Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
Modified Fat Blends
Background of the Invention
This invention relates to fat blends and methods for their manufacture and
use.
Hayes et al., 53 3. Clin. Nutr. 491, 1991, and Khosla and Hayes, 55 M. J.
Clin. Nutr. 51,
1992 (not admitted to be prior art to the present application) describe the
effect of various fat
blends formed from five different plant oils on plasma cholesterol and
lipoprotein levels in non
human primates. The response to specific saturated fatty acids was assessed in
three species of
monkey known to differ in their susceptibility to atherosclerosis and in their
plasma cholesterol
response to consumption of saturated fat.
Pronczuk et al., 26 Lipids 213, 1991, describe experiments on the effect of
various animal
fats (butter, tallow, lard, and fish oil) upon three species of monkey. They
state that substitution
of fish oil for corn oil decreases plasma cholesterol despite the fish oil
diet containing more
saturated fatty acid than the corn oil diet.
Mensink and Katan, 323 New England Journal of Medicine 439, 1990, and Zock and
Katan 33 J. Lipid Research 399, 1992 describe the deleterious effect of
dietary trans fatty acids
on depressing high density (HDL) and increasing low density (LDL) lipoprotein
cholesterol
levels in healthy subjects. Such a shift in the serum lipoprotein profile is
thought to be
atherogenic.
Hegsted et al., 17 American Journal of Clinical Nutrition 281, 1965, describe
the effects
of dietary fat on serum cholesterol in man. Test oils were used primarily
incorporating them into
recipes for many products such as waffles, muffins, cakes, cookies, pie
crusts, biscuits, salad
dressings, and spreads for bread.
Summary of the Invention
Applicant has discovered that specific blends of animal fats and vegetable
oils or fish oils
are effective in maintaining low serum cholesterol levels in mammals, such as
humans and other
primates. These blends are useful not only as dietary constituents that
favorably modulate
plasma cholesterol but also provide advantageous use in varioras cooking
procedures, such as in
deep-fat frying and baking, in dairy products e.g_, frozen deserts (such as
ice cream) or yogurt,
creams, cheeses, spreads (such as butter/margarine blends), in diet drinks, in
foods for
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specialized diets (~, hospital foods), and in other blended products, ~, salad
dressing, peanut
butter, and margarines.
In this invention, the saturated fat portion of the blend is derived from
animal fat and is
initially reduced in its cholesterol content by use of a non-hydrogenation
procedure, much as
described by Marschner et al. in U.S. Patents 4,804,555 and 4,99f,072, and
other equivalent
methods known in the art, for example, employing supercritical fluid
extraction, or extraction
using cyclodextrans. Such fats are termed herein cholesterol-reduced. A
cholesterol-reduced
animal fat is defined as one in which the amount of cholesterol in the fat is
reduced to less than
40, preferably less than 20 milligrams cholesterol per 100 grams of fat, or
one which contains
less than 50%, preferably less than 10%, of its original cholesterol content.
Critical also in the
invention, however, is the blending of the cholesterol-reduced animal fat with
vegetable oil such
that the final ratio of two key fatty acids (which can be determined by
standard procedures) is
between about 2 and 9. These key fatty acids are linoleic acid (refereed to as
18:2) and myristic
acid (referred to as 14:0) and the ratio is the percentage of diet~~ry energy
calories (abbreviated as
% energy) contributed by the linoleic acid divided by the % energy contributed
by the myristic
acid; i.e., 18:2 divided by 14:0. This % energy ratio is equivalent to the
weight/weight ratio of
these two fatty acids in the diet because both fatty acids have the same
metabolic energy yields.
