference serum 1 Stored LDL
c
Fresh LDL
b
Fresh LDL Stored LDL
5 0.36
0.47 0.76
0.63 0.42
0.85 0.46
0.67 6
0.52 7
0.35 0.84
0.62 0.54
8 0.36
0.84 0.69
a
The processing of LDL pools was as detailed in Table 3. Reactivity is expressed as optical density values calculated by subtracting the OD
414
values corresponding to the reactivity of a given oxLDL pool with an unabsorbed aliquot of a reference oxLDL-containing sera and the OD
414
corresponding to the reactivity of the same oxLDL pool with a pre-absorbed aliquot of the same reference serum.
b
Oxidized within 2 weeks of blood collection
c
Pool 5 was oxidized 5 weeks after blood collection; pool 6 was oxidized 5.5 weeks after blood collection; pools 7 and 8 were oxidized 9 weeks after blood collection.
pools of oxLDL were used as antigens. Fig. 4 repro- duces graphically the values mean 9 1 S.D. obtained
with these two quality control samples; the coefficients of variation calculated from these values were of 14.4
for the low control and 3.9 for the high control. These CV compared favorably with between-run CV
calculated with the same samples using a single LDL pool 7.4 and 3.0, respectively; n = 5.
4. Discussion
