© 1997 American Heart Association, Inc.
Articles |
Vitamin E/Lipid Peroxide Ratio and Susceptibility of LDL to Oxidative Modification in Non–Insulin-Dependent Diabetes Mellitus
From the First Department of Internal Medicine, National Defense Medical College, Tokorozawa, Saitama, Japan.
Correspondence to Hiroshi Yoshida, MD. From June 1, 1996, through April 30, 1998: c/o Daniel Steinberg, MD, PhD, Room 1080, Department 0682, Basic Science Bldg, Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0682; after May 1, 1998: First Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359, Japan.
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Abstract The presence of conventional risk factors cannot sufficiently account for the excess risk of atherosclerosis in patients with non–insulin-dependent diabetes mellitus (NIDDM). Oxidative modification of LDL has been implicated in the pathogenesis of coronary atherosclerosis. Thirty-five patients with NIDDM, 20 nondiabetic, hypertriglyceridemic subjects (HTG-control), and 21 diabetic, normotriglyceridemic subjects (NTG-control) were enrolled in this study. Oxidative susceptibility of LDL was determined by monitoring formation of conjugated dienes. Mean lag time of LDL oxidation and vitamin E/lipid peroxide of LDL was lower in patients with NIDDM (43.2±3.9 minutes and 1.6±1.3) than in HTG-control (48.8±3.2 minutes and 2.3±1.2, respectively) and NTG-control subjects (54.2±6.1 minutes and 3.0±1.8, respectively). Mean LDL particle size in patients with NIDDM and HTG-control subjects (24.4±0.9 and 24.7±0.7 nm, respectively) was smaller than in NTG-control subjects (25.9±1.0 nm). Multiple stepwise regression analyses ascertained that the vitamin E/lipid peroxide of LDL is a major determinant of LDL oxidation lag time. These results suggest that LDL in patients with NIDDM is more susceptible to oxidative modification primarily because of a reduced level of vitamin E/lipid peroxide of LDL. The enhanced susceptibility of LDL to oxidation may be a pivotal factor underlying the increased incidence of vascular disease in patients with NIDDM.
Key Words: oxidative susceptibility • LDL • lipid peroxide • vitamin E • non–insulin-dependent diabetes mellitus
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Introduction |
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Diabetes mellitus is associated with a markedly increased risk of atherosclerotic cardiovascular disease. Multiple cardiovascular risk factors, eg, central obesity, insulin resistance, hypertension, a positive family history of premature atherosclerotic disease, and dyslipidemia, coexist in subjects with NIDDM. The dyslipidemia in NIDDM is characterized by hypertriglyceridemia, low levels of HDL cholesterol, and the presence of small, dense LDL particles, which is regarded as the atherogenic lipoprotein phenotype that is also associated with an increased risk of cardiovascular disease in nondiabetic subjects.1 2 3 Subjects with IDDM also have an increased risk of cardiovascular disease despite the absence of central obesity, insulin resistance syndrome, and small, dense LDL particles and the presence of normal or high levels of HDL cholesterol. Tsai et al4 showed that lag time of copper-induced LDL oxidation is decreased in patients with IDDM compared with control subjects, and the increased susceptibility of LDL to oxidation is consistent with a role for lipoprotein oxidation in the pathogenesis of atherosclerosis in IDDM. Rabini et al5 reported increased LDL oxidizability in patients with NIDDM when phenylhydrazine was used as an inducer for in vitro oxidation and levels of thiobarbituric acid–reactive substances of LDL to evaluate LDL oxidizability. However, the mechanism by which LDL oxidizability increases in NIDDM is poorly understood.
Oxidized LDL reportedly plays an important role in atherogenesis.6 Oxidative modification of LDL is a prerequisite for the macrophage uptake and cellular accumulation of cholesterol that leads to the formation of atherosclerotic plaque and yields many modified molecules with diverse effects, such as chemotactic factors for monocytes and T lymphocytes and endothelial cell adhesion molecules specific for these cell types.6 7 The small LDL particles that are predominant in patients with NIDDM are more susceptible to oxidative modification than large LDL particles.8 9 Therefore, it is likely that the LDL in NIDDM is prone to oxidative modification. Regnström et al10 reported that susceptibility to LDL oxidation is associated with severity of coronary atherosclerosis. The in vitro oxidation may reflect in vivo oxidation, since the resistance of LDL toward in vitro oxidation has been found to be correlated with the extent of coronary atherosclerosis. Because oxidized LDL may contribute to the atherogenic process in many ways, susceptibility to oxidation may, at least in part, determine the atherogenicity of LDL.
