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Comparison of three methods for measuring LDL resistance against copper-induced oxidation.

Oxidation of LDL in the subendothelial space of the arterial wall initiates a series of events leading to enhanced uptake of LDL by scavenger receptors on macrophages and subsequent foam cell formation. Therefore, LDL oxidation is supposed to play a role in the development of atherosclerosis (1, 2).

The most uniformly accepted procedure for measuring the resistance of LDL to in vitro oxidation is determination of the lag time (LT) for conjugated diene (CD) formation, initiated by catalytic amounts of transition metal ions (3). Another method is based on the oxidation of parinaric acid (PA), a fluorescent and oxidation-sensitive polyunsaturated fatty acid (FA) probe, incorporated into LDL (4). A third method monitors fluorescence development (FD) during LDL oxidation (5), caused by reaction of protein amino groups with aldehydes generated during decomposition of peroxidized FAs (6).

The three methods described above measure different stages of the oxidation process. Oxidation of the PA probe represents one of the earliest stages of oxidation. The formation of CDs represents an intermediate stage of the oxidation process, and finally FD represents a late stage of the oxidation process.

The methods for measurement of LDL oxidizability were evaluated with respect to reproducibility and potential for automation. Their relationship with LDL constituents, as determinants of LDL oxidation, was also studied.

Subjects were 98 normolipidemic type 2 diabetic patients with good glycemic control (mean hemoglobin A1c, 6.2% [+ or -] 1.0%). LDL was isolated by ultracentrifugation between densities 1.019 and 1.063 kg/L, as described elsewhere (7). PA was purchased from Molecular Probes.

Other reagents used were obtained from Merck or Sigma. Total cholesterol, free cholesterol, triglycerides, and phospholipids were analyzed using enzymatic test kits (Boehringer Mannheim). The cholesterol ester content of LDL was calculated as total minus free cholesterol. LDL lipid constituents were standardized for LDL protein quantified by a modified Lowry procedure (8), with bovine serum albumin as the calibrator.

The mean diameter of the LDL particles was measured by the high performance gel-filtration chromatography method we described recently (7). Lipid peroxides, analyzed as malondialdehyde equivalents, were measured by HPLC with fluorescence detection after sample pretreatment and derivatization with thiobarbituric acid, according to the method of Tsai et al. (9). The FA composition of LDL was assessed by capillary gas chromatography, as described previously (10, 11). [alpha]-Tocopherol and coenzyme Q10 were simultaneously determined by reversedphase HPLC.

LDL oxidation was performed at 37[degrees]C. The molar ratio of copper ions to LDL was equal in all oxidation experiments. Before oxidation, salts and EDTA were removed by desalting on a 5-mL dextran column (Pierce). The three LDL oxidation methods used are described below.

LT was measured by monitoring CD formation (3) at 3-min intervals on a Hitachi U2000 spectrophotometer at 234 nm. The calculated LT was independent of the amount of LDL between 0.025 and 0.100 g/L LDL-protein. We routinely used a final concentration of 0.04 g/L LDL-protein. The final copper concentration was 15 [micro]mol / L.

The oxidation of PA after its incorporation into LDL (4,12) was monitored on a Cobas Bio centrifugal analyzer (Roche). The fluorescent PA probe (0.9 [micro]mol/L) was incorporated into LDL (0.07 g LDL-protein/L) by incubation for 10 min at 37[degrees]C. During automated analysis, LDL preparations were further diluted to 0.02 g/L LDL-protein and 0.26 [micro]mol/L PA with phosphate-buffered saline.


The fluorescence decay (emission wavelength, 450 [+ or -] 45 nm; excitation wavelength, 324 nm) was monitored every 2 min for 1 h.

Measurement of the increase in autofluorescence intensity during LDL oxidation (5) was also adapted to the Cobas Bio analyzer. During a 5-h period, the fluorescence was measured every 10 min at 450 [+ or -] 45 nm with excitation at 360 nm. The final LDL-protein and copper concentrations were equal to the CD method.

Throughout the present study, results are expressed as the mean [+ or -] SD. Relationships between Us were evaluated by linear regression analysis. Spearman correlation coefficients were calculated to evaluate relationships between variables. Because multiple comparisons were made, significance was set at P = 0.01.

The mean CD-LT was 49.0 [+ or -] 6.6 min. Corresponding values for PA-LT and FD-LT were 20.4 [+ or -] 4.3 and 66.7 [+ or -] 8.0 min, respectively. As illustrated in Fig. 1A, PA-LT values were highly correlated to those obtained by the CD method [y = (0.49 [+ or -] 0.04)x - (3.53 [+ or -] 2.16); r = 0.78; [S.sub.y|x] = 2.85 min]. FD-LTs were also highly correlated to CD-LTs [y = (1.02 [+ or -] 0.07)x + (16.67 [+ or -] 3.22); r = 0.86; [S.sub.y|x] = 4.25 min], as shown in Fig. 1B.

Within-run CVs, determined on an LDL pool, were 2.0% (n = 6),2.5% (n = 12), and 1.5% (n = 12) for the CD, PA, and FD methods, respectively. Between-run reproducibility was determined for the entire procedure, including isolation of LDL. Between-run CVs were 7.9% (n = 17), 11% (n = 15), and 3.6% (n = 9) for the CD-LT, PA-LT and FD-LT determinations, respectively.

