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*
Graduate Program in Nutrition,
Department of Dairy and Animal Science,
**
Department of Veterinary Science, The Pennsylvania State University, University Park, PA 16802,
Department of Community and Preventive Medicine, University of Rochester, Rochester, NY 14627 and
The Mary Imogene Bassett Research Institute, Cooperstown, NY 13326
2To whom correspondence should be addressed at S-136 Henderson Building, Nutrition Department, University Park, PA 16802. E-mail: pmk3{at}psu.edu
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • dietary total fat • dietary saturated fat • LDL oxidation • conjugated dienes • lipid peroxidation • humans
|
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The quantity of LDL trapped in the artery wall is
directly proportional to the concentration of circulating lipoproteins
(Schwenke and Carew 1989
). The trapped LDL are modified
by a superoxide-dependent process, and lipid oxidation is initiated
within the LDL particle (Witztum and Steinberg 1991
).
Convincing evidence suggests that oxidative modification of LDL plays
an important role in the pathophysiology of atherogenesis
(Steinberg 1997
). In recent years, numerous molecular
mechanisms have been proposed to explain the different oxidation
pathways that lead to modification of LDL (Steinberg 1997
).
One of the earliest steps in the generation of oxidatively modified LDL
is the peroxidation of its polyunsaturated fatty acids
(PUFA).3
The oxidative breakdown products of these fatty acids, such as
malondialdehyde and 4-hydroxynonenal, form covalent bonds with
apolipoprotein B (apo B) by specifically modifying lysine residues
(Palinski et al. 1989
, Steinbrecher 1987
). Macrophages internalize modified LDL at a faster rate
than they internalize native LDL via a specific scavenger
receptor-mediated pathway (Goldstein et al. 1979
).
In vitro studies have shown that acetyl-LDL receptors do not
recognize unmodified LDL (Parthasarathy et al. 1987
,
Sparrow et al. 1989
), nor is uptake via these receptors
downregulated by internal macrophage cholesterol content (Brown and Goldstein 1983
). Accumulation of lipids can lead to a
conversion of macrophages into lipid-laden foam cells, typical
constituents of fatty streaks and atherosclerotic plaques. Thus, in
addition to lowering the levels of circulating LDL, decreasing the
susceptibility of LDL to oxidative modification may also impede the
development of atherosclerosis.
One effective strategy for reducing atherosclerosis and coronary heart
disease (CHD) risk is to alter plasma lipid and lipoprotein profiles by
manipulating the type and amount of dietary fat. Recent studies have
reported that diets rich in monounsaturated fatty acids (MUFA) result
in LDL that are less readily oxidized than LDL isolated from subjects
who consume diets rich in PUFA (Berry et al. 1991
,
Bonanome et al. 1992
, Parthasarathy et al. 1990a
, Reaven et al. 1991
, Reaven et al. 1993
, Schwab et al. 1998a
). Dietary saturated
fatty acids (SFA) adversely affect plasma lipids, lipoproteins,
hemostatic factors (Mitropoulos et al. 1994
,
Tholstrup et al. 1994
, Yu et al. 1995
) as
well as susceptibility of LDL to oxidation (Mata et al. 1996
). However, a recent study reported that replacing SFA with
either MUFA or PUFA in diets that provide less total fat (i.e., 
30%
energy) did not appreciably affect LDL oxidative susceptibility as
measured only by lag time (Schwab et al. 1998b
). We are
unaware of any studies that have examined the effects of decreasing
dietary total fat and SFA, whereas holding MUFA and PUFA levels
constant, on LDL oxidative status. This is of relevance because current
dietary guidelines for cardiovascular disease recommend decreasing both
dietary SFA and total fat. Thus, the present study was conducted to
evaluate whether a step-wise reduction in total fat and SFA could
change the lipid composition of LDL, and as a result, change LDL
oxidative susceptibility without the potential confounding effects of
different levels of MUFA and PUFA in the experimental diets, because
the MUFA and PUFA levels were constant in the three test diets.
|
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Data presented herein are from subjects studied at The
Pennsylvania State University Research Center. Relevant methods used in
this study are described below. Subjects.
