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Journal of the American College of Nutrition, Vol. 18, No. 5, 451-461 (1999)
Published by the American College of Nutrition

Antioxidant Supplementation Effects on Low-Density Lipoprotein Oxidation for Individuals with Type 2 Diabetes Mellitus

James W. Anderson, MD, Maya S. Gowri, PhD, Jan Turner, RD, Laura Nichols, RN, Veda A. Diwadkar, MSc, Ching K. Chow, PhD and Peter R. Oeltgen, PhD

Metabolic Research Group, VA Medical Center and Department of Internal Medicine, College of Medicine (J.W.A., M.S.G., J.T., L.N., V.A.D.), University of Kentucky, Lexington, Kentucky
Department of Nutrition and Food Science (C.K.C.), University of Kentucky, Lexington, Kentucky
Department of Pathology (P.R.O.), University of Kentucky, Lexington, Kentucky

Address reprint requests to: James W. Anderson, M.D., Medical Service, 111C, VA Medical Center, 2250 Leestown Road, Lexington, KY 40511


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Objective: This study compared susceptibility to oxidation of low-density lipoproteins (LDL) of non-diabetic and diabetic (Type 2) men and examined the response of diabetic men to antioxidant supplementation ({alpha}-tocopherol, ß-carotene and ascorbate).

Research Design and Methods: Twenty adult non-diabetic and 20 diabetic men were recruited. Oxidation of LDL was assessed by four different assay systems, and the extent of oxidation was assessed by four different measurements. Diabetic men received eight weeks of placebo ("baseline"), twelve weeks of antioxidant supplements ("treated") and eight weeks of placebo ("post-treatment"). Supplements provided 24 mg of ß-carotene, 1000 mg of ascorbate and 800 IU of {alpha}-tocopherol daily.

Results: With Cu oxidation at 37°C, thiobarbituric reactive substances (TBARS) formation was significantly higher (p=0.032) and loss of free amine groups was significantly greater (p=0.013) in the LDL from diabetic subjects than controls. Antioxidant supplementation of diabetic subjects significantly decreased all parameters of LDL oxidation with Cu at 30°C and 37°C. At 30°C the lag phase increased from 55 to 129 minutes (p<0.0001); conjugated diene formation decreased from 1.23 to 0.62 OD units (p<0.0001); TBARS formation decreased from 78 to 33 nmoles MDA/mg LDL protein (p<0.0001); and free amine loss decreased from 41 to 12% (p<0.0001). With Cu oxidation at 37°C, similar changes occurred.

Conclusions: These studies indicate that the LDL from diabetic subjects are more susceptible to oxidation than LDL from non-diabetic subjects. Supplementation of diabetic subjects with antioxidant vitamins significantly decreases susceptibility of LDL to oxidation by Cu. These studies are consistent with epidemiological and intervention studies suggesting that antioxidant vitamin use significantly decreases risk for coronary heart disease.

Key words: antioxidant, diabetes mellitus, LDL, {alpha}-tocopherol, ß-carotene, ascorbic acid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Individuals with diabetes mellitus are at significantly higher risk for atherosclerotic cardiovascular disease (ASCVD) than are non-diabetic individuals [1]. Many risk factors—such as dyslipidemia [2], hypertension [3], and obesity [4]—probably contribute to the increased prevalence of ASCVD in diabetes. Recent evidence [2,5–9] suggests that an increased susceptibility to oxidation of low-density lipoproteins (LDL) may contribute to this diathesis.

The role of oxidation of LDL in the pathogenesis of atherosclerosis is attracting increasing attention [10,11]. LDL can be oxidized in vitro by all the major cells in the vascular wall. Oxidized LDL is processed by a scavenger receptor of macrophages, leading to cholesterol ester accumulation. These lipid-laden macrophages become foam cells which, in time, create fatty streaks in blood vessel walls. In addition, oxidized LDL has many other atherogenic effects in the vascular subendothelium; these particles enhance expression of adhesion molecules and promote macrophage differentiation, which facilitates further oxidation of minimally modified LDL [10,11].

