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Technical Briefs |
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This work was presented previously in abstract form: Cole TG, Parikh N. High-throughput analysis of oxidizability of LDL suitable for use in clinical studies [Abstract]. Clin Chem 1998;44:A84.
a author for correspondence: fax 314-362-4782, e-mail Thom{at}imgate.wustl.edu
The susceptibility of lipoproteins to oxidation is thought to be
a critical step in a complex process culminating in arterial lipid
deposition; however, the role of the oxidizability of lipoproteins in
atherosclerosis has not been demonstrated in any large-scale clinical
trials (1)(2)(3)(4). Although several measurements of the
oxidizability of LDL have been developed, the most commonly used method
is the measurement of the rate of formation of conjugated
hydroperoxides of polyunsaturated fatty acids in response to exposure
to copper ions, which leads to the formation of conjugated dienes (CDs)
(5). Three major parameters are used to describe the
oxidizability of LDL by the CD assay: (a) the lag time (LT)
during which lipoprotein-associated antioxidants are consumed, thereby
sparing the LDL lipids from oxidation; (b) the maximum rate
of CD formation (Max V); and (c) the total amount of CD
formed (
CD).
In practice, the CD assay requires the sequential measurement of absorbance at 234 nm in a spectrophotometer over a prolonged period of time, often >3 h. Because the automated cell carrier of most spectrophotometers holds a maximum of only six cuvettes, the analysis of large numbers of specimens, such as for large-scale clinical trials, is difficult and time-consuming. In addition, the analysis of replicate specimens or the inclusion of quality-control materials generally is precluded by the small number of specimens that can be analyzed simultaneously. We have developed methods that allow the isolation of LDL from a large number of specimens and the analysis of the oxidizability of this isolated LDL. Several factors were necessary for these goals to be attained: (a) the use of an ultracentrifuge rotor with capacity for a large number of specimens (Type 25; Beckman Instruments); (b) the recent availability of a 96-well microtiter plate reader that reads absorbance at 234 nm (PowerWave 200 reader; Bio-Tek Instruments); and (c) the development of a disposable 96-well microtiter plate that is transparent at 234 nm (Costar UV-Plate; Corning Costar).
Blood was collected into Vacutainer Tubes containing liquid EDTA (1.5
g/L blood final concentration) from healthy volunteers after a
12- to 14-h fast, and the plasma was removed immediately after
separation of cells by centrifugation at 1700g at
4 °C. LDL was either prepared immediately from fresh plasma or 1-mL
aliquots of plasma were stored in cryovials with no other added
chemicals at -80 °C by a two-step ultracentrifugation procedure.
Specimens were protected at all times from direct light, and all
solutions were kept cold and contained 10 µmol/L EDTA. Frozen
specimens were thawed at room temperature on a rotator, shielded from
light by aluminum foil. To a 1.0-mL aliquot of plasma, 298 µL of a
solution of KBr (
= 1.225 kg/L) was added. An aliquot (650 µL) of
this plasma was transferred to a Beckman 8 x 51 mm polycarbonate
ultracentrifugation tube and overlaid with 350 µL of a solution of
KBr ( = 1.063 kg/L). The tubes were centrifuged in the outer ring of
the Beckman Type 25 rotor at 85 925gavg
for 18 h at 10 °C in a Beckman L8–55 ultracentrifuge. If >44
tubes were centrifuged simultaneously, the middle ring was used to
accommodate the extra tubes and the time of centrifugation was extended
to 20 h. After centrifugation, a Beckman CentriTube tube slicer
was used to separate the top layer containing VLDL and LDL from the
bottom layer containing HDL and other plasma proteins. The top layer
(200 µL) was transferred to another 8 x 51 mm polycarbonate
centrifuge tube, overlaid with 800 µL of NaCl ( = 1.006 kg/L), and
centrifuged as above. After centrifugation, the LDL was concentrated in
an orange-colored layer at the bottom of the tube. The tube was sliced
with the CentriTube slicer such that the top 800 µL was removed and
discarded. The concentrated LDL was transferred to a clean cryovial,
brought to a final volume of 500 µL with 8.77 g/L NaCl, and
applied to a prepacked PD-10 Sephadex G-25 M column (Pharmacia Biotech)
to remove residual salts and water-soluble antioxidants such as
bilirubin, uric acid, and ascorbic acid (6). The desalted
LDL was eluted with 10 mmol/L phosphate-buffered saline (PBS), pH 7.4.