A total of 27 healthy, free-living subjects, ages 23–67 y (mean = 37 y) participated in the study. Of these, 16 were male (2 were African-American) and 11 were female [8 were premenopausal (1 was African-American) and 3 were post-menopausal]. Subjects’ baseline total cholesterol (TC) levels (5.56 ± 0.91 mmol/L) were between the 25th and 85th percentile for age, race and gender. Plasma triglycerides (TG, 1.14 ± 0.34 mmol/L) and HDL-cholesterol (HDL-C) (1.32 ± 0.28 mmol/L) were between the 10th and 90th percentile, respectively.
Study design.
DELTA employed a randomized (subject assignment and the order of diet
periods), double-blind, three-period crossover study design
(Ginsberg et al. 1998
). Each experimental diet period
was 8 wk, followed by a break of 4 to 6 wk during which the subjects
consumed their habitual diets. All meals and snacks were provided to
the subjects during the experimental diet periods. Subjects ate two
meals each weekday in the dining facility at the Research Center. Other
weekday meals, snacks and all weekend meals were packaged and eaten by
participants at a time and place of convenience. All food portions were
weighed or measured; fat-containing items were weighed to the ±0.1
g and all other items were weighed to the ±1.0 g. Diets were prepared
individually for each participant based upon energy needs so that body
weight was maintained during each experimental diet period (subjects
were weighed 5 d each week).
Blood samples were collected once per week from subjects that had fasted for 12 to 14 h during wk 5 and 7 in each diet period. All samples were processed immediately. The mean values of lipids, lipoproteins, fatty acids, vitamin E, and LDL oxidative susceptibility at wk 5 and 7 are reported.
Test diets.
Three experimental diets were fed: an average American diet (AAD) that
provided 34% of energy from fat and 15% from SFA, a Step-1 diet that
provided 29% of energy from fat and 9% from SFA, and a low saturated
fat (Low-Sat) diet that provided 25% of energy from fat and 6% from
SFA, respectively (Table 1
). In addition, all experimental diets provided 
13% and 7% of energy
from MUFA and PUFA, respectively, and 16% of energy from protein.
Dietary carbohydrate varied among the experimental diets and was
calculated to provide 50, 55 and 58% of energy in the AAD, Step-1 and
Low-Sat diets, respectively (Table 1)
. Five different energy levels
were prepared and matched to participants’ energy requirements (6276,
8368, 10,460, 12,552, 14,644 kJ, respectively). Unit foods (628 kJ
each) that were compositionally similar to the test diets were used as
energy adjusters for subjects who required additional energy to
maintain weight.
|
Blood for the lipid and lipoprotein analyses (10 mL) was collected into
silicone-gel-coated tubes for subsequent plasma preparation. Plasma was
obtained by centrifugation at 1500 x g for 30 min.
Aliquots (0.5 mL) were stored in cryovials at -80°C until the end of
the study when samples were analyzed. The analyses were done at the
Mary Imogene Bassett Research Institute in Cooperstown, New York. TC
and HDL-C were assayed by an enzymatic method using cholesterol
esterase and cholesterol oxidase (Roche Diagnostic Systems, Nutley,
NJ). For HDL-C detection, non-HDL lipoproteins were
precipitated by treatment with 50,000 MW Dextran sulfate (Sigma
Diagnostics, St. Louis, MO). TG were assayed by initial hydrolysis with
lipase followed by detection of glycerol (Sigma Diagnostics
Triglyceride Reagent, St. Louis, MO). Calibration of cholesterol and TG
was validated by participation in the CDC Lipid Standardization
Program. LDL-C levels were calculated using the Friedewald equation
(Friedewald et al. 1972
). Apo B and apolipoprotein
A-I (apo A-I) were determined by rate nephelometry on a Beckman
Array using Beckman reagents, calibrators and controls (Beckman
Instrument Company, Brea, CA).
Preparation of LDL for oxidation studies.