The LDL-oxidation hypothesis is further supported by emerging data that {alpha}-tocopherol intake decreases susceptibility to coronary heart disease. Epidemiological studies [12–15] suggest that higher intakes of {alpha}-tocopherol are associated with lower risk for CHD, and prospective clinical trials [16,17] supports this hypothesis. A-tocopherol is the major antioxidant transported in the LDL particle [18]. Intake of {alpha}-tocopherol supplements significantly reduces the in vitro oxidation of LDL, manifested by an increased lag phase (an indirect measure of the total antioxidant content of LDL) and decreased formation of conjugated dienes and thiobarbituric acid reactive substances (TBARS) [18,19]. While these effects are well documented in non-diabetic subjects, less information on the effects of antioxidant supplementation on the in vitro oxidation kinetics of LDL for diabetic subjects is available [20,21]. ß-carotene, like {alpha}-tocopherol, is a fat-soluble vitamin which is transported in LDL and reduces the extent of LDL oxidation [22]. Ascorbate is a potent water-soluble antioxidant and also acts to preserve {alpha}-tocopherol levels [23]. The effects of combination of three antioxidants on LDL oxidation kinetics in diabetic subjects is not well defined.

This study had two purposes. First, we compared the in vitro oxidation kinetics of LDL from diabetic subjects with those of control subjects. Second, the intervention clinical study was designed to determine the effects of antioxidant supplementation on the oxidation kinetics of LDL for men with Type 2 diabetes mellitus. Diabetic subjects received a placebo for eight weeks; subsequently, diabetic subjects received an antioxidant supplement containing ß-carotene, ascorbate and {alpha}-tocopherol for twelve weeks; finally, diabetic subjects were followed post-supplementation for eight weeks. Measurements were made after eight weeks of placebo (baseline), after twelve weeks of antioxidant vitamin supplementation and after a further eight weeks of placebo administration.

This study confirmed and extended two previous studies of antioxidant supplementation in diabetic subjects [20,21] and indicated that diabetic subjects had statistically significant alterations in certain parameters of in vitro oxidation kinetics of LDL when compared to non-diabetic subjects. After antioxidant supplementation, the susceptibility to oxidation of LDL from diabetic subjects was substantially decreased. These observations suggest that antioxidant supplementation may decrease risk for ASCVD for diabetic individuals.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Type 2 diabetic subjects who met the inclusion criteria were recruited for the study. Inclusion criteria were onset of diabetes after age 35 with at least five years of treatment without use of insulin; well-established diabetes with multiple fasting plasma glucose values >=7.8 mmol/L [24]; age, 40 to 70 years; body mass index <33 kg/m2; and serum creatinine, <2.0 mg/dL. Subjects were excluded for the following reasons: history of ketoacidosis or onset of diabetes before age 35; smoking; use of vitamin supplements; treatment with glucocorticoids, thyroid hormone, lipid-lowering agents, or other drugs known to affect serum lipids; proteinuria with urine protein excretion >250 mg/d; and serum {alpha}-tocopherol levels >28 µmol/L. Twenty control subjects who were age- and gender-matched to diabetic subjects were also recruited. A control subject had LDL oxidation studies on every occasion that LDL oxidation studies were performed on diabetic subjects; because control subjects were required to be available on fairly short notice throughout the study, we were not able to match diabetic and control subjects as closely for age and serum triglyceride concentrations as desirable.

Study Protocol
This was a single-blind, placebo-controlled study. Twenty diabetic subjects meeting the enrollment criteria had baseline measurements and were instructed on a weight-maintaining ADA diet without vitamin and mineral supplements. They were placed on placebo and followed for eight weeks; they had detailed studies of antioxidant status and oxidation status at the completion of this control period. They continued their diet and received {alpha}-tocopherol, ß-carotene and ascorbate supplements for the next twelve weeks. At the completion of this antioxidant treatment period, subjects had repeat studies of antioxidant and oxidation status. The placebo was distributed for the final period, and the subjects were followed for another eight weeks; measurements of glycemic control and serum lipids were performed on all subjects at completion of the study.

Twenty normal subjects were also enrolled in the study and were used as controls for LDL oxidation studies. They were not supplemented with antioxidants. The control subjects also had measurements of LDL oxidation and antioxidant status.

