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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1049-1056.)
© 1995 American Heart Association, Inc.

Articles

Lowering of HDL_2b by Probucol Partly Explains the Failure of the Drug to Affect Femoral Atherosclerosis in Subjects With Hypercholesterolemia

A Probucol Quantitative Regression Swedish Trial (PQRST) Report

Jan Johansson; Anders G. Olsson; Lott Bergstrand; Liselotte Schäfer Elinder; Sven Nilsson; Uno Erikson; Jörgen Mölgaard; Ingar Holme; Göran Walldius

From the King Gustaf V Research Institute, Karolinska Hospital, Stockholm (J.J., L.S.E., G.W.); Research Centre of General Medicine, North Western Health Board, Stockholm County Council (J.J.); Department of Internal Medicine, University Hospital, Linköping (A.G.O., J.M.); Department of Diagnostic Radiology, University Hospital, Uppsala (L.B., S.N., U.E.), Sweden; and the Life Insurance Companies Institute for Medical Statistics, Ullevål Hospital, Oslo, Norway (I.H.).

Correspondence to Jan Johansson, MD, PhD, Research Centre of General Medicine, Borgmästarvillan–Karolinska Hospital, S-171 76 Stockholm, Sweden.

	Abstract

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Abstract The aim of the Probucol Quantitative Regression Swedish Trial (PQRST) (n=303) was to investigate whether probucol (0.5 g BID) added to diet and cholestyramine (8 g BID) could retard progression or induce regression of femoral atherosclerosis in hypercholesterolemic (>6.86 mmol/L) subjects. Probucol did not induce regression over the 3-year trial period as estimated by change in lumen volume on quantitative arteriography of a 20-cm segment of the femoral artery. In this report we studied in a representative subgroup (n=72) whether the reduction in HDL concentrations induced by probucol could explain the failure of the drug to be effective. We analyzed the effects of treatment on HDL particle size subclasses. Probucol lowered the relative level of HDL_2b, comprising the largest HDL particles, by 53% and the protein concentration of HDL_2b by 67%. The protein reduction in HDL was mainly confined to the apolipoprotein A-I moiety. The change in lumen volume correlated significantly with change in HDL, ie, HDL cholesterol (r=.34, P<.01), HDL₂ cholesterol (r=.37, P<.01), HDL_2b protein (r=.44, P<.001), and the relative HDL_2b value (r=.51, P<.001). The corresponding values for relative HDL_2b distribution calculated on the active (n=35) and placebo (n=37) groups separately were also significant (r=.39 and .32, respectively; both P<.05). The correlation between drug-induced change in the relative HDL_2b concentration and change in atherosclerosis was independent of the alteration in triglyceride concentration and could not be explained by treatment interaction. HDL_2b lowering was highly significantly correlated to probucol concentration. We suggest that the lowering effects of probucol on HDL and particularly on the HDL_2b fraction at least in part explain why regression of femoral atherosclerosis was not obtained by the drug.

Key Words: high-density lipoprotein particle size • atherosclerosis • arteriography • cholestyramine and antioxidation

	Introduction

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Probucol is a plasma lipid–lowering drug that in contrast to other such agents not only decreases the concentration of LDL cholesterol but also HDL cholesterol.¹ Probucol also has antioxidant properties² that have been suggested to contribute to the retardation of atherosclerosis progression in rabbits and nonhuman primates.^{3 4} However, little is known about the effects of probucol treatment on the development of atherosclerosis in humans. Therefore, the PQRST,⁵ a 3-year, double-blind, randomized trial in hypercholesterolemic individuals, was performed.

Basic treatment with diet and cholestyramine for 3 years was associated with an increase in lumen volume, suggesting regression of femoral atherosclerosis.⁶ However, the addition of probucol to the basic treatment did not improve the development of femoral atherosclerosis.

One major question in the PQRST was to evaluate whether the lowering effect of probucol on HDL cholesterol despite the antioxidative properties and serum cholesterol–lowering effect of probucol could adversely affect atherosclerosis development. This question is important considering the negative correlation for HDL with the progression of atherosclerosis and cardiovascular diseases found in prospective cohort studies^{7 8 9 10} and intervention trials.^{11 12 13 14 15 16}

Results from recent investigations using separation of HDL lipoproteins by particle size have indicated that the "antiatherogenic" effect of HDL is confined to the large HDL particles, especially subclass HDL_2b,^{17 18} comprising the very largest HDL particles in the HDL density interval. In contrast, a high plasma level of HDL_3b, represented by small particles, is associated with the progression of atherosclerosis.^{17 18 19} Therefore, it was of interest to monitor HDL particle size changes by treatment with cholestyramine and probucol in the PQRST and to follow the concomitant alterations in atherosclerosis development. Change in atherosclerosis was estimated by measurement of changes in lumen volume (the primary end point of the trial) as determined by quantitative arteriography.⁵ This study was performed in a representative subsample of the PQRST participants.

