Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 57(4 & 5), 411-418

@ Harcourt Brace & Co Ltd 1997

S a t u r a t e d f a t t y acids and LDL

r e c e p t o r modulation in humans and
monkeys
K. C. Hayes, P. Khosla, T. Hajri, A. Pronczuk
Foster Biomedical Research Laboratory, Brandeis University, Waltham MA 02254, USA

Summary It has been known for 40 years that dietary saturated fat (SAT FAT) increases plasma cholesterol,
including LDL-C and HDL-C. In humans, where LDL-C is typically > 90 mg/dl this SAT FAT effect largely reflects
changes in LDL-C pool size. The original human studies suggested that LDL-C expansion during SAT FAT
consumption reflected reduced LDL clearance (LDL receptor activity) in hyperlipemics and increased LDL production
rates in normolipemics (LDL-C < 100 mg/dl). This dual explanation is supported by data from several animal models
where specific saturated fatty acids (SFAs) have been the focus. However, the situation is complicated by the fact that
polyunsaturated fatty acids (PUFAs) oppose SFAs, i.e. PUFAs decrease LDL-C and increase LDL receptor (LDLr)
activity, so the effect of SAT FAT intake may represent the combined influence of increased SFAs and decreased
PUFAs. In fact, careful scrutiny of primate data suggests a negligible effect of saturated fat on LDL clearance (and
receptor activity) in the absence of dietary cholesterol when PUFA intake is adequate (5-10%en) and the lipoprotein
profile is relatively normal (LDL-C < 90 mg/dl), i.e. normolipemic situations at the time of dietary intervention. In such
cases increases in LDL-C due to SFAs (particularly 12:0+14:0) appear to reflect LDL overproduction associated with a
shift in cholesterol from tissues to the plasma cholesteryl ester (CE) pool (both LDL-C and HDL-C) without altering
whole-body cholesterol balance. The reason for this shift, which is accompanied by an increase in the plasma
oleic/linoleic CE ratio, is unknown but may reflect a decreased rate of CE hydrolysis by the liver. When individuals or
animals are rendered hyperlipemic by other factors (e.g. chronic caloric and dietary cholesterol excesses in humans or
by cholesterol feeding in animals) specific SFAs (particularly 16:0) can contribute to decreased LDL; activity initiated
by a primary factor, such as dietary cholesterol. However, LDLr down-regulation by dietary cholesterol greatly exceeds
any contribution from SFAs.

INTRODUCTION while gerbils and hamsters develop a major increase in

HDL-C. 4 In other words, SFAs increase plasma cholesterol
It is generally appreciated that dietary saturated fatty
in the dominant lipoprotein pool of the species in
acids (SFAs) raise total plasma cholesterol (C, both LDL-C
question, if at all.
and HDL-C). A current school of thought suggests that
In humans fed SFAs the expanding LDL-C pool could
the major effect of SFAs is to depress LDL receptors (LDLr)
reflect several factors: 1) increased production of LDL; 2)
and decrease clearance of LDL-C from plasma. 1-3 How-
decreased LDL clearance causing a 'backup' in plasma, or;
ever, this conclusion may be premature when one con-
3) simply having LDL particles produced and cleared at
siders the general response across species and the
normal rates, but each particle packed with more choles-
particulars of data generated in human subjects. In fact,
terol per particle. When these particles circulate, each
the fundamental role of SFAs is probably not on LDLr
would carry more cholesterol to expand the plasma
activity but is much broader in scope. For example, SFAs
LDL-C pool. A combination of all three mechanisms is
increase plasma cholesterol in most species with the
possible, but most studies have focused on production
increase being distributed according to the LDL or HDL
and clearance by tracing LDL apoB with 1125radiolabelling.
profile of the species under investigation. In humans
To better appreciate the published findings, it is
the primary increase is in LDL-C. In macaque monkeys it
instructive to consider which fatty acids affect the LDL-C
represents a balanced increase in LDL-C and HDL-C,
pool most demonstrably within the context of two basic
Correspondence to: K. C. Hayes, Tel. 001 781 736 2051; Fax. 001 781 736 2054 factors that influence the LDL-C response, in general.