Supplements
The supplements consisted of antioxidant capsules containing ß-carotene, 6 mg/capsule, ascorbate, 250 mg/capsule, and {alpha}-tocopherol ({alpha}-tocopheryl acetate), 200 IU/capsule (Roche Vitamins, Inc., Nutley, NJ). Subjects were asked to take two capsules twice daily to obtain a total daily dose of 24 mg/d of ß-carotene, 1000 mg/d of ascorbate and 800 IU/d of {alpha}-tocopherol. Placebo capsules were identical in appearance and contained 750 mg of sucrose per capsule. Capsules were dispensed for each four-week period. During each visit, once every four weeks, subjects returned the bottle with unused capsules to assess compliance and received an additional bottle.

Measurements
Serum glucose levels were determined using an enzymatic (glucose oxidase) method [25]. Glycosylated hemoglobin values were determined using an affinity binding assay (Abbott Laboratories, North Chicago, IL) [26]; normal values were 4.4% to 6.4%. All serum lipid measurements were performed through the Lipid Research Laboratory at the VA Medical Center, Lexington, KY as previously described [27]. The Abbott Spectrum II Analyzer (Abbott Diagnostics, North Chicago, IL) was used for lipid measurements. Serum levels of {alpha}-tocopherol [28], ß-carotene [29] were measured by high performance liquid chromatography; {alpha}-tocopherol levels of LDL fractions from diabetic subjects at eight and 20 weeks were also determined. LDL protein concentrations were measured by the method of Lowry et al. [30].

LDL Isolation
Fifty mL of whole blood was collected in 0.1% EDTA and plasma separated by centrifugation. LDL was isolated by sequential ultracentrifugation [31]. Plasma density was raised to 1.09 g/mL by the addition of KBr and transferred to Beckman centrifuge tubes along with saline EDTA (0.1% EDTA). Samples were centrifuged at 50,000 rpm at 4°C for eleven hours using a VTi 50 rotor in a Beckman Model L8-80 ultracentrifuge. The density of the VLDL+LDL fraction recovered by tube slicing was adjusted to 1.3 g/mL, layered under saline EDTA and centrifuged at 50,000 rpm for 2.5 hours at 4°C. The LDL recovered by tube slicing was dialyzed extensively against four liters of phosphate-buffered saline (PBS, pH 7.4) overnight to remove EDTA and salts. After dialysis LDL was stored at 4°C under nitrogen. All LDL isolations were performed on the day of sample collection and all oxidation studies were performed on the next day after overnight dialysis of samples.

Oxidation Studies
The susceptibility of LDL to in vitro oxidation was assessed using four different oxidation techniques.

1. Copper Oxidation at 30°C: LDL, 100 µg protein/ml in PBS, was incubated with five µM of copper at 30°C for two hours by a modification of the technique of Esterbauer [32] and absorbance read every five minutes at 234 nm in a spectrophotometer.

2. Cu Oxidation at 37°C: LDL, 50 µg protein/mL, was incubated with five µM of copper at 37°C for two hours with measurements made every ten minutes. In both Cu oxidation procedures, oxidation was stopped by placing the tubes on ice followed by the addition of one mM of EDTA and 40 µM of BHT.

3. AAPH Oxidation: 50 µg of LDL protein/ml was incubated with four mM of AAPH for two hours at 37°C and oxidation stopped by placing the tubes on ice followed by the addition of one mM of EDTA and 40 µM of BHT; measurements were made every ten minutes [33].

4. Hypochlorite Oxidation: Oxidation was carried out on ice by the addition of 25, 50, 100 and 200 µL of two mM NaOCl freshly diluted in PBS to 100 µg of LDL protein/mL. After 15 minutes, oxidation was stopped by the addition of two mM EDTA and 40 µM BHT [34].

Measure of Oxidative Damage
Conjugated diene formation was measured spectrophotometrically at 234 nm. Lag phase measurements during in vitro oxidation serve as an indicator for resistance to oxidation. The lag phase was calculated by measuring the intercept of the tangent of the slope of absorbance curve in the propagation phase with the baseline [32] and was expressed in minutes. TBARS measurement, one estimation of lipid peroxidation, was done by a modification of the method of Kosugi and Kikugawa [35].