Diabetes mellitus is a complex metabolic disease that has been strongly linked to a variety of atherosclerotic complications. Because oxidative modification of LDL has been implicated as a major factor in the pathogenesis of coronary atherosclerosis, we compared the oxidative susceptibility of LDL in patients with NIDDM with that in healthy control subjects (NTG-control subjects) and nondiabetic subjects with hypertriglyceridemia (HTG-control subjects) in an attempt to clarify the mechanisms underlying premature atherogenesis in NIDDM.
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Methods |
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Subjects
A total of 76 nonsmoking men and postmenopausal women, aged 28 to 70 years (48.2±15.1 years), served as subjects in this study. Premenopausal women were not included since estrogen affects plasma lipids, LDL particle size, and oxidative susceptibility of LDL.11 12 None were hypertensive, none had renal or liver dysfunction, and none were taking any supplemental vitamins or hypolipidemic drugs. The prevalence of small, dense LDL and the subclass pattern B has been shown to be twice as high in men with NIDDM as in normolipidemic, nondiabetic men.2 Because the plasma TG level of NIDDM subjects in the Feingold et al2 study was 123±12.1 mg/dL, we decided to exclude subjects with fasting plasma TG levels >300 mg/dL. At those high levels, chylomicron, chylomicron remnant, genetic abnormalities in lipoprotein lipase, and inconsistent diet could affect LDL characteristics and LDL oxidizability.
Of the 76 subjects, 35 had NIDDM. Each had a history of two fasting blood glucose values >140 mg/dL and an HbA1c level >6%; 21 were being treated orally with a hypoglycemic agent (glibenclamide, 1.25 to 5 mg/d) and did not receive insulin therapy during the course of the study. Glibenclamide has not been reported to affect LDL oxidizability. The duration of diabetes in the subjects was 2 to 11 years (mean, 7.6 years). None of the study subjects had a clinical history of coronary heart disease or cerebrovascular disease. Simple retinopathy was present in 4 patients, but remarkable diabetic retinopathy (proliferative type) was not present. Serum creatinine levels were <1.5 mg/dL, and none of the study subjects had proteinuria; therefore, subjects with remarkable diabetic nephropathy were excluded.
The remaining 41 subjects were used for comparison purposes. The
HTG-control group consisted of 20 nondiabetic,
hypertriglyceridemic subjects of similar
age and body mass to the NIDDM subjects; this group had a fasting
plasma TG level of 
150 mg/dL (Table 1
) . The
NTG-control group consisted of 21 healthy, normolipidemic subjects of
similar age and body mass to the NIDDM subjects; this group had fasting
plasma cholesterol and TG levels of 
220 and 150 mg/dL,
respectively (Table 1). The gender ratios in the three groups were
approximately the same (Table 1).
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Blood Sampling and Preparation of LDL
The height, body weight, and blood pressure of each subject were
recorded. Fasting (12 hours overnight) blood samples were collected
in vacuum containers containing EDTA (1 mg/mL) and placed in an ice
bath. Plasma was immediately separated by
centrifugation (1500g for 20 minutes at
4°C) and subsequently stored at -80°C until preparation of LDL.
Neither sucrose nor glycerol was used in storing the plasma at
-80°C. LDL was isolated from the plasma by sequential
ultracentrifugation between densities of 1.019 and
1.063 g/mL, following the method of Havel et al.13 Storage
of plasma at -80°C for 2 months before isolation of LDL had no
detectable effect on in vitro LDL oxidation with copper. The lag time
and propagation rate of LDL prepared from fresh plasma (n=6) were
48.4±0.6 minutes and 9.2±0.9 nmol
diene·min-1·mg-1 protein,
respectively, and 48.3±0.9 minutes and 8.9±1.3 nmol
diene·min-1·mg-1 protein,
respectively, on LDL prepared from the same plasma that had been stored
at -80°C for 2 months; the differences were not significant.