That is, Iinoleic and myristic acids are metabolized in man to release
approximately the same
number of calories on a gram for gram basis. Thus, the ratio of percentages of
dietary calories
contributed by the fatty acids can be simplified to the weight ratio of the
fatty acid components
in the cholesterol-reduced fat blend. This ratio of fatty acids provides an
index of metabolic
value for the fat blend, i.e., it predicts the impact of the injected fat on
the plasma cholesterol
level when consumed by human or animal. The overall effect on lowering of
serum cholesterol
concentration represents the combined impact of a favorable fatty acid ratio
plus the benefit
gained by removal of cholesterol from the animal fat. The combination of a
suitably elevated
18:2/14:0 fatty acid ratio and the use of cholesterol-reduced fat act
synergistically upon
cholesterol levels in an animal consuming the claimed blends.
In addition, applicant has discovered that such blends of fats are
particularly useful in
deep-fat frying, and other uses described above (and listed in Table 5 below),
compared to the
individual fat or oil alone. Unmodified vegetable oils (i.e., non-
hydrogenated) can only be used
with difficulty in such processes since they are susceptible to oxidation upon
being heated and
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agitated. The oxidation products are thought to be hazardous to health. Addis
and Warner, In
Free Radicals and Food Additives, eds. Aruoma and Halliwell, Ch. 5, 1991;
Addis and Hassel, In
Food Safety Assessment, eds. Finley et aL, Ch. 30, 1990; Park and Addis, In
Biological Effects
of Cholesterol Oxides, eds. Peng and Morin, Ch. 3, 1991; Addis and Park, In
Biological Effects
of Cholesterol Oxides, Ch. 4, 1991; and Zhang and Addis, 55 J. Food Sci, 1673,
1990. Addition
of anti-oxidants to such vegetable oils is of relatively limited utility when
the oil is heated to
temperatures in excess of 100°C, and in particular at temperatures
between 140°C-170°C (which
are used in deep-fat frying). The combination of the vegetable oil and animal
fat provides a
blend which has advantageous oxidation properties, such that the blended
polyunsaturated fatty
acids are less readily oxidized in the presence of saturated fatty acids even
when heated at the
high temperatures described above. For example, the blend has an increased
stability to
oxidation when heated to 100°C, or greater, in air, at least 25%
greater than the vegetable oil
component heated separately from the animal fat component. In addition, the
metabolic value of
the animal fat i.e., its effect on cholesterol metabolism is significantly
improved by blending of
the oil with the fat.
Thus, in a first aspect, the invention features a blended cholesterol-reduced
animal fat and
vegetable oil which are proportionally combined in relative amounts to provide
a fat blend
including a linoleic acid content (L) and a myristic acid content (M) in a
weight ratio between 2
and 9 inclusive ( i.e., L:M).
In related aspects, the invention features a method for making a blended fat
composition
by combining the above-mentioned fat and oil to provide the desired ratio
described above, and a
method for using the blend for cooking, for example, in deep-fat frying or the
other uses
described above. The blending of the fat and oil is performed by standard
procedure. Another
related aspect of the invention features a method for hardening vegetable and
fish oils by
addition of cholesterol-reduced animal fat to produce solid shortenings and
rnargarines. In yet
another aspect, the blend is formed by combining one part by weight vegetable
oil or cholesterol-
reduced fish oil with between one and ten parts by weight cholesterol-reduced
animal fat.
In preferred embodiments, the animal fat is highly cholesterol-reduced, i.e.,
the
cholesterol concentration has been reduced between 90% and 99% of its original
concentration;
the ratio of fats is selected to provide a blend more stable to oxidation upon
heating at
temperatures in excess of 100°C (and preferably m~re stable at
temperatures between 140°C-
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170°C); the animal fat is tallow, lard, milk fat, mutton fat, chicken
fat, egg fat, or turkey fat; the
cholesterol-reduced animal fat contains between 3 and 40 mg cholesterol/100
grams tallow,
between S and 40 mg cholesterol/100 grams butter fat, and between 2 and 30 mg
cholesterol/100
grams lard; and the vegetable oil is one including linoleic acid, ,e.~" it is
safflower oil, sunflower
oil, corn oil, soybean oil, cottonseed oil, peanut oil, canola oil, olive oil
or palm olefin. In
addition, cholesterol-stripped fish oil can be blended with other cholesterol-
stripped saturated
animal fats to stabilize the fish oil against air oxidation at room-
temperature (20-25°C).