411
412 Hayes et al
KEY TO METABOLIC FACTORS
IMPACTING LIPOPROTEIN SETPOINT
1. LDL, activity is key
4 2oo
VLDL LP~"~TG 2. VLDL output
3a. LDL formation
~,.j-~v I N 3b 3b. HDL formation
/~VER v ~ 3a~ HDL 4. LPL removes TG
$. CETP adds to LDL
/ CHOLESJER_OLLDL_I ) IDL / 6. CE formation
BLOCKS LD~ ~ /CETP 7. Bile acid synthesis
L L
Fig. 1 Metabolicfactors (indicated as 1-7) contributing to the
lipoprotein setpoint, or profile, of an individual, are depicted. In
humans, where LDL-C predominates, the LDLr activity (1) is critical
as it ultimately affects LDL clearance and pool size. But LDL also
indirectly depends on the rate of VLDL 'substrate' production by the
liver (2) and its conversion to IDL (3a) and HDL (3b) when acted
upon by lipoprotein lipase (LPL) in adipose and muscle (4). If the
LDLr is up-regulated (as in rats), then IDL clears directly to the liver
with minimal LDL formation. An accumulation of IDL-C or LDL-C is
augmented by active transfer of cholesteryl esters from HDL by
CETP (5). Once delivered to the liver, the free cholesterol can be
'removed' by re-esterification via ACAT (6) (probably only in
species that accumulate hepatic CEs) or converted to bile acids (7)
for excretion (extremely efficient in rat livers). Hepatic cholesterol
accumulation 'blocks' the LDLr causing the plasma pool of LDL to
expand. The balance among all these variables establishes the
lipoprotein setpoint. Dietary fat and cholesterol influence many of
these parameters.
The primary factor is the inherent lipoprotein profile (set-
point) of the host (Fig. 1), and the second relates to those
dietary factors (e.g. 18:2 and dietary cholesterol) that
modify this setpoint or alter its 'threshold'. This combi-
nation of genetics and diet modulate the impact of SFAs
on cholesterol metabolism. 4,s Without first reconciling
these two variables, experimental results are typically
confusing and often uninterpretable.
The lipoprotein setpoint has been discussed elsewhere, 5
but briefly it reflects the various metabolic parameters
dictating the plasma lipoprotein profile of an individual.
An elevated setpoint would encompass hyperlipidemia
with elevated LDL-C, triglycerides (TGs), and low HDL-C,
whereas someone with a low setpoint would have a total
cholesterol (TC) less than 180 mg/dl, low LDL-C and TGs
with normal to elevated HDL-C. The setpoint is deter-
mined, in part, by dietary factors, a lower setpoint being
favored by dietary soluble fiber, vegetable protein, and a
low-cholesterol intake. Although both SFAs and polyun-
saturated fatty acids (PUFAs) can affect the setpoint, we
have previously underscored the specific relationship
and importance of dietary intake of linoleic acid (18:2
n-6), but abbreviated as 18:2) and the 18:2 threshold
(Fig. 2).4.e The intake of 18:2 seems especially critical
because it affects how specific SFAs influence cholesterol
metabolism. When the available 18:2 is below its required
'threshold' for an individual or population, cholesterol-
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 57(4 & 5), 411-418
LDL-C
(mg/dl)
24o 14:0 M I N I M A L 18:2 T H R E S H O L D BELOW W H I C H LDL, A C T I V I T y IS EXCEEDINGLY
VULNERABLE A N D L D L - C RISES I N RESPONSE T O C E R T A I N S A T U R A T E S
18o MAXIMUM HEPATIC LDLr A C T I V I T Y
36:0 INDUCIBLE BY n 6 FUFA (UppER "[HRESHOLD)
leo
14o
loo 18:0 L
8o
Al~ taty ,~Arry A O ~ ~
EQUAL A ~ T m MAX. T ~ L D
MO~-OR- LVSS
OFl~en
eo I I ~ I I J I I ~ I I I
4o 1 2 3 4 5 6 7 8 9 10 11 12
20
o
Fig. 2 The scheme depicts the putative dynamics between the
relative importance of linoleic acid (18:2, as percent dietary energy)
and modulation of LDL-C, which in turn is based on antagonism
between 18:2 and primarily 14:0. Only 18:2 is perceived as exerting
a positive influence on (i.e. increases) LDL receptor (LDLr) activity
in humans. By shifting the typical 18:2 intake from low (3%en) to
moderate (7%en) the hepatic clearance of LDL-C is increased
(lowering LDL-C and plasma cholesterol) to counter the cholesterol-
elevating influence of 12:0+14:0, which is probably via LDL
overproduction (see text). Consumption of 16:0 exerts a negative
effect (increases LDL-C) only if the 18:2 intake is too low (below
threshold, as in cholesterol feeding), probably because VLDL
production increases causing increased LDL formation (see Fig. 1).
Other factors affecting cholesterol metabolism presumably affect
the 18:2 threshold requirement, accounting for individual and
population differences in the threshold.
emia readily develops as SFAs are increased. However,
once the 18:2 threshold is exceeded, it becomes increas-
ingly difficult to elicit a SFA response, and adding more
18:2 to the diet fails to decrease TC appreciably.
These points can be demonstrated in the hamster
where the fatty acid (FA) influence on LDL receptors has
been investigated. 1,2 Based largely on this hamster model
fed a substantial amount of cholesterol, current dogma
suggests that SFAs (12:0, 14:0, 16:0) raise cholesterol pri-
marily by decreasing LDL-receptor (LDL~)activity, thereby
expanding the LDL-C pool. 3 Our working hypothesis
would argue that dietary cholesterol depresses the LDLr
(raising the lipoprotein setpoint) and thereby exacerbates
(or indirectly elicits) the SFA effect. On the other hand,
dietary 18:2 can lower the lipoprotein setpoint by
enhancing LDLr activity.2'5 A common misconception
that emanates from discussions of the Spady-Dietschy
hamster model is that SFAs alone are responsible for the
decreased LDL-receptor activity. However, only in the
hamster fed chow with 0.12% added cholesterol is this
SFA effect demonstrable, as pointed out by the authors
themselves/ In the absence of dietary cholesterol in
chow-fed hamsters (and several other species, as well) it is
difficult to demonstrate a difference between SFAs and
polyunsaturated fatty acids (PUFAs) or monounsaturated
fatty acids {MUFAs) on LDL-C or LDLr activity, suggesting
that SFAs per se do not depress LDL~ activity. Nonetheless
in certain species, such as the gerbil and cebus monkey
(and probably in certain humans), a SFA-induced increase
in LDL-C pool size does occur (in response to specific fatty
acids) without the interaction of cholesterol feeding. Even
© Harcourt Brace & Co Ltd 1997
 Saturated fatty acids and LDL receptor modulation in humans and monkeys 413