	Methods

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Materials and Design
The subject material, inclusion/exclusion criteria, and design of the study have been described in detail elsewhere.⁵ In summary, 1496 men and women younger than 71 years of age with hypercholesterolemia, both asymptomatic subjects and individuals with a history of cardiovascular disease, were referred for evaluation. Subjects with a serum total cholesterol concentration greater than 6.86 mmol/L and a serum triglyceride concentration less than 4.0 mmol/L were instructed to follow a lipid-lowering diet for 3 months. Subjects with remaining hypercholesterolemia were then prescribed 4 to 8 g cholestyramine BID and probucol placebo BID for 2 months. Those responding with an 8% or greater reduction in total cholesterol continued with cholestyramine and were given 500 mg probucol active BID for another 2 months. The subjects responding with a further 8% reduction in total cholesterol underwent femoral arteriography. Those with visible atherosclerosis on the x-ray films were randomized.^{5 6} A total of 303 subjects entered the double-blind phase of the trial and were treated with either cholestyramine plus probucol placebo (placebo group) or cholestyramine plus probucol active (active group) for 3 years. A total of 249 subjects had an arteriography performed 3 years later.

The present study is focused on 72 patients included consecutively at the end of the recruitment phase of the study. In these subjects HDL particle size analysis was performed throughout the study. Thirty-five of the subjects belonged to the active group and 37 to the placebo group. Serum concentrations obtained after diet intervention but before the drug-testing period constituted baseline values for various calculations.

One rationale for choosing this baseline was the knowledge that the two agents have major but opposite effects on HDL cholesterol concentrations. Furthermore, by having a baseline from a relatively late phase of the prerandomization period, the latency to the arteriography investigation was shortened, and laborious HDL particle size analyses did not have to be performed on subjects who were to be excluded.

The study was approved by the Ethics Committees at the involved institutes and universities. The subjects were informed and gave their consent before entering the study.

Lipoproteins
Venous blood was drawn in the morning after an overnight fast. Blood for preparation of HDL GGE analysis was drawn into ice-cooled disodium EDTA tubes (1.4 mg/mL). The major lipoprotein fractions were separated by a combination of ultracentrifugation and precipitation in accordance with the Lipid Research Clinics Protocol²⁰ as previously described.²¹ In short, VLDL was separated from LDL and HDL by preparative ultracentrifugation at density=1.006 kg/L. LDL and HDL were separated by precipitation of the LDL fraction with heparin (2 mol/L)/manganese (5%). The LDL concentration was calculated by subtraction of the HDL portion from the total concentration before precipitation. HDL₃ was separated by ultracentrifugation at density=1.125 kg/L²² and HDL₂ cholesterol calculated by subtracting the value of HDL₃ from that of total HDL. Cholesterol²³ and triglyceride²⁴ concentrations were determined in the VLDL, LDL, and HDL fractions. Only cholesterol was determined for HDL₂ and HDL₃. In each run the cholesterol²³ and triglyceride²⁴ analyses were standardized against two frozen control sera of different concentrations. The control sera were double-checked monthly against reference methods for cholesterol²⁵ and triglyceride²⁶ analyses for detection of possible drift in methodology or control sera over time.

The mean overall percent COV (%COV=d²/2n, where d is the difference in concentration between two measurements of the same variable and n is the number of samples analyzed) when duplicate samples were run separately through the preparation, isolation, and analysis procedures were serum cholesterol, 4%; serum triglycerides, 7%; VLDL cholesterol, 9%; VLDL triglycerides, 8%; LDL cholesterol, 6%; LDL triglycerides, 11%; HDL cholesterol, 5%; HDL triglycerides, 10%; and HDL₃ cholesterol, 6%.

Plasma apoA-I and B concentrations were analyzed by competitive radioimmunoassay (Pharmacia Diagnostics AB). The combined between- and within-run %COV was 6% for apoB and 7% for apoA-I.