Oxidative damage to apolipoprotein B was assessed by trinitrobenzene sulfonic acid (TNBS) reactivity measurements. Reactive or free amino groups of LDL were estimated with TNBS reactivity by the method described by Steinbrecher [36]. The concentration of reactive or free amino groups was determined using valine as a standard by a modification of the technique described previously [37]. Briefly 25 µg of LDL was mixed with one mL of 4% NaHCO3 (pH 8.4) and 50 µL of 1% TNBS was added to it. After one hour incubation at 37°C, 100 µL of 1N HCl and 100 of 10% SDS were added and absorbance read at 340 nm. The concentration of reactive or free amino groups was determined with valine as a standard.

Mouse Peritoneal Macrophage Monolayers Cultures
Peritoneal macrophages were harvested from unstimulated mice in Hank’s balanced salt solution by peritoneal perfusion as described previously [34]. The peritoneal perfusate from mice were pooled and centrifuged at 1100 rpm for 10 minutes at 4°C. The cell pellet was washed once with 20 mL of Dulbecco’s modified Eagle’s medium (DMEM). The recovered cells were resuspended in DMEM containing 10% (v/v) fetal calf serum (FCS), penicillin (100 units/mL) and streptomycin (100 µg/mL) at a final concentration of 0.75x106 cells/mL. Two mL aliquots of this cell suspension were dispensed per well into 6-well tissue culture plates and incubated for two hours at 37°C in a humidified CO2 (5%) incubator. After two hours, each well was washed twice with two mL of DMEM without serum to remove nonadherent cells. The monolayers were incubated at 37°C with 5% CO2 for 18 hours in two mL of DMEM containing 10% FCS. The monolayers were washed twice with DMEM before use for experiments.

Labeling Minimally Modified LDL with DiI
LDL was minimally modified by storage at 4°C for six months [38]. Modification was assessed by TBARS and TNBS measurements. TBARS concentrations were approximately two to three nmol MDA/mg LDL protein and free amine reduction was approximately 20% to 30%. Minimally modified LDL was labeled with DiI, a fluorescent probe, [39] and the specific activity determined using a DiI standard curve.

Uptake of Minimally Modified LDL
Macrophage monolayers were treated with 10, 25, 50 µg/mL of DiI-LDL protein per well and incubated at 37°C with 5% CO2 for two hours. After the incubation period, DiI was extracted from the cells [39] and fluorescence determined using a fluorescent spectrophotometer with excitation and emission wavelength set at 520 and 566 nm, respectively. The DiI from cells was quantitated using a DiI standard curve.

Statistical Analysis
Group means were compared by one-way ANOVA. Means were considered significantly different at p<0.05 by two-way unpaired or paired t tests. Twenty control men were compared to 20 diabetic men for baseline values. For untreated vs. treated diabetic men, 18 paired comparisons were made.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subject Characteristics (Table 1)
Baseline measurements were available on 20 diabetic and 20 control men. Diabetic men were significantly older (p<0.001), on average, than the subjects recruited for control measurements. Diabetic men had slightly, but not significantly, higher BMIs than controls. Baseline serum cholesterol and LDL-cholesterol concentrations were very similar in control and diabetic men. Average serum HDL-cholesterol concentrations were significantly lower (p=0.012) in diabetic men and fasting serum triglyceride concentrations were significantly higher (p=0.011) in diabetic than control men. Eighteen of the 20 diabetic men successfully completed the study. One withdrew to have surgery and another was noncompliant to supplement use. Compliance to supplements was judged to be excellent in 18 subjects, based on pill counts and serum ß-carotene levels.


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  		Table 1. Baseline Characteristics of Subjects

 
Clinical Responses (Table 2)
Body weights did not change significantly over the 28 weeks; entry weights (0 weeks) averaged 90.4 kg and final weights (28 weeks) were 90.5 kg. Anti-diabetes medications were not changed during this period and glycemic control, reflected by fasting plasma glucose and glycohemoglobin values, did not change significantly. Fasting plasma glucose concentrations averaged 9.8 and 10.1 mmol/L for initial and final values, respectively. Glycohemoglobin concentrations were 8.0% initially and were 8.1% at the completion of vitamin supplementation (20 weeks). Similarly, subjects were encouraged to maintain the same diet and their serum lipid concentrations were stable over the study period. Serum cholesterol concentrations averaged 4.8 and 4.7 mmol/L at initial and final visits, respectively. Initial and final values for serum triglycerides, LDL-cholesterol and HDL-cholesterol, also, were almost identical.