Determination of Oxidative Susceptibility of LDL
Each LDL sample was dialyzed against PBS, pH 7.4, at 4°C for
24 hours in the dark to remove the EDTA. Oxidative susceptibility of
LDL was determined immediately after the dialyzations with EDTA-free
PBS by continuously monitoring the production of conjugated
diene according to the method of Esterbauer et al.14 LDL,
adjusted to 50 µg protein/mL with PBS, was incubated with 2 µmol/L
CuSO4 in PBS (final volume, 2 mL) at 37°C. Conjugated
diene formation during LDL oxidation was monitored by changes in
wavelength absorbance at 234 nm in a spectrophotometer (Shimadzu 160A,
Shimadzu, Tokyo, Japan) equipped with a six-position automatic changer.
The changes in absorbance were recorded every 10 minutes for 4
hours after initiating oxidation with copper. Lag time and propagation
rate were determined as previously described.14 The lag
time (in minutes) of LDL oxidation was defined as the intercept of the
tangent of the slope in the absorbance curve during the propagation
phase. The propagation rate was calculated from the slope of the
tangent, using a molar extinction coefficient for conjugated diene of
e234=29
500·M-1·cm-1 and was expressed as
nanomoles of diene formed per minute per milligram of protein.
Previous studies showed that EDTA-treated plasma stored at -80°C is
stable for several weeks and that freezing plasma at -80°C before
isolation of LDL has no significant effect on lag time or propagation
rate during incubation of LDL with copper.15 16
Evaluation of LDL Particle Size
LDL subfractions were separated at 10°C by 2% to 16%
nondenaturing polyacrylamide-gradient gel electrophoresis (PAA
2-16, Pharmacia, Uppsala, Sweden) as previously
described.17 18 The gels were prerun in a buffer
containing 90 mmol/L Tris plus 80 mmol/L boric acid, pH 8.3,
supplemented with 3 mmol/L EDTA at 125 V for 20 minutes with a GE-2/4
electrophoretic chamber (Pharmacia). Samples were made dense with a
solution consisting of 40% sucrose and 0.02% bromophenol blue, and 5
to 10 µL was applied to each line on the gels. Electrophoresis was
initiated by applying voltage to the chamber in the following sequence:
15 V for 15 minutes, 70 V for 20 minutes, and 125 V for 24 hours. For
fixation before protein staining, gels were exposed to 10%
sulfosalicylic acid for 1 hour immediately after electrophoresis. The
gels were stained in 50% methanol plus 10% acetic acid containing
Coomassie brilliant blue R-250. After destaining in 20% methanol plus
9% acetic acid, gels were scanned at 596 nm on a laser densitometer
(LKB Ultrascan XL). The calibration curve determined from the
high-molecular-weight standards was applied to each peak to estimate
the LDL particle diameter. LDL migration distances
(Rf) were measured relative to apoferritin. LDL
diameters were estimated from the migration distances of latex beads
(38 nm), thyroglobulin (17 nm), and apoferritin (12.2 nm). The
estimated diameter for the major peak in each scan was termed the peak
particle diameter. A quadratic equation proposed by Williams et
al,18 LDL diameter=exponential (3.816-1.554
Rf+0.241 Rf2), was
used to determine the LDL particle diameter (nm).
Measurements of Plasma Lipids, Apolipoproteins, and LDL
Composition
Plasma or LDL total cholesterol, TG, free
cholesterol, and phospholipid concentrations were measured
by an enzymatic method using commercially available enzymatic agents
(Hoffmann-La Roche Ltd, Basel, Switzerland). The cholesteryl ester
concentration was calculated as 1.67x(total
cholesterol-free cholesterol).19
HDL cholesterol in whole plasma was measured after
precipitation of apo B–containing lipoproteins with dextran sulfate
and magnesium chloride. Plasma apo A-I, A-II, B, C-II, C-III, and E
concentrations and LDL apo B concentration (coefficient of variation,
4.5%) were measured using the immunoturbidimetry method (Daiichi Pure
Chemicals Co Ltd, Tokyo, Japan). LDL protein concentrations were
determined by the method of Lowry et al20 using bovine
serum albumin as a standard. The LPO content of LDL was
measured using a commercially available agent (Determiner LPO, Kyowa
Medics, Tokyo, Japan), which is based on a calorimetric assay using the
reaction of a leucomethylene blue derivative with lipid hydroperoxides
in the presence of heme compounds.21 The seeding LPO in
LDL was tested before in vitro oxidation.