HDL Gel Electrophoresis
HDL GGE subclasses were analyzed by a modification¹⁷ of the technique described by Blanche et al.²⁷ In short, HDL was separated as a plasma fraction within the densities of 1.070 and 1.21 kg/L and subjected to electrophoresis on polyacrylamide gradient gels (PAA 4/30, Pharmacia). The proteins were stained with amido black and scanned at wavelength 570 nm (Fig 1). The absorption of the gel itself was subtracted from the curves of the HDL samples. The relative areas under the curve were as follows: corresponding to HDL_2b, 9.71<diameter ()<12.90 nm; HDL_2a, 8.77<<9.71 nm; HDL_3a, 8.17<<8.77 nm; HDL_3b, 7.76<<8.17 nm; and HDL_3c, 7.21<<7.76 nm. The absolute concentration in milligrams of protein per milliliter for each subclass was derived by multiplying the relative estimates for the HDL GGE subclasses by the total protein concentration of the isolated HDL fraction. The protein concentration of HDL was analyzed according to Lowry et al.²⁸

View larger version (18K): [in this window] [in a new window]   Figure 1. Plots show densitometric scans of HDL separated on 4% to 30% polyacrylamide gradient gels. Absorption of the gel itself forms the rounded bottom line. The protein of HDL is represented between this bottom line and the curve above. A, Scan of HDL from a subject with relatively low HDL2b and relatively high HDL3b concentrations, a combination that has been suggested to form an "atherogenic" HDL profile.17 18 19 This pattern is more favorable in B. C, Scan from a third subject who has an unusually high HDL2b concentration.

Duplicate plasma samples (n=40) were taken at the same venipuncture but in different vacutainer tubes; and preparation, isolation of HDL by ultracentrifugation, GGE, protein staining of gels and destaining, planimetry, and determination of plasma total HDL protein were run separately. The %COV values were HDL_2b, 9%; HDL_2a, 6%; HDL_3a, 6%; HDL_3b, 6%; and HDL_3c, 12%. These figures include the 4% COV for the protein determination.²⁷ The COV values for the relative estimates were thus considerably lower. The protein migration of the standard molecules on the gels was virtually identical over the study period, indicating consistent particle size separation properties. Calculating the relative migration distances (R_f) from thyreoglobulin (MW=669 kD) in relation to that of bovine serum albumin (MW=67 kD) of the three protein markers ferritin (MW=440 mW), catalase (MW=32 kD), and lactate dehydrogenase (MW=140 kD) gave the %R_f values (mean±SD) of 23.8±1.2, 54.1±1.0, and 68.4±0.8 for ferritin, catalase, and lactate dehydrogenase, respectively.

Serum probucol concentrations were analyzed by high-performance liquid chromatography as previously described.²⁹ Since serum probucol resides in the lipophilic compartment of the lipoproteins, its concentration is strongly positively correlated to the serum triglyceride level.³⁰ Therefore, the probucol value was adjusted by dividing the mean serum probucol concentration by the corresponding serum triglyceride concentration.

Change in lipoprotein concentrations was defined as the mean value from 1, 2, and 3 years after randomization minus the level at baseline, if not otherwise stated.

Arteriography
Arteriographic indexes of atherosclerosis before randomization and after 3 years of treatment were compared.^{31 32 33} Two angiographic series of a 20-cm segment of the superficial femoral artery were performed with a 10-minute interval. The angiographic procedure was highly standardized to minimize methodological variation. The arteriograms were digitized, and lumen volume was calculated from the computerized images. An increase in lumen volume was considered to reflect reduced amounts of atherosclerosis.⁶

Statistical Methods
All data handling and statistical analysis were carried out with SAS version 6.08 by the Sema Group InfoData AB, Stockholm. Representativeness of the 72 subjects was tested by comparison with the remaining individuals of the PQRST on whom total and HDL₂ cholesterol but not HDL GGE measurements were performed. Mean values and SDs were calculated and Student's t tests applied for comparison between groups. For categorical data, ² tests were used. Spearman rank correlations between change in lumen volume and change in lipoprotein concentrations from baseline were calculated.

A graphic illustration of standardized regression coefficients between lumen volume change and change in HDL variables with 95% confidence intervals was used. The number of subjects with a lumen volume increase versus decrease was given for each consecutive quartile of HDL_2b change and compared according to a ² test for trend. The possible influence of treatment regimen (active or placebo) and possible interaction between treatment and change in HDL variables were tested in multiple regression analyses. Possible influences by various factors on change in lumen volume were tested with partial regression analysis and multiple regression analysis.