Blood for plasma and LDL preparation (30 mL each) was collected into
vacutainer tubes containing 0.1 mL of 0.5 mol/L EDTA. Plasma was
isolated by centrifugation at 600 x g for 10 min
immediately after blood draw. Butylated hydroxytoluene (BHT) was added
to all samples to a final concentration of 18 nmol/L. A portion of the
plasma was placed into four amber vials (0.5 mL each) and stored at
-80°C for subsequent fatty acid and 
-tocopherol analyses. The
remainder was immediately used for LDL isolation. LDL (d = 1.026
to 1.063 kg/L) were isolated by density gradient ultracentrifugation
(Kleinveld et al. 1992
, Redgrave et al. 1975
). Briefly, plasma (4 mL) was adjusted to a density of 1.21
kg/L with solid KBr. Then, a density of 1.063 kg/L (3 mL) and 1.019
kg/L (3 mL) solution adjusted with KBr and NaCl, and saline
(d = 1.006 kg/L) (2.5–3 mL) were layered on to the
plasma in a 14-mL tube. Duplicate plasma samples were centrifuged in a
Beckman SW40 swinging bucket rotor at 188,000 x g for
22 h at 15°C. The LDL fraction was collected and LDL protein was
measured. The rest of the LDL were dialyzed for 24 h at 4°C (in
the dark) against N2 purged 0.01 mol/L phosphate buffered
saline (PBS) containing 0.1 g/L of chloramphenicol. LDL samples from
all subjects in each period were dialyzed at the same time. The PBS (2
L) was changed three times.
Oxidation of LDL.
LDL oxidative susceptibility was assessed using two different methods:
measurement of conjugated diene (Abbey et al. 1993
,
Esterbauer et al. 1989a
) and lipid peroxide formation
(Jiang et al. 1992
). LDL oxidation analyses for each
feeding period were completed within 18 h after dialysis of LDL.
(i) Conjugated dienes: The kinetics of conjugated diene formation were
determined by continuously monitoring the change in absorbance at 234
nm according to the method of Esterbauer et al. (1989a)
.
Briefly, LDL (100 µg of LDL protein) were diluted to 1 mL with PBS.
Oxidation was initiated by adding CuCl2 (final
concentration 0.01 mmol/L). Absorbance at 234 nm was recorded at 3-min
intervals for 2.5 h at 37°C on a Beckman (Beckman
Instruments, Inc., Fullerton, CA) Model 50 UV spectrophotometer fitted
with a heater and a six-position automatic sample changer. The
maximum oxidation rate was determined by measuring the slope
(
A234/min) of the linear portion of the curve. The maximum amount of
conjugated dienes formed in LDL was determined using the difference
between the maximum absorbance during the decomposition phase and the
absorbance at the start of the lag phase. Quantities of conjugated
dienes formed were calculated using the molar absorptivity of 2.95
x 104 (mol/L)-1 cm-1. (ii)
Lipid peroxide assay: Lipid peroxides were measured during
Cu2+-induced oxidation of LDL. This assay is based on the
principle that peroxide-mediated oxidation of Fe2+ to
Fe3+ at low pH in the presence of the ferric-complexing
dye xylenol orange forms a Fe3+-xylenol orange complex that
absorbs at 560 nm (Jiang et al. 1992
). Briefly, 200 µg
of LDL protein were incubated with Cu2+ (10 µmol/L) for 3
and 6 h. The quantity of lipid peroxide formed was calculated
using the molar absorptivity of 4.52 x 104
(mol/L)-1 cm-1.
LDL fatty acid composition.
Fatty acid composition of LDL was determined by gas chromatography.
Lipids were extracted from LDL (400 µL, 400 µg protein) using
the method of Folch et al. (1957)
. The fatty acids in
the lipid extract were methylated using boron trifluoride/methanol
(Lepage and Roy 1988
, Morrison and Smith 1964
) and quantified using a Hewlett-Packard 5890 gas
chromatograph (Analytical Instrument Recycle, Inc., Golden, CO)
equipped with a Supelco SP-2330 capillary column (Supelco, Bellefonte,
PA) with helium as a carrier at a flow rate of 30 mL/min. Temperature
was set at 150°C for 8 min, then increased to 190°C at 2°C/min
and remained at 190°C for 20 min. Both injector and detector
temperatures were 250°C (Supelco Bulletin 855A, 1994). To quantify
LDL fatty acids, 70 µg of a C17:0 internal standard was added to each
sample before extraction.
Plasma and LDL vitamin E.