The vitamin E (
-tocopherol) content of LDL was measured
by high-performance liquid chromatography using
a method described by Bieri et al.22 LDL was precipitated
using ethanol, and vitamin E was subsequently extracted with hexane.
The hexane phase was evaporated under N2 gas, and the
residue was dissolved in ethanol. Vitamin E was separated by
reverse-phase high-performance liquid
chromatography using a C18 column (25x0.46
cm, 5-µm particle size; TSK gel ODS-80Ts) eluted with
ethanol/distilled water (92:8, v/v) at 1.0 mL/min as the mobile phase
and monitored at 295 nm in an ultraviolet detector (UV-8000, Tosoh,
Tokyo, Japan).
General Biochemical Analyses
Hepatic enzymes were measured using available kits
(Boehringer Mannheim, Tokyo, Japan). Glucose and
creatinine were measured enzymatically, also using
available kits (Determiner, Kyowa Medics). Serum insulin levels were
measured by an immunoradiometric assay method (Dainabot Co Ltd, Tokyo,
Japan). HbA1c levels were measured by high-performance liquid
chromatography using a TSK gel Glyco HS column
(Tosoh).
Statistics
Data are presented as the mean±SD. Differences among
the three groups (NIDDM, HTG-control, and NTG-control subjects) were
examined by one-way analysis of variance. Significant
differences (P<.05) between two groups were established
using Scheffe's test for multiple comparisons. Associations between
different parameters were determined by Pearson
product-moment correlation coefficients. Multiple stepwise
regression analysis was performed to evaluate the relative
impact of LDL character parameters and other
parameters of glucose metabolism on lag time of
LDL oxidation.
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Results |
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General Characteristics, Lipids, and Lipoproteins
Clinical characteristics of the three groups of subjects used in this study are given in Table 1
. LDL cholesterol values
were estimated according to the Friedewald equation.23 The
three groups were comparable with respect to age, gender ratio, and
body mass index. Subjects with NIDDM had significantly higher TG,
fasting blood glucose, and HbA1c levels and significantly lower HDL
cholesterol and apo A-I concentrations than NTG-control
subjects. NIDDM subjects also had significantly higher fasting blood
glucose and HbA1c levels than HTG-control subjects. However, fasting
serum insulin levels did not differ among the groups. HTG-control subjects had significantly higher apo A-II and E levels than NTG-control and NIDDM subjects. HTG-control subjects also had significantly higher TG and apo B levels than NTG-control subjects. HDL-cholesterol and apo A-1 levels in HTG-control subjects appeared to be lower than those in NTG-control subjects, but the differences were not significant.
LDL Chemical Composition and LDL Particle Size
Overall comparisons in LDL chemical composition and LDL particle
size among the three groups of subjects are presented in Table 2. Levels of vitamin E, LPO, and each of the lipids are
expressed as the ratio to apo B. Concentrations of total
cholesterol, free cholesterol, cholesteryl
ester, TG, phospholipid, and vitamin E/apo B were not significantly
different among the groups. However, subjects with NIDDM had about
double the LPO/apo B levels of NTG-control subjects, and this
difference was significant. In addition, subjects with NIDDM had
significantly lower vitamin E/LPO levels than HTG-control and
NTG-control subjects, and vitamin E/LPO levels in HTG-control subjects
were significantly lower than those in NTG-control subjects.
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LDL peak particle diameter in subjects with NIDDM and HTG-control subjects was significantly smaller than that in NTG-control subjects; the difference between NIDDM and HTG-control subjects was not significant.
Oxidative Susceptibility of LDL
Alterations in LDL oxidation parameters (lag time,
propagation rate) in vitro in each group of subjects are shown in Fig 1. The lag time of LDL oxidation in subjects with NIDDM
was highly significantly shorter than that observed in NTG-control
subjects (43.2±3.9 versus 54.2±6.1 minutes, P<.001). The
mean lag time of LDL oxidation in HTG-control subjects (48.8±3.2
minutes) was not significantly different from that in either of the
other two groups and appeared to be shorter than that in NTG-control
subjects, but not significantly (P=.06). Propagation rates
of LDL oxidation among the three groups of subjects were not
significantly different from each other. Maximum levels of conjugated
dienes attained during the propagation phase were 427±39, 433±46, and
419±40 nmol/mg LDL protein in NIDDM, HTG-control, and NTG-control
subjects, respectively. The maximum oxidation levels did not differ
significantly among the three groups.