	Results

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Subjects
The 72 subjects did not differ in baseline clinical characteristics from either the residual 231 of the randomized population in the PQRST (not shown) or from the residual 177 subjects who had femoral arteriography performed at the start and end of the trial (Table 1). Lumen volume of the femoral artery at baseline was also not significantly different from that of the entire PQRST population. Baseline variables were compared between the active and placebo groups (Table 1), and no differences were found.

View this table: [in this window] [in a new window]   Table 1. Baseline Clinical Characteristics of Subjects in the PQRST

Lipoproteins
In the placebo group total cholesterol was lowered because of a decrease in LDL cholesterol concentration. No significant effect on HDL cholesterol was found (Table 2). Total serum triglyceride level did not change in either group.

View this table: [in this window] [in a new window]   Table 2. Mean Change From Baseline in Clinical Characteristics During Treatment for Probucol Active and Placebo Groups

In the active group the cholesterol level decreased significantly in all three major lipoprotein fractions, ie, VLDL, LDL, and HDL. In the active group total cholesterol was lowered more than in the placebo group (-2.87 versus -1.95 mmol/L, respectively; P<.001). This was accounted for by a more pronounced cholesterol lowering in all three lipoprotein fractions, particularly in HDL cholesterol (-0.48 versus -0.04 mmol/L, P<.001). The apoB level decreased highly significantly in both treatment groups (Table 2). No significant between-group difference was found for this variable.

HDL Particle Size and ApoA-I
In the active group total HDL protein concentration decreased by 25%, partly because of a decrease in apoA-I of 22% (Table 2). In the placebo group a small increase in apoA-I and a small decrease in the total HDL protein concentration, both significant, were found. The difference in change in total HDL protein concentration between the two groups was 301 mg/L (P<.001). The corresponding difference for apoA-I was 341 mg/L (P<.001).

The most pronounced changes in the relative distribution of HDL particle size were seen in the active group, in which HDL_2b decreased from 16% to 7.8% of the total HDL content and HDL_3a increased from 29.3% to 37.7% (both P<.001). The response to drug treatment on the concentration of these two HDL subclasses was significantly different between the active and placebo groups (both P<.001).

For the absolute HDL GGE levels (milligrams of protein per liter) the most pronounced effect was seen for the largest HDL_2b and smallest HDL_3c particles in the active group, which decreased in concentration by 67% and 41%, respectively (Table 2). In the placebo group significant decreases were seen for the HDL_3b and HDL_3c subclasses, with 15% and 22% decreases, respectively.

Serum Probucol Concentrations
Serum probucol level in the active group was 73±23 µmol/L (mean±SD) during the 3-year treatment period. The concentration was stable over time. The relation between serum probucol concentrations and changes in lipoprotein levels were studied during both the drug test period (n=72) and the trial phase (active group, n=35) (Table 3).

View this table: [in this window] [in a new window]   Table 3. Spearman Rank Correlation Coefficients

After serum probucol concentration was adjusted for triglyceride level, highly significant inverse correlations were found with changes in HDL cholesterol, HDL₂ cholesterol, apoA-I, and subclass HDL_2b (P<.01 for all) (Table 3). These findings were consistent and found both after 2 months of drug testing and during the 3-year trial period.

Arteriographic Data
After 3 years of treatment lumen volume increased significantly in the placebo group (P<.05, Table 2). There was a significant increase in lumen volume in favor of the placebo-treated group. Lumen volume increased with a mean value of 218 mm³ (4%) compared with a decrease of 146 mm³ in the active group (P<.05 for between-group comparison).

Relations Between Change in Lipoproteins and Arteriographic Variables
Change in lumen volume did not correlate with changes in total cholesterol and triglyceride concentrations in serum, VLDL, or LDL in any treatment group or in all individuals pooled. No significant correlations were found between serum probucol or triglyceride-adjusted probucol concentration and change in lumen volume.

The change in total HDL cholesterol concentration correlated significantly with change in lumen volume (r=.34, P<.01) for the 72 subjects. This correlation was confined to HDL₂ cholesterol (r=.37, P<.01) and more specifically to the absolute HDL_2b level (r=.44, P<.001) and the relative HDL_2b concentration (r=.51, P<.001). The correlation between the change in relative HDL_2b concentration and change in lumen volume was also significant when calculated for the active and placebo groups separately (r=.39 and r=.32, respectively; both P<.05).