Blends of this invention are believed to be advantageously less thrombogenic
than the
animal fat component alone, and potentially can be used as antioxidants in
food formulations.
Since excess 18:2 consumption is potentially deleterious, the proposed blends
can be used to
raise serum HDL levels and reduce serum LDL susceptibility to oxidation, and
to reduce risk of
cancer, arthritis and other adverse prostaglandin responses.
Other features and advantages of the invention will be apparent from the
following
description of the preferred embodiments thereof, and from the claims.
Description of the Preferred Embodiments
The drawings will first briefly be described.
Drawings
Figs. 1-10 are graphical representations of the amount of oxidation of various
fats heated
separately, compared with the same amounts of fats combined and heated as fat
blends,
measured by spectrometry at between 110 and 300 nm; specifically, Fig. 1 shows
results
obtained with a 1:1 beef tallow: corn oil mixture heated at 100°C for
nineteen hours (line (a)}
and of the same beef tallow and corn oil heated separately and then combined
after heating (line
~)) 9
Fig. 2 shows the effect of heating at 100°C for nineteen hours on the
level of oxidation
products of a blend of beef tallow and corn oil at a 9:1 ratio (line (a}), and
of the same fat and oil
heated separately (line (b});
Fig. 3 shows the oxidation products of beef tallow heated alone at
100°C for nineteen
hours (control);
Fig. 4 shows the oxidation products of corn oil when heated at 100°C
for either seventeen
or forty-one hours, either alone (1X} or diluted ten-fold (0.1 X) in mineral
oil;
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Fig. S is a graph showing the amount of oxidation products detected at 230
nanometers
for corn oil mixed with varying amounts of mineral oil;
Figs. 6, 7, 8, and 9 are graphs showing the effect of heating undiluted (1X)
(or ten-fold
diluted in mineral oil (0. 1X)) soybean, canola, sunflower, and peanut oil
respectively for
seventeen hours at 100°C;
Fig. 10 is similar to Fig. ~, but shows the effect of heating peanut oil for
thirty-eight
hours rather than seventeen hours;
Figs. 11-15 are histograms showing the effect of various dietary fatty acid
ratios on
cholesterol levels in gerbils and hamsters; specifically Fig. 11 depicts the
cholesterol response in
gerbils fed a purified diet containing 40% energy as cholesterol-reduced
butter (B, left group) or
cholesterol-reduced tallow (T, starting with 6th column) with increasing
amounts of safflower oil
(left to right) to produce a final blend with the 18:2/14:0 ratio indicated on
the lower axis. The
nasal cholesterol (maximal lowering effect) is reached when the ratio is
approximately 5.0 for
tallow and 8.0 for butter. As discussed below, a lower ratio is effective with
a butter blend since
more can be added to compensate for its high 14:0 content, and once a
"threshold", level of 18:2
is reached that level of 18:2 will have the desired cholesterol lowering
effect;
Figure 12 depicts the plasma cholesterol response in gerbils fed 40% energy as
coconut
oil (having a high saturated fatty acid content) or safflower oil (having a
high polyunsaturated
fatty acid content) and various forms of butter: 1) with its natural
cholesterol load (263mg);
2) cholesterol-reduced (shown as "stripped", to l2mg per 100 gr); or
cholesterol-reduced butter
with cholesterol added back at 225mg per 100 gr of fat. The benefit of
reducing cholesterol
levels is evident;
Figure 13 compares the effect on gerbil plasma cholesterol levels of lard
stock with that
of cholesterol reduced lard, or the latter with 22 mg cholesterol added per
100 gr fat. A lard
olefin fraction is also compared in its effect;
Figure 14 compares the effect on gerbil cholesterol levels of tallow stock,
tallow
fractions, or cholesterol-reduced tallow with or without 225 mg cholesterol
per 100 gr fat;
Figure 15 shows that the low (inadequate) level of 18:2 in stock butter fat
contributed
about +26mg/dl plasma cholesterol, whereas the contribution from the
cholesterol in butter was
about +30mg1d1. When both factors were adjusted, by reducing cholesterol
levels and adding
18:2, (column 5), the response was -66mg/dl, i.e., more than the -56mg/dl
predicted by adding
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the two individual factors together. The improved butter fat now approaches
safflower oil in its
metabolic profile. These data demonstrate the synergistic effect of
cholesterol reduction and an
improved 18:2/14:0 ratio which together form the basis of this invention;
The data generated in Figs. 16-19 was obtained using vegetable oil-based,
cholesterol-
free, diets.