The concentration of -tocopherol in plasma and LDL was measured by
HPLC employing a Supelco LC-18 column (Supelco) and a UV detector (290
nm) (Lang et al. 1986
, Yang and Lee 1987
). 
-Tocopherol (80 µg) was added to each sample (0.4
mL plasma or 400 µg LDL protein) as an internal standard before
lipid extraction. Lipid extracted from plasma or LDL was dissolved in
0.3 mL methanol/alcohol (1:1) for HPLC analysis. A 100-µL sample was
injected onto the HPLC, and the column was eluted isocratically with
methanol/H2O (97.1:2.9) at a flow rate of 2 mL/min. The
-tocopherol concentration was calculated using the ratio of the peak
area of the compound over the peak area of the internal standard.
LDL lipids and proteins.
Cholesterol in isolated LDL was assayed by an enzymatic method using
cholesterol esterase and cholesterol oxidase (Sigma Diagnostics
Cholesterol Reagent). TG were assayed by initial hydrolysis with lipase
followed by detection of glycerol (Sigma Diagnostics Triglyceride
Reagent). LDL phospholipids (PL) were measured by an enzymatic
colorimetric method using phospholipase D, choline oxidase and
peroxidase (Wako Pure Chemical Industries, Ltd., Richmond, VA). LDL
protein was measured using reagent kits obtained from Sigma Chemical
Co. based on the Lowry method (Lowry et al. 1951
).
Statistics analysis.
SAS was used for statistical analyses (SAS Institute Inc., Cary, NC).
All data were expressed as means ± SD. Three-way
ANOVA was used to test the effects of diet, feeding period and gender.
Tukey’s multiple comparison test was used to determine the effects of
diet treatment. Pearson correlation analysis was used to test the
correlations between LDL oxidation potential and plasma total and
LDL-C and apo B levels. Regression analysis was used to test
effects of plasma LDL-C and apo B on the potential oxidation of LDL
and differences among the regression coefficients for each diet
treatment. The significance of all tests is at = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Subjects (n = 27) completed all three phases of the
study. All subjects maintained their body weight during the study.
Plasma TC was about 4% (P = 0.14) lower when subjects
consumed the Step-1 diet and 9.5% (P = 0.01) lower
when subjects consumed the Low-Sat diet compared with those when
subjects consumed the AAD (Table 2
). Plasma LDL-C was 8% (P = 0.16) lower when subjects
consumed the Step-1 diet and 11% (P = 0.03) lower when
subjects consumed the Low-Sat diet compared with those when
subjects consumed the AAD. Plasma HDL-C was 8% (P
= 0.11) lower when subjects consumed the Step-1 diet and 14%
(P = 0.02) lower when subjects consumed the Low-Sat
diet compared with those when subjects consumed the AAD. Plasma apo A-1
was 6% (P = 0.21) lower when subjects consumed the
Step-1 diet and 10% (P = 0.03) lower when subjects
consumed the Low-Sat diet compared with those when subjects
consumed the AAD. The reductions in TC, LDL-C and HDL-C and apo
A-1 were only significant when the Low-Sat diet was compared with
the AAD (P < 0.05). The decrease in apo B levels and
the increase in TG in response to the lower fat and saturated fat diets
were not significant (P = 0.47 for apo B and
P = 0.41 for TG, ANOVA), although the trends [e.g.,
+11.5% (P = 0.24) for TG after both the Step-1 and
Low-Sat diets compared with the AAD] were consistent with those
changes that were significant in the larger study (Ginsberg et al. 1998
). The ratios of TC to LDL-C and LDL-C to
HDL-C were not significantly different among three experimental
diet periods (P = 0.75 and 0.93, respectively, ANOVA)
(Table 2)
.
|
When subjects consumed the Low-Sat diet, their LDL contained less
18:2 and 16:0, and more 18:1 and total MUFA than those when subjects
consumed the AAD (P < 0.05) (Table 3
). The percentage of 18:1 and total MUFA was higher in LDL from subjects
when they consumed either the Step-1 or Low-Sat diets compared with
LDL isolated from subjects when they consumed the AAD (P
< 0.05). The percentage of PUFA was lower in LDL from subjects
when they consumed the Low-Sat diet than LDL isolated from subjects
when they consumed the AAD (P < 0.05). Total fatty
acids per mg of LDL protein did not differ among the three diet
periods. No gender and feeding period (seasonal) effects on LDL fatty
acid composition were found (data not shown).