In the placebo group the HDL_3a alteration (both relative and absolute) correlated highly significantly and inversely with the change of lumen volume (r=-.51 and r=-.46, respectively; both P<.001). The corresponding correlations for the active group were not significant.

Correlations between changes in concentrations for HDL variables and lumen volume are shown in Fig 2 with 95% confidence intervals on standardized regression coefficients. The interval for the changes in HDL cholesterol, HDL₂ cholesterol, and HDL_2b concentrations are located on the right-hand side of the zero line and indicate significant positive regression coefficients with lumen volume change. Not-depicted particle size fractions had their confidence intervals centered around the zero line, indicating nonsignificant correlations with lumen volume change.

View larger version (16K): [in this window] [in a new window]   Figure 2. Plot shows standardized regression coefficients between lumen volume change of the femoral artery and change in HDL variables with 95% confidence intervals. chol indicates cholesterol; TG, triglyceride; and Apo, apolipoprotein.

The 72 subjects were placed into quartiles according to the magnitude of their treatment-induced change in relative HDL_2b concentrations and the number of individuals with lumen volume increase compared with those showing a decrease according to ² analysis (P=.0007, Fig 3). There was a gradually higher proportion of subjects with an increase in lumen volume for each consecutive change in HDL_2b quartile. When the figure was split according to active or placebo treatment, opposite distributions were found. In the placebo group most subjects were in the third and fourth quartiles, indicating an HDL_2b increase, and 26 of the 37 patients (70%) had an increase in lumen volume. By contrast, most subjects in the active group fell into quartile one or two, indicating an HDL_2b decrease, and only 12 of the 35 subjects (34%) had a lumen volume increase. In a similar model the corresponding ² tests for trend value for HDL cholesterol and HDL₂ cholesterol in the entire PQRST population were P=.015 and P=.026 (n=249), respectively.

View larger version (31K): [in this window] [in a new window]   Figure 3. Bar graphs show lumen volume increase vs decrease for quartiles of change in relative HDL2b concentration for all subjects (n=72), the active group (n=35), and the placebo group (n=37). 2 test for trend was P=.0007.

There was no significant drug interaction between treatment group and change in HDL cholesterol, HDL₂ cholesterol, and the absolute HDL_2b level on the relation between the corresponding variables and change in lumen volume. The correlation between change in relative HDL_2b concentration and change in lumen volume remained significant (P<.02) after adjustment for treatment group.

In a multiple regression model with change in lumen volume as the dependent variable, sex, age, change in LDL cholesterol, and change in VLDL triglyceride concentration were entered as independent variables (all P<NS). However, the addition of HDL_2b as an independent variable had a significant effect (P<.0075).

Since there is a known inverse correlation between HDL and triglycerides,^{34 35} we wanted to explore the possible influence of change of VLDL triglycerides on the correlation between HDL and lumen volume. In partial correlation analysis it was found that change in VLDL triglycerides did not influence the correlation between the changes in lumen volume and HDL_2b for the 72 subjects or the correlation between the changes in HDL cholesterol and lumen volume for the 249 subjects. The partial correlation coefficients were virtually the same as those for the bivariate correlation, ie, .52 (P<.0001) for the correlation of HDL_2b to lumen volume and .16 (P<.02) for the correlation of HDL cholesterol to lumen volume.

	Discussion

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In the present study performed on a representative subgroup of the PQRST subject material, we basically obtained the same results as for the entire subject population. The significant decrease in atherosclerosis, ie, increase in lumen volume, in the placebo group treated with diet and cholestyramine followed the expected response to lipid regulation.^{16 36} When probucol was added to cholestyramine, it decreased lumen volume (the clinical trial primary end point) compared with the increase seen in the placebo group. To find possible explanations for these effects, we related the changes in lipoproteins by treatment to change in lumen volume.

The analysis showed that the changes in the concentrations of HDL cholesterol, HDL₂ cholesterol, and particularly particle size subclass HDL_2b were highly significantly correlated with the change in lumen volume of the femoral artery. No significant drug interaction bias was found, and the correlation of HDL_2b to lumen volume remained significant after adjustment for treatment group. Thus it is reasonable to assume that there is a causal and biologically meaningful link between change in HDL_2b and lumen volume change; ie, the more pronounced the HDL_2b elevation, the more marked the regression of atherosclerosis and vice versa. Notably, the results mainly illustrate effects related to preclinical atherosclerosis since two thirds of the population were free from cardiovascular disease and only 13% had intermittent claudication.