Table I LDL apoB kinetics for humans fed different dietary fats

Study design (ref) LDL-C LDL apoB LDL apoB LDL apoB
mg/dl pool size FCR PR
mg/kg pools/day mg/kg/day
Turner et aV (< 150 mg cholesterol/day, P/S 0.2 vs 8, 18:2@ 4 vs 32%en)
normolipemics (n = 7, x-over)
butter 122" 33 0.35 11.5*
safflower 98 ~ 28 0.39 10.5
hyperlipemics (n = 8, x-over)
butter 271 * 72* 0.23 15.4*
safflower 219 51 0.27* 12.7
Shepherd et al 8 (400 mg cholesterol/day, P/S 0.25 vs 4, 18:2@ 5 vs 24%en)
(n = 8, x-over)
milk fat 134 36 0.32 11.5
corn oil 104" 31" 0.35* 10.8
Cortese et al 9 (340 mg cholesterol/day, P/S 0.30 vs 25%en as fat)
(n = 8, x-over)
High-SAT FAT 210" 37* 0.26 9.6*
Low-SAT FAT 175 26 0.30* 7.7
(365 mg cholesterol/day, P/S 0.12 vs 3.8, 18:2@ 2.5 vs 25%en)
(n = 3, x-over)
butter 164" 38* 0.37 15.3
sunflower 129 31 0.35 11.8

*significant difference attributed to dietary fat; tsignificant difference between normolipemics

and hyperlipemics fed safflower oil.

in these species (as pointed out below), it is not always
clear from the study design whether SFAs alone are to
blame, or whether a simultaneous decrease in PUFAs is a
factor.
When evaluated in isolation, both human and non-
human primate studies are inconclusive on this subject.
Even collectively they fail to provide strong support for
direct modulation of LDLr by SFAs and, in fact, appear to
disfavor any such conclusion when details of the various
designs are scrutinized.
HUMAN STUDIES
Turner et al 7 were among the first to illustrate the concept
that the lipoprotein setpoint affects apoB kinetics when
dietary fat saturation is manipulated in humans (Table 1).
Two dietary fat extremes were fed to individuals with
different lipoprotein setpoints, i.e. normolipemics (n= 7)
or hyperlipemics (n = 8). Fat contributed 40% en, with one
diet based on butterfat, the other safflower oil. Because
the ratios between polyunsaturated and saturated fatty
acids (P/S ratios) represented extremes (at 0.2 vs 8 ), the
lower TC during the safflower oil diet could represent
the effect of high PUFAs or removal of SFAs, or both. It is
not possible to make that distinction from the design.
Dietary cholesterol was < 150 mg/day, minimizing any
complicating effect it might have had. When consuming
the safflower oil diet, both groups of subjects decreased
their LDL-C by 20%, and LDL apoB by 15% (nor-
molipemics) or 28°/o (hyperlipemics), but only the hyper-
lipemic group revealed a 15% decrease (P< 0.05) in LDL
apoB fractional catabolic rate (FCR) when fed butter fat,
reflective of a LDLr clearance problem. However, in both
groups during the butter fat period the production rate of
LDL apoB (PR) increased significantly by 10% (normo)
and 20% (hyper). This suggests that excess synthesis, not
clearance, was the predominant problem associated with
dietary saturated fat (SAT FAT), with impaired clearance
possibly being secondary to an initially elevated lipo-
protein setpoint (hyper group only).
A similar dietary fat challenge was conducted by
Shepherd et al,8 and the design also included a crossover
between mflkfat and corn oil (Table 1). During corn oil
intake, LDL-C declined by 22% and LDL apoB pool size by
14% coupled with a 9% increase in FCR without affecting
the production rate. However, 400 mg cholesterol was
consumed/day, which would support the notion that
altered (depressed) LDG by cholesterol allowed for up-
regulation and increased FCR by PUFAs, as delineated by
the hamster model. 1-3 This study emphasizes the impor-
tance of monitoring dietary cholesterol when interpreting
results, even in humans.
A third human study in hypercholesterolemic subjects
adds to our understanding. 9 Two groups of subjects were
evaluated, again for LDL apoB kinetics (Table 1). The first
group (n=8) was used to compare high and low fat
intakes. A high-SFA diet (46% en, P/S 0.3 based on butter
and meat fat) depressed the FCR and increased the LDL
apoB pool size and production rate relative to the same
fat composition (P/S 0.3) at low-fat intake (20% en). This
result identified the possibility that absolute intake of a
SFA or 18:2, or even total fat intake, represent potential