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  		Table 2. Clinical Response in Diabetic Subjects (Average±SEM)

 
Serum and LDL Vitamin Responses (Table 3)
Baseline serum values for ascorbate, {alpha}-tocopherol and ß-carotene were similar in control and diabetic men. Serum {alpha}-tocopherol concentrations were slightly, but not significantly, lower in diabetic men (19.3 µmol/L) than in control men (20.9 µmol/L). {alpha}-tocopherol concentrations in LDL were identical for control and diabetic men (7.4 nmol/mg protein). Serum ß-carotene and ascorbate concentrations were slightly, but not significantly, higher in diabetic than control men. Antioxidant vitamin supplementation produced a significant, twofold increase in {alpha}-tocopherol concentrations in serum and LDL and an elevenfold increase in ß-carotene concentrations in serum. After discontinuation of supplementation, serum and LDL {alpha}-tocopherol concentrations returned to pre-treatment levels. Serum ß-carotene remained approximately twofold higher than baseline at 28 weeks or at eight weeks after discontinuing supplementation.


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  		Table 3. Serum and LDL Antioxidant Vitamin Concentrations1

 
Copper Oxidation at 30°C (Table 4)
The lag phase, maximum conjugated diene formation, TBARS production and decrease in free amine groups (TNBS) were similar in control and diabetic subjects. Antioxidant vitamin supplementation was accompanied by significant changes in all these parameters (p values for ANOVA of <0.0001). The lag phase increased from 56 minutes (baseline) to 129 minutes (supplementation, p<0.0001). The maximum formation of conjugated dienes decreased from 1.23 to 0.62 OD units (p<0.0001). The formation of TBARS decreased from 78 to 33 nmol MDA/mg protein (p<0.0001). The loss of free amine groups (TNBS) decreased from 41% to 12% (p<0.0001).


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  		Table 4. Oxidation Kinetics of LDL from Control and Diabetic Subjects1

 
Fig. 1 illustrates the time course of conjugated diene formation with LDL oxidation for five representative diabetic subjects before and after antioxidant supplementation. The curve resulting from averaging values for 18 subjects before and after supplementation was serpentine and not representative of the response seen when individual responses were compared; rather than present individual curves, the curves of five subjects that show the representative responses are presented. Prior to supplementation, during 120 minutes of observation, the conjugated diene formation followed the expected pattern of lag phase and propagation. However, after supplementation, the lag phase was prolonged, and the propagation phase did not develop fully during 120 minutes. To measure the lag phase accurately, the incubation period was extended from 150 to 180 minutes to allow completion of the propagation phase. Over the 120 minutes of incubation, conjugated diene production after antioxidant vitamin supplementation was approximately one-third that observed before supplementation.



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  		Fig. 1. Conjugated diene formation with Cu oxidation at 30°C of LDL from diabetic subjects. Samples from five diabetic subjects before (x) and after ({triangleup}) antioxidant supplementation for 12 weeks are illustrated.

 
Copper Oxidation at 37°C
At 37° Cu oxidation, LDL samples from diabetic subjects showed several differences indicating an increased susceptibility to oxidation. The lag phase was about 10% shorter for diabetic than control subjects. Oxidation of LDL from diabetic subjects produced significantly greater amounts of TBARS (102 nmol/mg) compared to LDL from controls (86 nmol/mg, p=0.032). Oxidation of LDL from diabetic subjects also resulted in significantly (p=0.013) greater losses of free amine groups (56%), compared to oxidation of LDL from control subjects (43%). Maximum conjugated diene formation was similar for control and diabetic subjects.

Antioxidant vitamin supplementation was accompanied by significant alterations in all parameters of LDL oxidation (p values for ANOVA of 0.01 to 0.0006). The lag phase increased two-fold from 28 minutes (baseline) to 59 minutes (supplementation, p=0.0003). The formation of conjugated dienes decreased from 0.75 to 0.60 OD units (p=0.034). The formation of TBARS decreased from 102 to 71 nmol/mg (p=0.0047). The loss of free amine groups (TNBS) decreased from 56 to 33% (p<0.0001). Fig. 2 illustrates the time course of free amine reduction with LDL oxidation. The loss of free amines was greater with diabetic than control subjects (p<0.05). Antioxidant supplementation was associated with a significantly slower rate of free amine reduction than for untreated diabetic men (p<0.0001).