|
The ratios of TC, PL and total lipid to protein in LDL from either the
Step-1 or Low-Sat diet periods were lower (P < 0.05) than those from the AAD period (Table 4
). No significant differences in the ratios of lipids-to-protein between
the Step-1 diet period and Low-Sat diet period were observed. The
ratio of TG-to-protein in LDL was not significantly different among the
three diet periods. Consumption of the Step-1 and Low-Sat diets
resulted in LDL that had significantly lower 18:2 to 18:1 and PUFA to
MUFA ratios than LDL from subjects when consuming the AAD (P
< 0.05). The 18:2 to 18:1 ratio was lower in LDL from subjects
after consumption of the Low-Sat diet vs. consumption of the Step-1
diet (P < 0.05). There were no significant differences
in plasma and LDL -tocopherol levels among the three diet periods.
There were also no feeding period (seasonal) or gender effects on LDL
lipids and -tocopherol (data not shown).
|
When subjects consumed the Step-1 diet and the Low-Sat diet, their
LDL had a lower oxidation rate and less conjugated diene formation than
did LDL from subjects when they consumed the AAD during
Cu2+-induced oxidation (P < 0.05)
(Table 5
). Lag time was not significantly different when comparisons were made
among LDL from subjects consuming the three test diets (P
= 0.72, ANOVA). The quantity of lipid peroxides produced in LDL
from subjects when they consumed the Low-Sat diet was significantly
lower than LDL from subjects when they consumed the AAD (P
< 0.05). The reduction in lipid peroxides produced in LDL after
consumption of the Step-1 diet was not significant compared with after
consumption of the AAD (P = 0.34). No feeding period
(seasonal) and gender effects were found (data not shown).
|
).Lipid peroxide formation was also positively correlated with plasma TC
(r = 0.50, P < 0.001), LDL-C
(r = 0.51, P < 0.001), and apo B
(r = 0.52, P < 0.001) when averaged
over all diets. Pearson correlation coefficients between plasma LDL and
conjugated diene (and lipid peroxide) formation in LDL and
P-values for each experimental diet are shown in Figure 1
. A
similar correlation was observed between plasma TC and conjugated diene
formation (P < 0.001) (data not shown). When subjects
switched from AAD to either Step-1 or Low-Sat diets, their plasma
LDL-C and LDL oxidation rate decreased in parallel (Fig. 2A and
B
).There was, however, no significant difference in LDL-C and
oxidation rate between the Step-1 and Low-Sat diet periods
(P = 0.41 and 0.53, respectively) (Fig. 2C
).
Similar results were found for the correlation between changes in
plasma apo B levels and changes in susceptibility to LDL oxidation
(Fig. 3A
).Collectively, these results demonstrate that increases in the quantity
of circulating LDL are associated with greater increase in LDL
oxidation even if individuals consume low-fat diets (Fig. 3A and 3B
).
|
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|
). Similar results were
observed with LDL oxidation at the same level of plasma LDL-C in
the three diet periods (Fig. 3B
). These results imply that
at the same level of plasma apo B (over the range of 0.8 to 2 g/L), LDL
isolated from subjects when they consumed either Step-1 or Low-Sat
diets was less susceptible to oxidation than LDL from subjects when
they consumed the AAD (P < 0.05). The change in LDL
oxidation over the range of LDL-C or apo B between Step-1 and
Low-Sat diets were not different (P = 0.81) (Fig. 3A and 3B)
. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, Hegsted et al. 1993
, Keys et al. 1965
, Mensink and Katan 1992
, Yu et al. 1995
). Public health advisories
from the American Heart Association, the National Cholesterol Education
Program and the American Diabetes Association have recommended that all
Americans over the age of 2 y reduce their intake of total fat and
SFA to less than 30% and 8 to <10% of energy, respectively. There
are many scientists who believe that greater decreases in SFA are
warranted, and some are concerned that additional reductions in dietary
fat will not significantly reduce LDL-C further and may lower
HDL-C and increase TG levels. Some investigators have shown that
very low fat diets increase TG levels and result in LDL particles
switching from pattern A to B–small, dense LDL particles
(Krauss and Dreon 1995
, Reaven et al. 1994
), which are more susceptible to oxidation (Austin 1994
, Chait et al. 1993
, Reaven et al. 1994
), while others reported that very low fat diets plus
exercise beneficially affected LDL quantity and quality (Beard et al. 1996
). In the latter study, serum cholesterol and
LDL-C levels and body weight were significantly decreased, and the
mean particle diameter of the LDL increased (P < 0.01)
in subjects on very low fat diets plus exercise (Beard et al. 1996
). The findings of the present study demonstrate that diets
low in SFA and total fat contribute to resistance of LDL to oxidation,
and in the larger cohort of the DELTA Study with 103 subjects
(Ginsberg et al. 1998
) we reported significant
(P < 0.01) reductions in plasma TC and LDL-C after
consumption of both the Step-1 and the Low-Sat diets.