© Harcourt Brace & Co Ltd 1997 Prostaglandins, Leukotrienes and Essential FattyAcids (1997) 57(4 & 5), 411-418
414 Hayes et al

Table 2 LDL apoB kinetics for monkeys fed different dietary fats

Study design (ref) LDL-C LDL apoB LDL apoB LDL apoB
mg/dl pool size FCR PR
mg/kg pools/day mg/kg/day
Khosla and Hayes 1° rhesus (n = 4)
no cholesterol
12:0 + 14:0 73 14 0.86 12.0
16:0 + 18:1 57 7* 0.79 5.5*
Khosla et al 1~ rhesus (n = 6, paired design)
0.07% cholesterol
Am Fat Blend 143 50 0.80* 39
0.03% cholesterol
Step 112:0 + 14:0 113 44 0.99 44
Step 116:0-rich 82* 36* 1.12 40
Khosla and Hayes ~3cebus (n = 10, x-over)
no cholesterol
16:0-rich 50 20 1.29 25
18:1 -rich 53 20 1.38 22
18:2-rich 48 21 1.22 26
Khosla and Hayes TM cebus (n = 12, x-over)
no cholesterol
16:O-rich 53 18 1.72 t 30
18:l-rich 47 17 1.71t 29
0.3% cholesterol
16:0-rich 136 t 40 t 1.06* 41t
18:l-rich 117 t 34 t 1.27 42 t
Khosla and Hayes ~ecebus (n = 11, x-over)
0.08% cholesterol
Am Fat Blend 76 *t 59 1.12 *t 33
0.04% cholesterol
Step 1-16:0 64 52 1.34 31
Step I-trans 18:1 61 53 1.35 34
Nicolosi et a117cebus (n = 5)
no cholesterol
coconut oil 126" 35* 1.10 39
corn oil 38 20 1.88* 38
0.1% cholesterol
coconut oil 155" 41" 0.71t 29
corn oil 58 t 22 1.45* 32
Stucchi et al ~8cynomolgus (n = 6-13)
0.1% cholesterol
coconut oil 468* 90 0.42 36
butter + oils 324* 70 0.44 30
corn oil 47 16* 1.20* 18*
Brousseau et al2° cynomolgus (n = 10, x-over)
0.1% cholesterol
12:0 + 14.0-rich 219" 112" 0.53 55*
18:1 -rich 191 81 0.51 41
18:2-rich 160 72 0.62* 42
Hunt et a121cynomolgus (n = 3-5)
0.01% cholesterol
SATs 95 na 0.75 19
POLYS 66* na 0.75 15*
0.06% cholesterol
SATS 194 na 0.49 25
POLYs 142* na 0.47 14*
0.50% cholesterol
SATs 248 na 0.17 26
POLYs 221" na 0.15 22*
Sorci-Thomas et a122African Green [LDLr mRNA]
no cholesterol (n = 4-5)
lard 68 88 2.4 na
safflower 59 80 1.9 na
0.4% cholesterol
lard 163 t 137 t 1.0" na
safflower 120 *t 101 ,t 1.2 na

*significant difference attributed to dietary fat; tsignificant difference due to dietary cholesterol.