To avoid potential errors caused by contamination by albumin and the
measurement of protein by the Lowry or dye-binding methods, the
apolipoprotein (apo) B concentration of the LDL preparations was
measured directly by nephelometry (Behring Diagnostics) after column
filtration. The purity of the LDL was assessed by lipoprotein
electrophoresis. On the final day of LDL isolation, the total time from
the slicing of the tubes to the application of the LDL to the
microtiter plate for analysis was ~5 h.
For a standard oxidation measurement, purified LDL was adjusted to 55 mg/L apo B with PBS containing 10 µmol/L EDTA. A stock solution of CuSO4 · 5 H2O (11 mmol/L) was diluted to 275 µmol/L with PBS without EDTA. Aliquots of LDL (250 µL, 12.5 µg) and CuSO4 (25 µL) were applied to a Costar UV-Plate; the final concentrations were 50 mg/L LDL apo B, 25 µmol/L Cu2+, and 9.1 µmol/L EDTA. The plate was inserted into the temperature-controlled PowerWave 200 reader and prewarmed to 37 °C, and readings were taken at 234 nm every 3 min for 3 h. The plate was shaken automatically for 15 s before each reading. For routine use, specimens were analyzed in duplicate, allowing the analysis of 46 specimens and 2 quality-control materials in each assay. The total time for analysis of 48 specimens from thawing to reporting of results was <2.5 days, with only ~12 h of technician time.
The KC4 program (revision 12) provided with the PowerWave 200 reader
calculated minimum and maximum absorbance values and the slope
(Absorbance/min) and the time of the inflection point for the
propagation phase for each kinetic curve. These values and the
absorbance readings for each well were exported to a QuattroPro (Corel)
spreadsheet that calculated the LT (min), Max V (µmol CD/min-g apo
B), and CD (µmol CD/g apo B). LT was defined as the time at the
intercept of the propagation phase with the baseline (5).
The amount of CD formed was based on the molar absorptivity of CD,
234 = 29 500 L/mol-cm (5).
Details of the spreadsheet calculations can be obtained upon
request.
The inclusion of EDTA throughout the isolation procedure, the
protection from light, and the constant refrigeration of the specimens
minimized the possibility of premature oxidation of LDL during the
various isolation steps. When these factors were not carefully
controlled, LT decreased significantly, although Max V and CD were
less affected.
The use of gel filtration columns for the final purification of the
isolated LDL removed the relatively high concentration of residual KBr
from the ultracentrifugation steps and also removed low-molecular
weight, water-soluble antioxidants, such as bilirubin, ascorbic acid,
and uric acid, which have been shown to be potent antioxidants
(5)(6)(7)(8). The LTs for isolated LDL that had not been processed
by gel filtration were approximately twofold longer than the LTs for
the same specimens after column filtration. These findings suggest that
the nonfiltered specimens contained antioxidants that were removed by
column filtration and were in agreement with the observations of and
Brussaard et al. (9) and Scheek et al. (10) (by
dialysis), and of McDowell et al. (7) (by gel filtration).
The columns also provided a rapid (~10 min) method for the
purification of LDL that did not lead to a loss of protein or
lipophilic antioxidants (
-tocopherol, lycopene, and ß-carotene),
which has been shown to occur as a result of dialysis (10).
After preliminary validation experiments, we chose a Cu2+ concentration of 25 µmol/L (~16 µmol/L free Cu2+) for use in the standard oxidation assay. At this concentration, all oxidation parameters were stable and did not show the concentration-dependent variability observed at lower Cu2+ concentrations. Although the "proper" ratio of Cu2+ to LDL for meaningful interpretation of the effects of physiologic, pharmacologic, or dietary perturbations on oxidation has been discussed, no consensus has been reached (6)(11)(12). The inclusion of 10 µmol/L EDTA in all solutions including the oxidation reaction mixture preserved the LDL and prevented oxidation during the relatively long isolation process required for the treatment of large numbers of specimens.