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In the present study, 21 of the 35 subjects with NIDDM were taking glibenclamide (1.25 to 5 mg/d). It was determined that glibenclamide had no major effect on in vitro LDL oxidation. The addition of glibenclamide to buffer solutions (n=6) did not seriously affect the susceptibility of LDL to copper-induced oxidation; lag times of LDL oxidation (control [no addition], 49.4±0.4 minutes; 0.1 µmol/L glibenclamide [equivalent to clinical doses], 48.8±0.7 minutes; and 1.0 µmol/L glibenclamide [equivalent to clinical doses], 50.1±0.6 minutes) were not significantly different among groups. Furthermore, LDL isolated from plasma preincubated for 3 hours at 37°C (n=6) with glibenclamide at clinical dose concentrations (1.0 µmol/L) was not more susceptible to oxidative modification (lag time, 38.5±1.3 minutes) than LDL from plasma preincubated without glibenclamide (lag time, 39.1±0.5 minutes); the difference was not significant.
Comparison Between LDL Oxidizability in NIDDM Subjects With Normal
and High TG Levels
We examined LDL oxidizability in subjects with NIDDM in further
detail by separating the subjects into two groups, those with normal TG
levels (<150 mg/dL, n=12) and those with higher TG levels (>150
mg/dL, n=23). Lag time and propagation rate of LDL oxidation, LDL peak
particle diameter, LDL vitamin E/apo B, LDL LPO/apo B, and LDL vitamin
E/LPO were not significantly different between the two groups (data not
shown).
Determinants of Oxidative Susceptibility of LDL
LDL in NIDDM is more susceptible to oxidation in vitro, as shown
in Fig 1, and vitamin E/LPO ratios in subjects with NIDDM were the
lowest among the three groups of subjects (Table 2). To clarify the
determinants of oxidative susceptibility of LDL, we examined the
coefficients of correlation between lag time of LDL oxidation and
various parameters in subjects with NIDDM; these
coefficients are presented in Table 3. Lag time
of LDL oxidation was significantly correlated with LDL vitamin E/apo B
(r=.467, P<.01), LDL vitamin E/LPO
(r=.595, P<.001) (Fig 2) , and
LDL peak particle diameter (r=.403, P<.05) and
negatively correlated with LDL LPO/apo B (r=-.529,
P<.01).
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Correlation coefficients between lag time of LDL oxidation and various
parameters in HTG-control and NTG-control subjects were
also calculated. Lag time of LDL oxidation was significantly correlated
with LDL vitamin E/LPO in HTG-control and NTG-control subjects
(r=.726, P<.01 and r=.479,
P<.05, respectively) (Fig 2). Lag time of LDL oxidation was
also significantly correlated with HDL cholesterol and apo
A-I in NTG-control subjects but not in NIDDM and HTG-control subjects.
No other correlations were significant.
Multivariate stepwise regression analyses using
lag time of LDL oxidation as a dependent factor were carried out to
discover the determinants of oxidative susceptibility of LDL in
patients with NIDDM and to investigate the mechanisms of enhanced
susceptibility of LDL to oxidation in NIDDM patients. We incorporated
data on LDL chemical composition, LDL peak particle diameter, fasting
insulin, fasting glucose, HbA1c, HDL cholesterol, and apo
A-I into the multivariate analyses as
independent variables of lag time of LDL oxidation, taking the
results of simple correlations between lag time of LDL oxidation and
different parameters into account. The
multivariate analyses were carried out
separately in NIDDM, HTG-control, and NTG-control subjects; results are
presented in Tables 4 and 5. LDL
vitamin E/LPO was the strongest determinant of lag time of LDL
oxidation in NIDDM (Table 4), HTG-control, and NTG-control subjects
(Table 5), although LDL peak particle diameter was independently
associated with lag time of LDL oxidation in NIDDM and NTG-control
subjects. In subjects with NIDDM, LDL LPO/apo B was also independently
associated with lag time of LDL oxidation.