Prostaglandins, Leuketrienes and Essential Fatty Acids (1997) 57(4 & 5), 411-418 © Harcourt Brace & Co Ltd 1997
 Saturated fatty acids and LDL receptor modulation in humans and monkeys
complicating variables between published study designs.
In addition, data were presented for a second group of
subjects (n = 3) where the LDL-C and apoB pool size were
increased by an extreme SAT FAT diet based on butter
(P/S 0.12, 2.5 %en as 18:2) vs an extreme PUFA FAT based
on sunflower oil (P/S 3.8, 25%en as 18:2). Too few subjects
were studied, but no effect was noted on FCR, and only a
trend for increased LDL apoB production rate was observed
with the butter diet. However, cholesterol intake at 365 mg/
day during the butter diet represented a potential bias.
Thus, the few h u m a n studies that evaluated the impact
of dietary fat on LDL turnover (LDLr activity) are compli-
cated by the extremes in SFAs vs PUFAs and the limited
type of SFAs fed (all butter-based). In essence, a large
divergence in 18:2 %en intake between the SFA and PUFA
diets, as well as the varied cholesterol load resulted in
no clear trend for SFA/PUFA effects, let alone any specific
saturated fatty acid effects, on LDL apoB FCR or LDL apoB
production rates.
NONHUMAN PRIMATES
To address certain shortcomings in the h u m a n studies
we conducted five studies in monkeys (two in rhesus and
three in cebus) where these variables were carefully
controlled.
The first experiment was in rhesus monkeys ~° where
the 18:2 0/oen was held relatively constant (4O/0en)between
two cholesterol-free diets in which the P/S ratios were
relatively low (0.17 and 0.35) and the major exchange was
between specific SFAs (12:0 + 14:0) for (16:0) + 18:1
(= 18:1 n-9, oleic acid). This was achieved by replacing a
coconut oil-soybean oil mixture with palm oil-soybean oil
providing 3 l%en from total fat. The results (Table 2)
revealed a trend for increased LDL-C during 12:0 + 14:0
consumption, coupled with a doubling of both the LDL
apoB pool and production rates without affecting LDLr
activity, i.e. FCR was not altered. These results suggested
that the major effect of specific SFAs (i.e. 12:0 + 14:0) was
to increase the production rate of LDL apoB rather
than affect clearance when the 18:2 %en was adequate
and constant between diets and no diet cholesterol was
present to down-regulate the LDL receptors.
A second rhesus study ~1 confirmed the first. In this
experiment all monkeys initially received an American fat
blend (38%en as fat, 16%en as SFAs with cholesterol at
180 mg/1000 kcal) and then were split into two groups to
receive one of two Step I diets (30%en, 10%en SFAs, with
75 mg/1000 kcal of cholesterol). In one of these reduced-
fat diets the SFAs were predominately 16:0, in the other
mostly 12:0 + 14:0. The LDL kinetics (Table 2) indicated
that the main diet effect was attributed to reduced cho-
lesterol consumption, which caused the FCR to increase
equally during both Step I diets. No differential effect of
© Harcourt Brace & Co Ltd 1997 Prostaglandins, Leukotrienes and Essential FattyAcids (1997) 57(4 & 5), 411-418
415
16:0 vs 12:0 + 14:0 was seen on FCR, but 12:0 + 14:0 did
result in a greater LDL-C and LDL apoB pool size because
the production rate of LDL apoB increased.
Because rhesus are less sensitive to saturated fat than
cebus monkeys, 12 the latter species was challenged with
40%en fat in cholesterol-free diets that contained satu-
rates, monounsaturates or polyunsaturates from palm
oil, high- 18:1 safflower oil, or high- 18:2 safflower, respec-
tively. 13 The 18:2 %en was 4, 6, and 29, respectively.
Somewhat unexpectedly LDL kinetics revealed absolutely
no differences in LDL-C, apoB pool size, or production
rate between these three dietary fatty acid profiles, again
in the absence of cholesterol and with adequate 18:2
intake in all cases (Table 2). It was concluded that when
the LDLr activity and lipoprotein setpoint are normal and
18:2 is at (or above) its required threshold at the time of
intervention (i.e. lipoprotein metabolism is unstressed),
no effect of a 16:0-rich SAT FAT (unlike 12:0 + 14:0-rich
fats) can be detected on LDL metabolism, even in a fatty
acid sensitive species.
As a follow-up study, cebus were again challenged
with either a 16:0-rich or 18:l-rich fat in the presence or
absence of 0.3% cholesterol using a crossover design. TM A
significant depression in LDLr activity occurred that was
attributable to cholesterol consumption per se (Table 2).
Furthermore, only when cholesterol was added to these
fats did LDL-C and apoB pool size increase significantly
in the 16:0-rich diet group relative to the 18:l-rich diet
group; and although the apoB production rate increased
with cholesterol feeding, the increase was similar for both
fatty acid profiles. The increases in LDL-C and apoB pool
size during the 16:0-rich fat were explained by the greater
depression in FCR (LDLr activity) associated with choles-
terol intake. Thus, only in the presence of dietary choles-
terol (which depresses LDLr activity and increases the
lipoprotein setpoint and 18:2 threshold) could one distin-
guish between 16:0 and 18:1. This is analogous to the
cholesterol-fed hamster model where 18:1 induced
greater LDL-C clearance than 16:0 or 14:0Y ,~5
The impact of dietary cholesterol on LDLr activity is