The present study underscores the importance of lowering SFA and total
fat intake in beneficially affecting LDL particles qualitatively (i.e.,
LDL compositional characteristics) and possibly quantitatively [the
consumption of the Step-1 and the Low-Sat diets resulted in a
nonsignificant 5% reduction in apo B levels (P = 0.47), although Ginsberg et al. (1998)
reported that the
consumption of both the Step-1 and Low-Sat diets resulted in a
significant decrease in apo B level in the larger cohort of the DELTA
Study with 103 subjects (P < 0.05)]. Both
quantitative and qualitative changes in LDL affect LDL oxidative
susceptibility. Earlier studies have suggested that the quantity of LDL
transported in the artery wall is directly proportional to the
concentration of circulating lipoproteins (Schwenke and Carew 1989
). We found that the susceptibility of LDL to oxidation was
significantly correlated with plasma TC and LDL-C levels, and
consumption of low SFA diets decreased both. Thus, our findings suggest
that reducing dietary SFA and total fat may reduce LDL uptake by the
artery wall and decrease the oxidative susceptibility of LDL in vivo.
Although the decrease in oxidation rate paralleled the decrease in
LDL-C, at any given concentration over the range of 2 to 5 mmol/L
of LDL-C (equivalent to 0.8 to 1.8 g/L of apo B), LDL from subjects
when they consumed the Step-1 or Low-Sat diets were always less
susceptible to oxidative modification than LDL from subjects when they
consumed the AAD (Fig. 3)
. These data suggest that individuals who
maintain a relatively low LDL-C level when consuming an AAD are at
a greater potential risk of CHD than individuals who have the same
LDL-C concentration but consume a diet low in total fat and SFA.
This may explain, in part, why CHD is clinically manifested in persons
with a desirable LDL-C level (i.e., <3.36 mmol/L). Furthermore,
previous studies have shown that diets high in PUFA result in LDL that
are more susceptible to oxidation than diets high in MUFA. The
difference in LDL oxidation rate was positively correlated with LDL
18:2 and 18:2 to 18:1 ratio, and was negatively correlated with LDL
18:1 and 18:1 to 18:2 ratio (Bonanome et al. 1992
,
Reaven et al. 1993
). Oxidation of unsaturated fatty
acids in LDL occurs both in the core lipids (i.e., cholesteryl ester
and TG) and on the surface lipids (i.e., PL) of LDL particles
(Reaven et al. 1993
). It is well-recognized that
dietary fatty acid composition and the amount of dietary fat affect the
fatty acids found in the circulation as well as in the membranes
(Dougherty et al. 1987
, Judd et al. 1989
). For example, Sinclair et al. (1994)
observed significant increases in the proportion and concentration of
plasma PL arachidonic acid [20:4(n-6)] and long-chain n-3 PUFA
[20:5(n-3) and 22:5(n-3)], and a slight decrease in 18:2(n-6) in
subjects on very low fat diets (10% energy) containing lean beef. In
the present study, the ratios of 18:2 to 18:1 and PUFA to MUFA in the
LDL from subjects when they consumed the Step-1 and Low-Sat diets
were significantly lower than they were in the LDL from subjects when
they consumed the AAD. Linoleic acid (n-6) in LDL from subjects when
they consumed the Low-Sat diet also was significantly lower
compared with those from subjects when they consumed the AAD. These
observations are consistent with the results of another study
(Raatz SK, Bibus D, Thomas W, Kris-Etherton P, unpublished data) in which a low-fat diet resulted in
significant decreases in total PUFA, total n-6 and 18:2(n-6) fatty
acids and significant increases in total n-3, 20:4(n-6), 20:5(n-3) and
22:6(n-3) fatty acids. Thus, we contend that the increased LDL