The results support recent findings obtained in studies of patients with coronary artery disease^{17 18 19} and intermittent claudication³⁷ showing that the inverse relation between HDL and atherosclerosis was mainly accounted for by "large" HDL particles, ie, particle size subclass HDL_2b. Since the PQRST was an intervention study over 3 years, the results extend the knowledge gained from the case-control and cross-sectional studies^{17 18 19 37} regarding the importance of HDL_2b. The finding suggests that HDL_2b plays an active role in protecting the vessel wall from atherosclerosis development.

Since probucol treatment lowers the HDL_2b concentration by more than 50%, it is possible that this lipid effect in part explains why the drug did not induce regression of atherosclerosis.⁶ The probucol concentration when adjusted for triglycerides was significantly inversely correlated to the decrease in HDL_2b and apoA-I concentrations (Table 3). Notable was the fact that the subjects taking probucol showed a decrease in HDL proteins mainly confined to apoA-I.

Recent studies have shown that HDL can act as an antioxidant³⁸ and that lipid peroxides in LDL can be transported to HDL, a process related to the content of the enzyme paraoxonase associated with HDL.³⁹ A reduction of HDL by probucol may thus compromise the capacity for scavenging peroxides. We have reported the highly significant effects of probucol in increasing the resistance of LDL to copper oxidation, both at the prerandomization² and during the 3-year trial phase,⁴⁰ but the effect of probucol on HDL antioxidative characteristics and paraoxonase remains to be studied.

Recent studies have emphasized the importance of the endothelial lining in the regulation of the tonus and diameter of the artery by synthesis of nitric oxide and other vasoactive substances.^{41 42} Oxidized LDL has been shown to impair the vasodilating function of endothelium-derived relaxing factor in vitro.⁴³ A recent study in humans comparing the coronary vasomotor response to acetylcholine injection before and after treatment with diet, lovastatin and cholestyramine, or lovastatin and probucol for 1 year showed a significantly decreased constriction in the probucol-treated group.⁴⁴ These results indicate that antioxidation therapy can improve endothelium-dependent relaxing functions. An interaction between lipoproteins and endothelial functions has also been reported. For example, the dilatation of the coronary arteries is attenuated in subjects with low HDL levels compared with those with high concentrations.⁴⁵ Therefore, in the PQRST variation in both HDL concentration and LDL oxidation may have contributed to opposite vasomotor effects. Thus it may be possible that the probucol-treated group had increased vasodilatation because of antioxidation protection, which nonetheless was counteracted by vasoconstriction caused by the pronounced decrease in HDL. To what degree these functional and endothelium-dependent properties affect arterial lumen volume and true changes in atherosclerosis cannot be elucidated by the present study, as discussed in detail in our article reporting the primary end point.⁶

In conclusion, the HDL_2b alteration by cholestyramine and probucol plus cholestyramine treatment shows an inverse correlation to atherosclerosis development as estimated by the change in lumen volume on quantitative arteriography of the femoral artery. In analogy, the pronounced HDL_2b lowering by probucol might explain why the subjects taking the drug did not show regression of atherosclerosis. Besides effects on atherosclerosis, change in vasomotor tone may be considered as a concomitant explanation for the found relation between change in lumen volume and changes in HDL. Given this caution the results indicate that the clinical effect on femoral atherosclerosis by modification of oxidative properties appears to be relatively minor compared with that of altering HDL levels in plasma.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein COV = coefficient of variation GGE = gradient gel electrophoresis PQRST = Probucol Quantitative Regression Swedish Trial

	Acknowledgments

 
This study was supported by a research grant from Marion Merrel Dow Inc, Kansas City, Mo; the Swedish Medical Research Council (n:o 06962); the Swedish Heart-Lung Foundation; the Knut and Alice Wallenberg Foundation; the Torsten and Ragnar Söderberg Foundation; King Gustaf V 80th Birthday Foundation; the Karolinska Institute Foundation; Prof Nanna Svartz' Foundation; the Foundation for Old Servants; the Albert and Gerda Svensson Foundation; Loo and Hans Ostermans Foundation; and the Swedish Society for Medical Sciences. We thank Inger Malmaeus, Björn Gustavsson, and the InfoAnalysis group at the Sema Group InfoData AB, Stockholm, Sweden, for the professional computer handling and statistical analysis.

Received July 12, 1994; accepted May 5, 1995.

	References

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