further reflected in a final cebus experiment 1~ that also
compared Step I diets (16:0-rich vs trans 18:l-rich con-
taining 30%en from fat, 0.03% cholesterol) with an
American fat blend (38% en as fat, 0.08% cholesterol, high
in 12:0 + 14:0). Again the importance of dietary choles-
terol (and possibly lower fat intake) was apparent on LDL
kinetics because both Step I diets reduced LDL-C and
the apoB pool size, while increasing the FCR: However,
the production rate of LDL apoB was unaffected by the
modestly lower cholesterol intake of the two Step I diets,
even though 12:0 + 14:0 was also substantially reduced
from the control diet period.
Collectively these data confirm that differentiating
between 16:0 and 18:1 depends on initial dietary choles-
416 Hayes et al
terol down-regulation of LDLr. In the absence of dietary
cholesterol, our LDL kinetic data indicate that certain
SFAs, namely 12:0 + 14:0, can increase LDL apoB, LDL-C,
and LDL apoB production without altering LDL apoB FCR
(LDLr activity). Thus, certain SFAs appear to alter LDL
metabolism independent of any depression in clearance,
probably by increasing apoB production. Furthermore,
these details can only be elaborated when cholesterol and
18:2 %en are carefully controlled and individual SFAs are
accounted for during the dietary fat exchanges.
OTHER M O N K E Y STUDIES
The background information detailed above facilitates
interpretation of other reports concerning LDL~ activity in
n o n h u m a n primates. For example, the initial report on
apoB kinetics in cebus m o n k e y s was conducted after
5-10 years of feeding either coconut oil (without added
18:2) or corn oil with or without 0.1% dietary cholesterol
(four diet groups). The P/S ratios were extraordinarily
polar at 0.02 vs 4.7 with 18:2 intake estimates of 0.6% vs
17%en (Table 2). A P/S ratio of 0.02 is extremely low and
approaches essential fatty acid deficiency, whereas a
P/S of 4.7 is extremely high and much above the 18:2
threshold. Even in the absence of dietary cholesterol,
the authors found that coconut oil Without added 18:2
greatly expanded LDL-C and LDL apoB pools. This was
linked to reduced LDL FCR (both LDL~ activity and non-
receptor uptake were depressed) without any change
in LDL apoB production. The altered LDL metabolism
described during coconut oil intake may have reflected a
lack of 18:2 coupled with a high intake of 12:0 + 14:0 or,
during corn off intake, a distortion caused by excess 18:2.
However, the latter seems unlikely because high 18:2
intake in normolipemic humans 7-9 and cebus monkeys ~3
had no effect on FCR. When 0.1% cholesterol was added,
neither fat group revealed a change in LDL production
rate, even though the added cholesterol depressed FCR
and expanded LDL-C slightly. A drawback with the design
is that without better control over 18:2 and no control
over individual variation among monkeys (no crossover
design), the true effect of specific SFAs or PUFAs cannot
be realized.
In a second study by the same authors 18 cynomolgus
monkeys were used to again compare coconut oil (12:0 +
14:0-rich) and corn oil with a butter-based diet rich in
16:0 + 18:0 (Table 2). The P/S ratios were 0.02, 4.9 and 0.54
while the %en from 18:2 was 0.7, 23, and 7, respectively.
M1 three diets contained 0.1% cholesterol. The marked
rise in LDL-C demonstrates the extreme sensitivity of
cynos to a dietary cholesterol load, at least in the presence
of saturated fat. As commented on elsewhere 19the degree
of dietary cholesterol overload depressed LDL~so severely
(raised the lipoprotein setpoint) that it essentially pre-
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997)57(4 & 5), 411-418
empted the possibility of distinguishing between the rela-
tive ability of specific SFAs to modulate LDLr activity. Only
the high 18:2 intake (23%en) associated with corn oil was
able to overcome the LDI~ depression (increasing FCR and
essentially normalizing LDL-C and LDL apoB kinetics).
In a better designed study also using cynomolgus
monkeys, these authors 2° fed 10 monkeys three different
fats (containing saturates, monounsaturates and poly-
unsaturates) in a crossover design. Exquisite care was
taken with fat composition, blending 5-6 fats per diet in
order to exactly exchange 10% en as 12:0 + 14:0 for 18:1
or 18:2 within purified diets containing 30%en as fat,
again with 0.1% cholesterol (230mg/1000kcal). As a
result of the careful fat formulation, the 18:2 %en was
within a realistic dietary range at 5.7 %en in the 12:0 +
14:0-rich diet, 4.7 %en for the 18:l-rich diet, and 17.7 %en
for the 18:2-rich diet. The P/S ratios were 0.4, 1.1, 3.4,
respectively. The much lower cholesterolemia (compared
to Stucchi et al~a) reflected by the lower LDL-C, clearly
demonstrates the power of 18:2 to lower LDL-C in the
cholesterol-fed cynomolgus. For example, the difference
of 0.7 vs 5.7 %en as 18:2 between the roughly comparable
12:0 + 14:0-rich diets in the two experiments, decreased
LDL-C more than 50% in the second instance. But more
important to our discussion is the clear demonstration
that the rise in LDL-C due to 12:0 + 14:0 (even in the pres-
ence of moderately severe cholesterol loading) was attrib-
uted to increased LDL apoB production, not depressed
FCR (i.e. not an LDL~ clearance problem) when compared
to the 18:l-rich (neutral) diet. Furthermore, only a high