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In all subjects, glucose was significantly correlated with lag time of LDL oxidation (r=-.38, P<.05), but this significant correlation did not persist in multiple stepwise regression analyses that included LDL vitamin E/LPO and LDL peak particle diameter as independent factors in the same manner as described above. LDL vitamin E/LPO was significantly correlated with the lag time of LDL oxidation in all subjects combined (r=.743, P<.001).
The formation of LPO of LDL might very well be dependent on or influenced by LDL vitamin E content. However, there were no significant correlations between LDL vitamin E content and LDL LPO in NIDDM, NTG-control, HTG-control, and all subjects combined (data not shown).
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Discussion |
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It is well established that diabetes is one of the major risk factors for atherosclerosis, and diabetic patients have a two- to fivefold higher risk of coronary heart disease than nondiabetic individuals.24 25 26 The common occurrence of risk factors in diabetic patients, such as hypertension, obesity, and hypercholesterolemia, in addition to age and smoking, does not completely explain the increased level of mortality and morbidity from coronary heart disease in diabetic patients.
With regard to plasma lipids and lipoproteins, it is also clear that high plasma TG levels, usually found in patients with diabetes, have consistently been shown to be a risk factor for coronary heart disease in diabetic individuals.24 This is in contrast to the controversy over the role of hypertriglyceridemia as a risk factor for coronary heart disease in nondiabetic populations. Many studies have demonstrated strong correlations between plasma TG levels and increasing density and decreasing size of the predominant LDL species. Furthermore, the plasma lipoprotein profile accompanying a predominance of small, dense LDL particles, which has been designated LDL subclass pattern B, is associated with an increased risk of coronary heart disease.3 27 28 Diabetes is marked by characteristic alterations in lipoprotein levels, including an elevation of TG and VLDL and a decreased HDL concentration. In addition, it appears that small, dense LDL subclass patterns are more common in diabetic patients, as seen in the present study and in other studies.2 29 Barakat et al30 demonstrated a correlation of both insulin and TG concentrations with LDL particle size and reported that small LDL particles may revert to the normal state according to a decrease in the levels of plasma TG and insulin. NIDDM is characterized by peripheral insulin resistance, pancreas 13 cell failure, and increased hepatic glucose production. The relationship between degree of insulin resistance and fasting plasma insulin level has been reported previously. However, the correlation is considered to be higher in nondiabetic subjects than in subjects with NIDDM. In other words, we think it is possible that the defective ß-cell function, which could exist in NIDDM, is manifested in the fasting insulin level such that fasting insulin levels are not significantly higher in NIDDM subjects compared with nondiabetic control subjects in the present study. Previous reports demonstrated that small, dense LDL is more susceptible to oxidative modification.8 9 In humans, oxidative susceptibility of LDL in vitro has been reported to correlate significantly with the degree of coronary atherosclerosis.10 Therefore, it is important to evaluate LDL particle size and oxidative susceptibility of LDL in examining the risk of coronary heart disease in diabetic patients.
Results in the present study indicate an increased susceptibility of LDL to oxidative modification in vitro and the predominant presence of small LDL particles in subjects with NIDDM. Although 21 of the 35 NIDDM subjects in the present study were taking glibenclamide, we found no significant effect of this drug on in vitro LDL oxidation data.
Subjects in the HTG-control group were selected to serve as a specific control for patients with NIDDM, who have higher plasma TG levels and, as expected, smaller LDL particles than NTG-control subjects. Higher TG levels and smaller LDL particles both have major and minor effects on the increased LDL oxidizability in patients with NIDDM. As observed in subjects with NIDDM, HTG-control subjects had smaller LDL particles than NTG-control subjects, but the lag time of LDL oxidation did not significantly differ between HTG-control subjects and NTG-control subjects. Furthermore, the lag time of LDL oxidation was not significantly different between HTG-NIDDM and NTG-NIDDM subjects. Therefore, the effect of diabetes on decreasing lag time of LDL oxidation appears to be independent of higher plasma TG.