The free cholesterol, cholesterol ester, phospholipid, and triglyceride content of LDL were not associated with any of the Us (data not shown). Correlations between Us and lipid peroxides, LDL antioxidants, FAs, and particle diameter are presented in Table 1. CD-LT, PA-LT, and FD-LT were positively associated with the LDL [alpha]-tocopherol content; however, only the association with PA-LT reached statistical significance (r = 0.27; P = 0.008). Positive and significant associations between coenzyme Q10 and both PA-LT and FD-LT (r = 0.34, P <0.001; and r = 0.29, P = 0.004, respectively) were also found. Negative correlations between the arachidonic acid concentrations and both CD-LT and FD-LT were observed (r = -0.40, P <0.001; and r = -0.47, P <0.001, respectively). An inverse significant association was also found between CD-LT and lipid peroxides (r = -0.27; P = 0.008).

The aim of this study was to compare three methods to measure the LT at different stages of in vitro oxidation of LDL. In the CD technique, additional indices of LDL oxidizability can be derived, such as total amount of dienes and their rate of formation (13). This additional information is not obtained with the PA and FD methods. However, because an inverse relationship has been found between LT and severity and progression of coronary atherosclerosis (14-17), the LT is considered the most distinctive index of LDL oxidizability.

The PA and FD assays offer practical advantages in terms of simplicity in comparison with the conventional CD method because we have adapted both methods to a Cobas Bio analyzer. We obtained the shortest Us with the PA technique, confirming that the PA probe, because of its surface localization, monitors an early stage of the oxidation process. The FD method is the most reproducible for determining LTs.

It has been shown that small, dense LDL subfractions are more readily oxidized than large, buoyant LDL subfractions (18, 19). When we measured the mean LDL particle diameter by high performance gel-filtration chromatography (7), a very precise and reproducible technique, we found no significant association with Us. This does not contradict the above mentioned studies, but it does demonstrate that a relationship between size and LT of LDL oxidation is observed only in subfractions isolated from the same subject and not in a cross-sectional study design.

Recently, Kontush et al. (20) found no significant correlations between CD-LT and LDL composition. In agreement with this study, we found no significant correlations between Us and the concentrations of cholesterol esters, free cholesterol, phospholipids, and triglycerides in LDL. We did find significant positive associations between coenzyme Q10 and both PA-LT and FD-LT (Table 1), although the concentration of this antioxidant in LDL is very low (<1 mol/mol). In this and several other studies (13, 21, 22), CD-LT and FD-LT were not related to [alpha]-tocopherol, the most abundant antioxidant in LDL. In general, significant relationships between [alpha]-tocopherol content and LT have been observed only after supplementation with pharmacological doses (22-24). In our study, however, the [alpha]-tocopherol content of LDL was significantly correlated to PA-LT. These results suggest that monitoring PA oxidation in LDL is a useful technique for analyzing early oxidation processes taking place in the surface monolayer of the LDL particle.

Polyunsaturated FAs are the main substrates for lipid peroxidation. We observed no correlation between linoleic acid and any of the Us. In contrast, arachidonic acid was inversely related to both CD-LT and FD-LT, showing that the presence of this FA increases the oxidative susceptibility of LDL. We found a much weaker correlation between PA-LT and arachidonic acid, presumably because the PA probe is itself highly unsaturated and thus extremely prone to oxidation.

Peroxidation of lipids in isolated LDL requires the presence of traces of preformed peroxides (25). The lipid peroxide concentrations were indeed inversely related to LTs; however, only the association with CD-LT reached statistical significance.

In summary, Us as measured by the three methods are highly correlated, but the magnitude of correlations found with LDL constituents are method dependent. Us measured by the reproducible FD assay can be an alternative for the conventional CD method. The PA technique seems to be particularly suitable for studying an early stage of the oxidation process and relationships between LDL oxidizability and antioxidants.

We thank Franca Groot for analyzing the fatty acids of LDL and Marcel Hennekes for lipid peroxide measurements.


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Peter G. Scheffer, [1] * Stephan J.L. Bakker, [2] Erik E. Musch, [1] Corrie Popp-Snijders, [1,2] Robert J. Heine, [2] and Tom Teerlink [1] (Departments of [1] Clinical Chemistry and [2] Endocrinology, Research Institute for Endocrinology, Reproduction and Metabolism, Academic Hospital Vrije Universiteit, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands; * author for correspondence: fax 31-20-4443895, e-mail p.scheffer@
Table 1. Correlation coefficients between LTs (a) and lipid
peroxides, LDL size, antioxidants, and total amount of
FAs. (b)


Lipid peroxides -0.27 (c) -0.16 -0.20
LDL particle size 0.12 0.19 0.04
[alpha]-Tocopherol 0.18 0.27 (c) 0.15
Coenzyme Q10 0.26 0.34 (d) 0.29 (c)
Saturated FAs
 Palmitic acid (16:0) 0.13 0.11 0.09
 Stearic acid (18:0) 0.12 0.20 0.09
Monounsaturated FAs
 Oleic acid (18:1) 0.19 0.15 0.10
Polyunsaturated FAs
 Linoleic acid (18:2) 0.23 0.25 0.20
 Arachidonic acid (20:4) -0.40 (d) -0.24 -0.47 (d)

(a) The LTs were measured by following the production of CDs (CD-LT),
by monitoring the fluorescence intensity after incorporation of PA
into LDL (PA-LT), and by monitoring the autofluorescence development
of LDL (FD-LT).

(b) n=98.

(c) <0.01.

(d) <0.001.
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Title Annotation:Technical Briefs
Author:Scheffer, Peter G.; Bakker, Stephan J.L.; Musch, Erik E.; Popp-Snijders, Corrie; Heine, Robert J.; T
Publication:Clinical Chemistry
Date:Feb 1, 2000
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