18:2, intake (18 %en) was able to counter the depressed
FCR that had resulted from the high dietary cholesterol
down-regulation of LDL receptors. Thus, while the pro-
duction rate of apoB was not different between 18:1 and
18:2, and only increased with 12:0 + 14:0, the clearance
rate (LDLr activity) was not different between 12:0 + 14:0
and 18:1, only improving with the 18:2-rich diet.
The importance of 18:2 is further demonstrated in
another study involving cynomolgus monkeys. 21 The
design represented a reasonable P/S comparison (0.5 vs
0.9) based on two different blends of the same four
fats (butter, beef tallow, safflower oil, corn oil), which pro-
vided adequate control over 18:2 at 8% vs 11%en (both
presumably above the 18:2 threshold at low cholesterol
intakes). Graded intakes of cholesterol were applied, but
the six diet groups (2 fats, 3 levels of cholesterol) con-
tained only 3 to 5 monkeys per group without any
crossover. Nonetheless, LDL-C and the apoB production
rate were both increased by the SAT FAT diet without any
effect on FCR (LDLr activity) relative to POLYs during a
neglible intake of cholesterol (0.01%). At higher dietary
cholesterol intakes (0.06, 0.50%) LDL-C steadily increased
with both fats, with the differential effect between satu-
rates and polyunsaturates being sustained (Table 2). ApoB
©Harcourt Brace & Co Ltd 1997
 Saturated fatty acids and LDL receptor modulation in humans and monkeys
production also increased with cholesterol feeding, but
the effect was moderated by PUFAs. Similar to all such
studies, FCR was depressed dramatically by cholesterol
feeding, but a differential effect between SFAs and PUFAs
was not observed. As with Brousseau et al,20 the implica-
tion is that SFAs have no effect per se on LDL apoB clear-
ance when 18:2 is adequate; and 18:2 adequacy depends,
in part, on the cholesterol load. Apparently at 0.06%
cholesterol and above, even 1 l%en from 18:2 was insuffi-
cient to overcome the cholesterol suppression of LDLr
activity in cynos. On the other hand, certain SFAs can
increase apoB production, and this stimulation may be
exacerbated when dietary cholesterol is added. Thus, the
primary explanation for an increase in LDL-C due to SFAs
in the absence of dietary cholesterol, and possibly even
the main effect of SFAs in the presence of dietary choles-
terol, would seem to be overproduction of LDL rather
than impaired LDLr clearance, with the overproduction
deriving largely from 12:0 + 14:0 intake.
The importance of the 18:2 threshold on the SFA effect
is further supported by data from African Green
monkeys = fed lard or safflower oil-rich diets (40% en from
fat) with or without 0.4% cholesterol (four diet groups).
Although the P/S ratio comparison (0.3 vs 2.2) was rather
exaggerated, the SAT FAT diet actually supplied a reason-
able 18:2 intake (6% en) compared to 24%en from 18:2
for the safflower-rich fat blend. Without added choles-
terol, no difference was noted in LDL-C, plasma apoB, or
an index of LDLr activity, i.e. hepatic LDL~ mRNA abun-
dance (Table 2). As expected, adding the rather large
cholesterol load depressed LDLr (mRNA), but still no
difference was detected between fats, i.e. no depression in
LDL~was attributable specifically to SFAs. Again, one must
conclude that in a normocholesterolemic situation with
unencumbered lipoprotein metabolism (lipoprotein set-
point normal) and even under a cholesterol load that
expands the LDL-C pool, a 16:0 + 18:0-rich fat source
(lard) does not exaggerate the depressed LDLr activity as
long as 18:2 intake is adequate (at or above threshold).
In summary, saturated fat does not appear to be a factor
in reducing clearance of LDL when lipoprotein metabo-
lism is normal. Normal lipoprotein metabolism in humans
depends, in part, on adequate 18:2 intake and minimal
(< 300 mg/day) intake of dietary cholesterol. On the other
hand, certain normally consumed saturated fats (rich
in 12:0 + 14:0) in the context of a low-cholesterol diet
are able to increase the circulating LDL-C pool in certain
species or individuals, the primary cause being an over-
production of LDL With increasing dietary cholesterol,
down-regulation of LDLr occurs, raising the lipoprotein
setpoint. Under these circumstances 18:2 is the only
fatty acid to consistently lower LDL-C by increasing the
fractional catabolic rate (LDL~ activity). However, ff LDLr
activity is already normal or if the dietary cholesterol load
© Harcourt Brace & Co Ltd 1997 Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 57(4 & 5), 411-418
417
is too large, an 18:2-induced decrease in LDL-C does not
occur at reasonable 18:2 intakes (up to 12%en). Under
most dietary circumstances SFAs probably do not affect
LDLr activity other than by displacing 18:2, i.e. depressing
18:2 intake below its critical threshold requirement.
ADDENDUM
The results of White and coworkers (this meeting) are not
consistent with our findings, but it should be noted that
two of their model assumptions need careful considera-
tion. The first pertains to triglyceride molecular structure
(TG-MS) and the second to the hamster's response to fatty
acids as being representative of humans.
Feeding mono-acyl TGs in order to focus on a single
fatty acid assumes that TG structure and the relative load
of a given fatty acid presented in this conformation have