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  		Fig. 2. Decrease in free amine groups in LDL with Cu oxidation at 37°C for control subjects ({blacksquare}) and diabetic subjects before ({circ}) and after (•) antioxidant supplementation for 12 weeks.

 
AAPH Oxidation
With AAPH oxidation of LDL, the differences between diabetic and control subjects and the effects of antioxidant supplementation were not as consistent. While the lag phase was slightly longer for diabetic subjects, the formation of TBARS was higher as with copper oxidation at 37°C. The loss of free amine groups also was slightly greater for diabetic than control subjects. Antioxidant supplementation produced favorable trends for TBARS formation but only the decrease in free amine loss differed significantly for untreated diabetic subjects (24%) compared to supplemented diabetic subjects (14%, p=0.0026).

Hypochlorite Oxidation
Hypochlorite oxidation was not associated with measurable TBARS production. No significant differences were noted between control and diabetic subjects or between untreated and supplemented diabetic subjects. However, the loss of free amine groups was lower in supplemented diabetic subjects (41%) than in untreated diabetic subjects (51%).

LDL Uptake by Macrophages (Fig. 3)
The uptake of LDL, which were "minimally modified" [38] by storage at 4°C for six months, was compared for control, untreated and antioxidant supplemented diabetic subjects. Eleven controls and eleven paired diabetic subjects were studied. Minimally modified LDL were labeled with DiI and incubated with mouse peritoneal macrophages for two hours. After incubation, DiI was extracted and measured spectroflurometrically. Under these conditions, uptake of LDL by diabetic subjects was consistently, but not significantly, higher than that of controls. Antioxidant vitamin supplementation was associated with a tendency to reduce uptake of LDL by macrophages (p=0.07).



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  		Fig. 3. Uptake by mouse peritoneal macrophages of minimally-modified LDL from control subjects ({blacksquare}) and diabetic subjects before (•) and after ({circ}) antioxidant supplementation for 12 weeks. LDL, 10 to 50 µg/mL, were incubated for two hours with cells; DiI was extracted, measured by spectrofluorometry and reported as µg LDL protein/mg cell protein.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The oxidation of LDL is a very complex process [10,11,22]. The diabetes state alters LDL size and composition [8,9]. To characterize the effects of antioxidant supplementation in the diabetic state, we utilized four oxidation techniques. The 30°C Cu oxidation was utilized because CD formation could be monitored continuously and visually through the lag and propagation phases [32]. The 37°C Cu oxidation was used because preliminary studies suggested this approach might show the greatest differences between control and diabetic subjects. The AAPH technique was used as a distinctly different oxidation probe since this compound generates free radicals in solution, rather than facilitating oxidation as with Cu. Finally, hypochlorite is a selective protein oxidant that spares lipids. As outcome measures, we assessed conjugated dienes [32]—an early lipid oxidation product—and TBARS—a late product. We assessed protein damage by estimating loss of free amine groups with TNBS [36].

In this study the oxidation kinetics of LDL from diabetic men with Cu at 37°C differed significantly from those of LDL from non-diabetic men. However, supplementation of diabetic subjects with antioxidant vitamins overcame these abnormalities and significantly decreased LDL susceptibility to oxidation in most parameters assessed. For Cu oxidation, at both 30°C and 37°C, all measured parameters were decreased. With AAPH oxidation, the loss of free amine groups, as measured with the TNBS method, decreased significantly with antioxidant vitamin supplementation.

Of additional interest, our study documented that antioxidant supplements decreased loss of free amine groups, as measured by the TNBS method, during in vitro oxidation of LDL. This protection was observed with three oxidation methods (Cu at 30°C and at 37°C and AAPH), and the tendency was seen with hypochlorite oxidation. These specific protective properties of antioxidant vitamin supplementation have not been well documented previously.