Figs. 16A and 16B are graphs of plasma cholesterol levels (mg/D1) plotted
against the
specific % energy derived from 14:0 (Fig. 16a), or % energy from 18:2 (Fig.
16b);
Fig. 17 is a graph of the observed plasma cholesterol levels (mgldL) in rebus
monkey
compared to that predicted from a multiple regression based on the °lo
energy from 14:0 and
18:2;
Fig. 18 is a graph of the ratio of % energy (18:2!14:0) to plasma cholesterol
levels in
humans; and rebus monkeys; and
Fig. 19 is a graph of the predicted serum cholesterol corr~.pared to observed
serum
cholesterol (mg/DL) in rebus monkeys.
Oxidation of Improved Fat Blends
One significant problem accompanying storage and cooling with a
polyunsaturated fat is
its susceptibility to air oxidation. The process of oxidation represents the
peroxidation of linoleic
and Iinolenic acids after which the fat becomes rancid and unpalatable.
Antioxidant chemicals
such as TBHQ (tertiary butylhydroquinone) may be added to a polyunsaturated
fat but these are
expensive and of limited utility in slowing peroxidation when the fat is being
heated and agitated
(e.g=, during deep-fat frying) because they are absorbed by the food being
fried and rapidly
depleted from the oil. Brooks, 2(12) Inform 1091, 1991. Some of the oxidation
products of
polyunsaturated fats are known to be toxic, atherogenic, and/or carcinogenic
as discussed by
Addis and coworkers in references cited above. Fats can be hardened by
hydrogenation, but this
process is costly and can also generate atypical fatty acid isomers that can
be harmful to humans.
We have discovered that the rate of peroxidation of polyunsaturated fats
(measured
spectrophotometrically during the course of accelerated oxidation at
100°C and above) can be
significantly reduced by dilution of these fats with a saturated fat, such as
beef tallow (or other
edible fat or fat substitute which is relatively resistant to oxidation). Such
dilution has the added
advantage of improving the dietary utility of the saturated fat providing that
the relative
CA 02444088 2003-10-21
''
concentrations of specific fatty acids within the blend are within a desired
range, as described
below.
Dilution of polyunsaturated fats including corn oil, soybean oil, peanut oil,
canola oil and
sunflower oil with two or mare volumes of saturated fats, significantly
reduces the specific rate
of peroxidation of the polyunsaturated fat (i-e., the rate of oxidation per
gram of the
polyunsaturated fat). For example, on a gram for gram basis, pure corn oil is
oxidized twice as
rapidly as corn oil which has been diluted with four volumes of beef tallow.
This discovery is
surprising since dilution of vegetable oils (containing natural antioxidants
such as the
tocopherols and the carotenes) into animal fats or other diluents containing
relatively low
concentrations or no antioxidants, would be expected to accelerate the rate of
peroxidation.