no effect on fatty acid utilization or lipoprotein (LP)
metabolism. Numerous reports indicate otherwise, i.e.
at least some structured TGs have unique effects when
supplied in large quantity, particularly those involving
saturated fatty acids. Certain structural arrangements of
TGs (e.g. fatty acid pairings) may be more important than
others, but we are unable at this point to designate the
relative importance of each. Thus, until experiments are
conducted to delineate specific attributes of given TG-MS,
it is only prudent that modeling experiments that explic-
itly or implicitly utilize animal models to influence our
thinking about human LP metabolism as affected by
dietary fatty acids, should present TG forms encountered
in human diets. Having said that, the applicability of
these data to the h u m a n situation is questionable. The
results presented are of interest for potential clues con-
cerning the impact of mono-acyl glycerides, but in the
end one is left with the discordant gap between these
results and actual data derived from humans consuming
natural fats containing greater or lesser amounts of the
specific fatty acids in question. In real diet situations
these fatty acids are normally found in combination with
other fatty acids on the same TG molecule. Keep in mind
that with rare exception humans (or any species) seldom
consume any fatty acid in its mono-acyl TG form. Thus,
the peculiar influence of a large intake of trimyristin in
these hamsters may represent the simultaneous selective
exclusion of 18:2, 18:3 (aqinolenic acid) or even 18:1 and
16:0, which are very likely needed concomitantly for the
synthesis of a specific phospholipid or regulatory diacyl-
glycerol. If one uses structured TGs to pursue the latter
hypothesis, the present experiments have merit, but as
examples of experiments implicitly designed to elucidate
the role of fatty acids in the normal physiology of human
(or mammalian) LP metabolism, they come up short.
In essence, the application of elegant molecular biology
in tissues that are without the confines of normal physi-
418 Hayes et al
o l o g y g e n e r a t e s results t h a t are m o s t difficult to i n t e r p r e t
at best, a n d p r o b a b l y n o t a practical e x a m p l e of h u m a n
LP m e t a b o l i s m as affected b y diet.
It is t r u e t h a t feeding real fats (containing m u l t i p l e fatty
acid mixtures) does n o t p e r m i t c o n c l u s i o n s specific to
i n d i v i d u a l fatty acids, b u t utilizing m u l t i p l e p e r m u t a t i o n s
a n d feeding r e g i m e n s w i t h carefully s e l e c t e d fat b l e n d s
does allow for t h e practical isolation or e m p h a s i s of indi-
v i d u a l d i e t a r y fatty acids (and fats) in a f a s h i o n similar to
t h a t e n c o u n t e r e d o n a daily basis. The latter e x p e r i m e n t a l
a p p r o a c h is s o m e w h a t tedious, b u t at least t h e h o s t
r e s p o n s e is w i t h i n t h e b o u n d s of n o r m a l p h y s i o l o g y a n d
has t h e p o t e n t i a l of real-life applicability to h u m a n s ,
a s s u m i n g t h a t t h e l i m i t a t i o n of t h e a n i m a l m o d e l u n d e r
i n v e s t i g a t i o n is a p p r e c i a t e d a n d a c k n o w l e d g e d in t h e
interpretation.
As i n d i c a t e d earlier, t h e p r e s e n t h a m s t e r e x p e r i m e n t s
cause one to w o n d e r w h e t h e r t h e effect of t r i p a l m i t i n
r e p r e s e n t s 16:0 p e r se or a n o v e r a b u n d a n c e of 16:0 in t h e
relative a b s e n c e of 18:2. Because this line of q u e s t i o n i n g
leads to a n infinite n u m b e r of h y p o t h e s e s a n d experi-
m e n t s in proof, it seems m u c h m o r e logical experimentally
to c o n t r o l a m a x i m u m of t h e s e possibilities from t h e
o u t s e t b y i n c l u d i n g at least t h o s e fatty acids k n o w n to
b e r e q u i r e d for n o r m a l p h y s i o l o g i c a l events, i n c l u d i n g
certain essential fatty acids a n d (surprisingly) e v e n a cer-
tain level of saturates. Note t h a t t h e s e diets c o n t a i n no
essential fatty acids, o n l y 18:1 a n d s a t u r a t e d fatty acids.
Because t h e m e t a b o l i s m of fatty acids (and t h e i r i m p a c t
on LPs) is d e m o n s t r a b l y affected b y o t h e r factors affecting
t h e l i p o p r o t e i n profile (e.g. diet cholesterol) it b e c o m e s
e x c e e d i n g l y i m p o r t a n t n o t o n l y to c o n t r o l t h e level of
cholesterol c o n s u m e d , b u t to define t h e b a s a l i m p a c t of
cholesterol i n g e s t e d b y t h e m o d e l w i t h i n t h e c o n t e x t of a
fatty acid load. For example, it h a s b e e n s h o w n t h a t all
s a t u r a t e d fatty acids (except 18:0) a p p e a r e q u a l l y choles-
t e r o l e m i c in t h e D i e t s c h y m o d e l once sufficient choles-
terol is fed to d o w n - r e g u l a t e LDL receptors. In h a m s t e r s
fed purified diets, this occurs at a b o u t 0.04% cholesterol,
so b o t h t h e 0.12% a n d 0.25% c h o l e s t e r o l l o a d s h e r e i n
w o u l d b e e x p e c t e d to o v e r s h a d o w , e v e n mask, a n y influ-
ence t h a t i n d i v i d u a l fatty acids m i g h t have.
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