oxidation rate in subjects when consuming the AAD in part is associated
with the higher 18:2 or PUFA content in the LDL. Despite a greater
oxidation rate and total dienes produced in subjects consuming the AAD,
lag time did not differ among the test diets studied (P
= 0.72). These latter results agree with a recent study conducted
by Schwab et al. (1998b)
who reported that replacing SFA
with unsaturated fat had no effect on LDL lag time in subjects fed
controlled diets that varied in type of fat (i.e., animal fat,
vegetable oils). In both studies, plasma antioxidants were either not
different (the present study) or not of sufficient magnitude to alter
the susceptibility of LDL to oxidation (Schwab et al. 1998b
). The differences observed in the rate of oxidation and
total dienes produced in the present study could reflect in part the
fatty acid composition of the LDL particle. The difference in LDL fatty
acid composition after consumption of the Low-Sat diet (i.e.,
decrease in 18:2 and increase in 22:6) may suggest competition among
lipid series [(n-3) and (n-6)] for the enzymes of elongation and
desaturation (Hwang et al. 1988
, Lands 1991
). When the relative supply of n-3 fatty acids is abundant,
these fatty acids are preferentially desaturated and elongated vs.
other fatty acids (Holman 1986
).
Some studies have suggested that HDL might protect LDL from oxidative
modification (Parthasarathy et al. 1990b
, Reaven et al. 1993
), raising questions about merits of high
carbohydrate diets that lower HDL. In the present study, consumption of
the Step-1 diet and the Low-Sat diet resulted in a reduction in
HDL-C level compared with consumption of the AAD (P
= 0.11 and 0.02, respectively). Since consumption of both the
Step-1 and the Low-Sat diets also resulted in a decrease in
LDL-C level (P = 0.16) the LDL-C/HDL-C ratios in
the Step-1 and the Low-Sat diet periods did not differ from the AAD
period (P = 0.93). The nonsignificant changes in the
LDL-C/HDL-C ratios in the subjects when consuming the Step-1 and the
Low-Sat diets may diminish the adverse effects of the decrease in
HDL-C levels on the susceptibility of LDL oxidation. Nonetheless,
consumption of the low-SFA diets resulting in a reduction in
HDL-C levels and an increase in TG are not thought to be
beneficial. In the recent Veterans Affairs HDL Cholesterol Intervention
Trial (Rubins et al. 1999
), the rate of coronary events
was reduced by raising HDL-C levels and lowering levels of TG
without lowering LDL-C levels. Studies have shown that a
low-fat diet plus exercise could prevent a decrease in HDL-C
level and result in a significantly greater decrease in TC and
LDL-C levels and TG (Yu-Poth et al. 1999
).
Importantly, a diet low in SFA but high in MUFA and consequently total
fat would result in an increase in HDL-C, decreases in LDL-C
and TG (Kris-Etherton et al. 1999
), and also favorably
affect LDL oxidative susceptibility (Morgan et al. 1998
). Thus, while a diet low in SFA and total fat beneficially
affects a number of important risk factors for CHD, a diet high in MUFA
may be preferable because of even greater beneficial effects.
Previous studies have shown that supplementation with antioxidants
(vitamin C and ß-carotene and -tocopherol) or feeding a diet high
in antioxidants from fruits and vegetables reduced LDL oxidation
(Esterbauer et al. 1989b
, Esterbauer et al. 1991
, Frei 1995
, Harats et al. 1990
, Jialal et al. 1991
, Jialal and Grundy 1992
) and lipid peroxidation (Miller et al. 1998
). -Tocopherol is the predominant antioxidant found in
LDL. Supplementation with -tocopherol has been shown to lengthen the
lag time before the onset of oxidation and reduce LDL oxidation rate
and conjugated diene formation (Jialal and Grundy 1992
).