Data from the multivariate regression analyses
(Table 6) indicate that LDL vitamin E/LPO is a more important
determinant than LDL peak particle diameter not only in NIDDM subjects
but also in HTG-control and NTG-control subjects. We (as well as many
other investigators) used copper ions to initiate peroxidation of LDL
in vitro, thereby enhancing LPO breakdown. It is likely that copper
ions cannot cause peroxidation in completely peroxide-free LDL. The
seeding LPO in LDL could be generated by endogenous
peroxidation with 15-lipoxygenase or arise from LPO in
dietary fats if they avoid destruction during fat digestion,
absorption, and transport. Griesmacher et al31 reported
that serum levels of thiobarbituric acid–reactive substances were
significantly increased in patients with diabetes mellitus. In the
present study, LDL LPO/apo B levels were significantly increased in
subjects with NIDDM (Table 2). The seeding LPO in LDL also could be
generated during the LDL isolation process. Even if the generation of
lipid peroxide in LDL occurred during the LDL isolation process, there
are no major problems in comparing LDL oxidizability, because all LDL
was treated in the same way and under the same condition during the
course of this study, from plasma sampling to LDL isolation and the
oxidation experiment. Oxidation of LDL begins with the abstraction of
hydrogen from polyunsaturated fatty acids; LDL fatty acid composition
contributes to the process of LDL oxidation. LDL fatty acid composition
was not determined, but this study showed that maximum levels of
conjugated dienes during propagation phase did not differ among the
three groups. If NIDDM subjects had greater amounts of LDL
polyunsaturated fatty acids than the other two groups, the maximum
oxidation level in NIDDM subjects could be higher than that in the
other two groups. Therefore, LDL fatty acid composition might not
differ among the three groups, and LDL fatty acid composition may not
have affected the results obtained in the present study. Oxidant
stress may be increased in diabetes, in part because of generation of
oxygen free radicals during protein glycation and glucose
auto-oxidation.32 It is conceivable that the hyperglycemia
might result in LDL being seeded with small amounts of LPO in vivo,
thereby rendering LDL more susceptible to oxidation in vitro. However,
glucose did not correlate with the lag time of LDL oxidation in
subjects with NIDDM, and LDL LPO/apo B was a weaker determinant of
oxidative susceptibility of LDL than LDL vitamin E/LPO (Table 4).
Although in all subjects combined glucose correlated significantly with lag time of LDL oxidation, this correlation did not persist in a multiple stepwise regression analysis. We were unable to demonstrate the correlation between glucose and lag time of LDL oxidation separately in any of the groups, possibly because of clustering of glucose data within each group. In addition, LDL vitamin E/apo B did not differ among NIDDM, HTG-control, and NTG-control subjects. Epidemiological studies suggest that populations with high plasma levels of vitamin E have a lower risk of coronary heart disease, even after corrections have been made for other known coronary risk factors.33 34 35 36 Previous studies have shown that vitamin E is one of the most effective antioxidants in LDL, and oral supplementation with vitamin E increases resistance of LDL to oxidation in vitro.37 38 39 40 One of the mechanisms by which vitamin E may contribute to the reduction in the risk of coronary heart disease is through antioxidant protection of LDL. Although the decreased content of vitamin E in LDL is expected in subjects with NIDDM, the present data showed no significant differences in LDL vitamin E/apo B mean levels among NIDDM, HTG-control, and NTG-control subjects. Although other antioxidants, such as ß-carotene and ubiquinol, are present in much lower concentrations in LDL than vitamin E, ß-carotene or ubiquinol/apo B may need to be evaluated.37 41 42
In LDL oxidation experiments with copper ions, vitamin E is a protector against oxidation, and LPO may act as an initiator or supporter of oxidation in the sense that LPO is a source for further breakdown after the addition of copper, which is consistent with the concept that in vitro metal-catalyzed breakdown of seeding LPO initiates lipid peroxidation in LDL.43 Therefore, the vitamin E/LPO ratio is a very important factor in the examination of LDL oxidizability. We consider that the elevation in LDL vitamin E/LPO increases the resistance of LDL to copper ion-dependent oxidation. The present data show that LDL vitamin E/LPO is a major determinant of oxidative susceptibility of LDL in all subjects. LDL particles in HTG-control subjects are small and tend to be more susceptible to oxidation than those in NTG-control subjects, as seen in the present study and other studies.8 9 LDL vitamin E/LPO in HTG-control subjects tends to be lower and thereby results in increased oxidizability of LDL as compared with that in NTG-control subjects. In all cases, the mechanism by which LDL vitamin E/LPO decreases in NIDDM and HTG-control subjects remains unclear and needs further study.