The response to AAPH oxidation was distinct from the response to Cu oxidation. Whereas the lag phase for diabetic subjects with Cu oxidation at 37°C was about 10% shorter, the lag phase for diabetic subjects with AAPH was 17% longer than for controls. Furthermore, antioxidant vitamin supplementation was associated with a further prolongation of the lag phase. Decreased fluidity of the LDL particle from diabetic subjects could contribute to these observations [40].

Since the original observations of Esterbauer and colleagues [18], several investigators [19,41] have noted that {alpha}-tocopherol supplementation significantly prolongs the lag phase during in vitro oxidation of LDL from nondiabetic subjects. Dieber-Rotheneder and colleagues [18] provided two healthy subjects with 800 IU/d {alpha}-tocopherol supplementation for 21 days and noted that the lag time was approximately 146% of values observed during the control period. Princen and colleagues [41] provided healthy, smoking, nondiabetic subjects with 1000 IU {alpha}-tocopherol/d for seven days and noted that the lag time was 141% of values for control subjects. Jialal and colleagues [19] provided 800 IU/d {alpha}-tocopherol for eight weeks to healthy nondiabetic subjects and noted that the lag time was 160% of control values. The prolongation of lag phase noted in our study (212% of control values) is substantially longer than reported by previous investigators when {alpha}-tocopherol supplements without other antioxidant vitamins were provided for nondiabetic subjects.

Two previous groups [20,21] document that {alpha}-tocopherol supplementation significantly decreased in vitro oxidation of LDL for diabetic subjects. Reaven and colleagues [20] provided 1600 IU of {alpha}-tocopherol daily for ten weeks, Fuller and colleagues [21] provided 1200 IU of {alpha}-tocopherol daily for eight weeks, and we provided 800 IU of {alpha}-tocopherol for twelve weeks. Different oxidation techniques were used by these investigators and by us so results are not easily compared. Nevertheless, it is worth noting that the lag times with Cu oxidation, compared to baseline, were 161% at 30°C [20] and 141% at 37°C [21] compared to 233% at 30°C (current study) and 211% at 37°C (current study). The greater increase in lag times in our study may be related to the combination of antioxidant supplements used or to different in vitro techniques.

This study using {alpha}-tocopherol, ascorbate and ß-carotene did not address the specific effects of each specific supplement. Investigators in two recent studies [42,43] used a combination of antioxidant vitamins similar to the combination we used; these investigators, however, studied non-diabetic subjects. Jialal and Grundy [42] used capsules (provided by Roche Vitamins, Inc.), which were similar in content and total dose to those we used, and noted a fourfold increase in serum {alpha}-tocopherol concentrations and a fifteenfold increase in serum ß-carotene concentrations after three months. Despite higher serum and LDL {alpha}-tocopherol and ß-carotene concentrations, the lag phase using the conjugated diene curve was 180% of baseline values [42], compared to the 212% of baseline values that we observed. Likewise, Abbey and colleagues [43] used a daily combination of {alpha}-tocopherol, 200 IU, ß-carotene, 18 mg, and ascorbate, 900 mg, for nondiabetic subjects. With this combination, serum {alpha}-tocopherol values increased by 55% and ß-carotene values increased fivefold. However, the lag time increased to only 129% of baseline after three months. Since these experimental protocols differed, it is difficult to make direct comparisons. However, our studies suggest that antioxidant supplementation in diabetic subjects may have a greater effect in protection of LDL from oxidation than previously reported for non-diabetic subjects.

Recent studies suggest that {alpha}-tocopherol contributes about 80% of the antioxidant capacity to the LDL particle [22]. ß-carotene seems to play only a small role [22]. However, since supplementation with ß-carotene was accompanied by a tenfold increase in LDL content of ß-carotene and only a twofold increase in {alpha}-tocopherol content, ß-carotene could have made an important contribution to the favorable changes observed. Ascorbate, being water soluble, is not carried in the LDL particle, but could contribute to the protection of LDL by preserving or recycling {alpha}-tocopherol [23].

In this study antioxidant supplementation had no measurable effects on glycemic control, glycohemoglobin concentrations or serum lipid concentrations. One previous study [40] suggested that {alpha}-tocopherol administration is associated with decreased glycosylation of hemoglobin and some serum proteins. Our clinical laboratory measurements of glycohemoglobin concentrations may not have had the sensitivity to detect small changes in glycohemoglobin concentrations. While some investigators [45] have reported decreased serum lipid concentrations after {alpha}-tocopherol administration, others [20,21,42] have not observed significant changes.