In the present study, the antioxidants (e.g., vitamin C and
ß-carotene) were held constant among the three diets. There were no
significant differences between the ratios of -tocopherol to PUFA
and to cholesterol in the LDL from subjects when consuming the Step-1
and Low-Sat diets and those in the LDL from subjects when consuming
the AAD. Plasma -tocopherol levels did not differ among the three
diet periods. Thus, the antioxidant levels in LDL do not account for
the differences in LDL oxidation rate when comparisons were made among
the three test diets. This also explains why the lag time, which mainly
depends on antioxidant levels, was similar among the three diet
periods. It could be argued that the addition of BHT to the plasma
prior to the isolation of LDL might have affected our results. This is
not likely since the LDL were dialyzed after isolation. Moreover, even
if some residual BHT was present, it is quite likely that the amount
would be similar among LDL isolated from subjects fed the different
diets since the LDL were dialyzed at same time for each feeding period.
Nonetheless, we have addressed this important issue by evaluating the
effects of BHT on LDL oxidative susceptibility in plasma samples that
were either exposed or not exposed to BHT prior to isolation of LDL.
There were no differences in lag time, oxidation rate, and formation of
conjugated dienes between plasma samples with and without BHT (data not
shown). Furthermore, we have recently used a water-soluble
antioxidant (Trolox; Aldrich Chemical Co., Milwaukee, WI) in another
human feeding study evaluating the effects of diet on LDL oxidative
susceptibility and found that the results were comparable to the
present study (Morgan et al. 1998
). Thus, we
contend that our LDL oxidation results are an accurate estimate
of the susceptibility of the particle to oxidative modification.
Questions have been raised about the implication of findings from in
vitro LDL oxidative susceptibility studies with respect to the
development of atherosclerosis. It is important to note that some
studies have measured the autoantibodies against oxidized LDL in serum
from patients (Iribarren et al. 1997
, Maggi et al. 1995
, Palinski et al. 1989
, Puurunen et al. 1994
) and have shown that the susceptibility of LDL to
oxidation in vitro (conjugated diene formation) was correlated with LDL
oxidation in vivo (antibodies against oxidized LDL) (Maggi et al. 1995
). Moreover, both conjugated diene formation and
autoantibodies against malondialdehyde-LDL were associated with
atherosclerosis (Iribarren et al. 1997
). Thus, we
contend that a change in LDL oxidation (as measured in vitro) may
provide useful information about LDL oxidation in vivo. However, the
important question that needs to be resolved is: what is the
relationship between LDL oxidative susceptibility and atherogenesis?
Despite some evidence in humans to suggest there is some relationship
(Iribarren et al. 1997
), studies conducted in rabbits
(Fruebis et al. 1994
, Fruebis et al. 1997
) have not demonstrated any relationship between LDL
oxidative susceptibility and atherogenesis.
In summary, the results of the present study emphasize how diets can influence both quantitative and qualitative changes in LDL. We have shown that reducing dietary SFA and total fat decreases plasma TC and LDL-C levels and changes particle composition, resulting in beneficial changes in oxidative susceptibility. These changes may decrease the atherogenicity of LDL. Our findings also reveal that even when LDL-C levels were low in individuals who consumed the AAD that these particles still were qualitatively different and more susceptible to oxidative modification than were particles from subjects when they consumed the lower fat diets. Because of this, it will be important to conduct further studies to gain a better perspective about how lowering LDL by diet affects in vivo oxidation status of the particle and its uptake by macrophages in the artery wall. Nonetheless, the results of the present study clearly indicate that a diet high in total fat and SFA has potentially adverse effects on circulating LDL via both quantitative and qualitative changes.
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ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: AAD, average American diet; apo A-1, apolipoprotein A-1; apo B, apolipoprotein B; BHT, butylated hydroxytoluene; CHD, coronary heart disease; DELTA, Dietary Effects on Lipoproteins and Thrombogenic Activity Study; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; Low-Sat, low-fat and saturated-fat diet; MUFA, monounsaturated fatty acids; PBS, phosphate buffered saline; PL, phospholipids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; TC, total cholesterol; TG, triglycerides.
Manuscript received December 16, 1999. Initial review completed February 8, 2000. Revision accepted May 21, 2000.
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