A number of lipoprotein modifications in diabetes affect cell interactions. Glycosylation (glycation) of lipoproteins has been shown to occur in diabetes, and previous studies have shown that glycated LDL is more susceptible to oxidation.44 45 The present study indicates that HbAlc, which is regarded as an index of degree in protein glycation, is not an independent factor affecting LDL oxidizability in subjects with NIDDM. Although we did not directly evaluate the degree of LDL glycation, it seems that LDL vitamin E/LPO is a stronger determinant than HbAlc, which is a substitute for LDL glycation. However, it may be that the increased LPO/apo B of LDL and decreased vitamin E/LPO of LDL observed in NIDDM subjects are caused by LDL glycation and consequential products. Becauseboth protein glycation and lipoprotein oxidation involve free radicals, it has been suggested that antioxidant supplementation could benefit patients with NIDDM by inhibiting these processes. Reaven et al46 reported that supplementation of vitamin E in NIDDM leads to enrichment of LDL and LDL subfractions and reduced susceptibility of LDL to oxidation but that vitamin E supplementation did not reduce glycation of plasma proteins. The combination of many kinds of antioxidants on protein glycation and susceptibility of LDL to oxidation should be investigated in more detail.
Recently, it has become clear that HDL potentially can limit oxidative modification of LDL; this antioxidative effect of HDL is not explained by chain-breaking antioxidants present in HDL and is likely to involve an enzymatic mechanism.47 48 49 50 Several enzymes are present on HDL. For example, paraoxonase is in close physical association with HDL in human serum, and paraoxonase is believed to be anchored to HDL lipids by its hydrophobic N-terminal end and also to be bound to apo A-I.50 51 In the present study (and in other studies), HDL cholesterol and apo A-I levels in subjects with NIDDM were lower than those in NTG-control subjects. It may be that the decreased levels of HDL and apo A-I, regarded as a protector against LDL oxidation, augment the oxidative susceptibility of LDL in patients with NIDDM. However, HDL cholesterol and apo A-I were positively correlated with lag time of LDL oxidation only in NTG-control subjects. In addition, the multivariate analyses data show that HDL and apo A-I were not independent determinants of LDL oxidation in any subjects.
Fruebis et al52 reported that a probucol analogue increased lag time approximately threefold yet found no effect on atherosclerotic lesions in LDL receptordeficient rabbits. On the other hand, Sasahara et al53 found that probucol inhibited hypercholesterolemiainduced atherosclerosis in a nonhuman primate (cholesterol-fed macaque monkeys), and the extent of protection against atherosclerosis correlated positively with the increase in lag time of LDL oxidation. Mean lag time of LDL oxidation in the present study was 20.3% lower in NIDDM subjects (43.2±3.9 minutes) than in NTG-control subjects (54.2±6.1 minutes). Whether a difference of 20.3% in lag time is large enough to be relevant to the development of atherosclerosis in humans is controversial at the present time. However, Regnström et al10 reported that the significant association found between proneness to LDL oxidation in vitro and the severity of coronary atherosclerosis supports a role for lipid peroxidation and oxidative susceptibility of LDL in the development of atherosclerosis in humans. Therefore, although the decrease of lag time in NIDDM subjects may or may not be involved in promoting atherosclerosis, the reduction of oxidative stress toward LDL by antioxidative therapy may be useful clinically in preventing atherosclerosis in patients with NIDDM.
In conclusion, we found that oxidative susceptibility in vitro is increased in subjects with NIDDM compared with nondiabetic normotriglyceridemic or hypertriglyceridemic subjects, in part through a decrement in LDL vitamin E/LPO. This study demonstrated that the LDL vitamin E/LPO ratio is a pivotal factor in modulating LDL oxidizability in subjects and that the reduced LDL vitamin E/LPO ratio could cause the increased LDL oxidizability found in patients with NIDDM. Therefore, the decreased LDL oxidizability in NTG- and HTG-control subjects is conceived to be the result of higher LDL vitamin E/LPO ratios as compared with NIDDM subjects. The mechanism by which LDL vitamin E/LPO decreases in NIDDM remains to be discovered.
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Acknowledgments |
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This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Health and Welfare of Japan (T.I.). We thank Ms Emiko Miyajima (National Defense Medical College) and colleagues for helpful assistance and Dr M.W. Schein (Rockville, Md) for editorial assistance.
Received February 13, 1996; accepted October 8, 1996.
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