Although somewhat controversial, there is growing evidence supporting the hypothesis that oxidation of LDL has a central role in coronary atherosclerosis [10,11]. If oxidation of LDL in the subendothelium of coronary arteries contributes to foam cell formation, to an unstable atherosclerotic plaque, to plaque rupture and resultant thrombosis [11], intervention modalities to decrease LDL oxidation may offer distinct benefits to individuals at risk for atherosclerosis. This study and previous studies [18–21,41–43] strongly indicate that {alpha}-tocopherol supplementation decreases susceptibility of LDL to oxidation. Epidemiological studies also support the hypothesis that higher dietary intakes of {alpha}-tocopherol are associated with decreased risk for coronary heart disease [13,14,46–48]. This remains a controversial subject [49], but the limited information from clinical trials [16,17] also support the hypothesis that increased intake of {alpha}-tocopherol is associated with lower risk for coronary heart disease.

The potential benefits of {alpha}-tocopherol intake for diabetic subjects have attracted additional interest. Hunt and Wolf [7] forwarded the hypothesis that hyperglycemia increases nonspecific oxidation (termed glycoxidation) of proteins and Lyons [50] reviews this subject. Several studies [6,8,51] indicate that LDL from diabetic subjects are glycated to a greater extent than LDL from nondiabetic subjects. Further evidence indicates that increasing the glycation of LDL increases the susceptibility to oxidation [9,52]. Recently, Jain and colleagues [45] demonstrated that small amounts of supplemental {alpha}-tocopherol (100 IU/d) significantly decreased the serum lipid peroxide content of diabetic subjects. Gisinger and colleagues [53] reported that {alpha}-tocopherol supplementation decreases thromboxane A2 production by platelets and, thus, should decrease susceptibility to thrombosis in the diabetic individual. Thus, {alpha}-tocopherol supplementation has a number of potential benefits for the diabetic subject.

While much of the protective effect of {alpha}-tocopherol appears to be related to its transport in LDL, other effects may be important. LDL in postprandial state appears to be more susceptible to oxidation than fasting LDL; our recent work indicates that LDL from Type 2 diabetic subjects has an even greater susceptibility to oxidation in the postprandial state than LDL from control subjects (Diwadkar V and Anderson JW unpublished observations). Part of the benefits of {alpha}-tocopherol administration may relate to protection of postprandial lipoproteins from oxidation in the subendothelial space. Another important factor in LDL oxidation relates to ambient HDL concentrations. HDL carry important antioxidant enzymes, paroxanase and platelet activating factor acetylhydrolase, and also serve to protect LDL from oxidation in other ways. HDL also appear to exchange undamaged phospholipids for oxidized phospholipids in LDL. Transport of {alpha}-tocopherol in HDL may enhance and preserve these protective antioxidant effects of HDL [54]. Our recent research [54] indicates that HDL2 from diabetic subjects are less protective against in vitro oxidation of LDL than are HDL from non-diabetic control subjects. {alpha}-tocopherol supplementation may have special benefits in preserving HDL antioxidant function in diabetic subjects.

In conclusion, in vitro LDL oxidation kinetic studies showed significant differences between LDL from diabetic compared to control subjects with Cu oxidation at 37°C. Supplementation of diabetic subjects with ß-carotene, ascorbate and {alpha}-tocopherol over a 12-week period was accompanied by significant changes in most measured parameters. After supplementation, susceptibility of LDL to in vitro oxidation was significantly decreased as indicated by an increased lag phase and decreased production of conjugated dienes and TBARS during Cu oxidation. Loss of free amine groups, as measured with the TNBS method, was decreased with all four oxidation methods suggesting that antioxidant vitamin supplementation decreases susceptibility of apo-B100 to oxidation, aggregation and fragmentation.


   ACKNOWLEDGMENTS
 
This study was supported, in part, by a grant from Roche Vitamins, Inc., (Nutley, NJ) and by the HCF Nutrition Foundation. We appreciate the technical assistance of Susan Bridges.

Received February 1, 1999. Accepted June 1, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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