The development of a disposable plastic 96-well microtiter plate with
minimal absorbance at 234 nm has made possible the simultaneous
analysis of a large number of specimens. To evaluate the well-to-well
variability between measurements, all wells of the Costar UV-Plates
were filled with either deionized water or a routinely processed LDL
specimen, and the absorbance at 234 nm was read. The mean absorbance
was 0.099 (CV = 4.8%; n = 96) for water and 0.406 (CV =
2.7%; n = 96) for the LDL specimen, which is similar to the
absorbance reported for specimens in quartz cuvettes in a standard
spectrophotometer (13). To evaluate the within-run
reproducibility of the assay on the microtiter plate, a large amount of
LDL was prepared and applied to each well of a microtiter plate, and
the oxidation assay performed. Table 1
describes the well-to-well reproducibility of the measurement
of the three oxidation parameters. The interwell reproducibility was
excellent, with a CV <3% for each of the oxidation parameters. To
evaluate the reproducibility of the LDL preparation method as
well as of the oxidation assay, 19 replicate preparations of LDL were
made by the standard method from the plasma of one subject. The
standard oxidation assay was performed on each LDL in quadruplicate on
a single microtiter plate. The CV of the LT was 2.3%; however, the Max
V and CD were more variable, with CVs of 5.9% and 4.8%,
respectively. Between-run imprecision was assessed using two
frozen quality-control specimens that were analyzed in each assay with
clinical trial specimens (Fig. 1
). The between-run CV for LT was 5.9% to 9.0% over 18 or 20
assays performed over 37 weeks. The CV for Max V and CD was 4.0% to
5.8%. Values for within-run and between-run imprecision are difficult
to find in the literature because of the limited number of specimens
that can be analyzed in a single assay in most systems. Princen and
co-workers (14)(15)(16) reported within-run and between-run CVs
similar to ours, but using a different assay format that used a
10-position automated spectrophotometer. Kleinveld et al.
(12) also reported CVs similar to ours.
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The collection of blood in Vacutainer Tubes containing liquid EDTA,
followed by rapid separation of plasma from cells by centrifugation and
freezing at -80 °C in plastic cryovials, appears to be sufficient
for the maintenance of the oxidizability of the specimen. Antioxidants
such as -tocopherol, ß-carotene, and lycopene have been found to
be stable in frozen plasma specimens prepared by this method
(17). To be practical for use in clinical trials, specimens
must be unaffected by freezing. To evaluate the effect of freezing on
the oxidizability of LDL, we collected EDTA plasma from four healthy
laboratory employees after a 12-h fast. Aliquots of plasma were either
used immediately or frozen in cryovials at -80 °C. LDL was prepared
by the standard method on the freshly collected plasma or after 1, 2,
and 4 weeks of storage, and the oxidizability of the isolated LDL was
determined. None of the oxidation parameters deviated from the fresh
specimen by >5% over 4 weeks. Fig. 1
shows the effects of freezing
for up to 37 weeks on plasma. No trends were observed in any of the
oxidation parameters for either pool, indicating a negligible
degradation of the specimens over at least 37 weeks of storage at
-80 °C. Van der Vijver et al. (18) have
established the stability of EDTA plasma stored for 18 months at
-80 °C without harmful effects on the oxidizability of LDL.
In summary, we have optimized the performance of the CD assay to determine the oxidizability of LDL for use in clinical trials. The Cu2+-catalyzed oxidation of LDL has been used previously by many investigators as an indication of the amount of antioxidants associated with LDL. In our method, plasma is collected easily and stored without extraordinary efforts, and purified LDL is prepared from large numbers of specimens. Up to 96 kinetic measurements of the formation of CDs are made simultaneously in a temperature-controlled environment. In this format, multiple replicate measurements can be made, increasing the precision of the assay and allowing the use of quality-control materials in each assay. Although the CD assay may never become a routine clinical assay for the assessment of the oxidizability of LDL, it is useful for research and can now be applied to specimens from large clinical trials and epidemiologic surveys.
Acknowledgments
We thank Mark Witham of Bio-Tek Instruments, Inc. (Winooski, VT) and Ken Brown of Bio-Resources, Inc. (St. Louis, MO) for providing access to preproduction versions of the PowerWave 200 instrument.
Footnotes
Core Laboratory for Clinical Studies, Department of Medicine, Washington University School of Medicine, Box 8046, 660 S. Euclid Ave., St. Louis, MO 63110
References
-tocopherol during blood collection and processing procedures. Clin Chem 1995;41:943-944.
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, QC1; ——–, mean of 20 assays;
, QC2; – – – –, mean of 18 assays.