Example 1: Heat Resistant Blends
Referring to Figs. 1-10, the effect of blending an animal fat acid (or mineral
oil) and
vegetable oil on the rate of oxidation of the fats is demonstrated. In each
experiment shown in
the figures, the relevant fat (fatty acid) acid mixture was heated at
I00°C for the time noted and
the optical density recorded. The amount of conjugated diene and triene fatty
acids formed by
peroxidation of polyunsaturated fats was measured at an optical density
between 210 and 250
nanometers. Heating the polyunsaturated fats (vegetable oils) separately from
the saturated fats
or fat substitutes, ~, beef tallow or mineral oil, shows significant oxidation
of the
polyunsaturated fatty acid components. Mixing at a ratio of 1:l provides a
small improvement in
the oxidation stability of the vegetable oil, and mixing at a ratio of 9:1
animal to vegetable oil
provides a significant improvement, i.e., lesser amounts of oxidation.
These findings, together with the data provided below for cholesterolemia
demonstrate
that the advantages of a reduced serum cholesterol level, and an improved
blend resistant to
oxidation, can both be achieved by using blended fat compositions containing
between two and
ten parts of a saturated fat source (such as tallow) to each part of
polyunsaturated fat (such as
corn or soybean oil).
The current practice of utilizing partially hydrogenated, i.e., hardened
polyunsaturated
fats, introduces the metabolically undesirable (i-e., atherogenic, see above)
trans fatty acids as
opposed to natural unsaturates. The present invention substitutes cholesterol-
reduced naturally
solid saturated fats mixed in a blend with natural liquid forms of
polyunsaturated oils or
CA 02444088 2003-10-21
~s
8
cholesterol-reduced fish oils to produce a solid at room temperature,
oxidation-resistant, heat-
stable, cholesterol-lowering fat blend, ~., in the form of a shortening. This
allows production
of a deep fry cooking fat with optimal physical and metabolic (cholesterol
control) properties,
while maintaining a completely natural (unhydrogenated) product.
Cholesterol Lowering Blends
Studies of the influence of dietary fat saturation and cholesterolemia are
almost 40 years
old. From the earliest observations (on the ability of vegetable oils to lower
plasma total
cholesterol relative to animal fats) to the more recent findings of the Lipid
Research Clinics
Coronary Primary Prevention Trial (involving more than 6000 subjects), it has
been clearly
established that saturated fats raise plasma cholesterol whereas
polyunsaturated fats lower it.
(National Research Council, Committee on Diet and Health Food and Nutrition
Board
Commission on Life Sciences: in Diet and I-Iealth: Implications for Reducing
Chronic Disease
Risk National Academy Press, Washington, DC 1989). These findings led to mass
introduction
of polyenes in the market place (since the 1950's) which doubled the typical
polyene
consumption from 1940 to 1985 from 2.5% energy to 5.4°l° energy
(Stephen et al., "Trends in
individual consumption of dietary fat in the United States", 52 Am. d. Clin.
Nutr. 457, 1990).
The rise in polyunsaturated fat intake has been associated with a peak and
decline in coronary
heart disease and serum cholesterol (Commission on Life Sciences, 1989,
supra). However,
despite this vast body of data, much confusion persists concerning the effects
of specific fatty
acids, and more importantly, about the underlying mechanism of their action on
LDL and HDL
dynamics.
Dietary fatty acid interrelationships are important because the LDL/HDL ratio
appears
critical to the atherogenic potential of the lipoproteins. Applicant believes
that a proper balance
in the fats and oils (specifically the fatty acids) consumed will enhance the
circulating lipoprotein
profile. Amongst the saturated fatty acids, those containing twelve to sixteen
carbons (12C-16C;
lauric, myristic and palmitic acid, respectively) were historically thought to
raise the plasma
cholesterol and LDL-C, whereas those containing less than 12C or the 18C,
stearic acid, were
considered neutral. The monounsahirated fatty acid oleic acid ( 18: I ), has
no effect on plasma
cholesterol when exchanged for carbohydrate, but exerts a cholesterol-lowering
effect (both LDL
and total) when exchanged for saturated fatty acids. Similarly, the major
polyunsaturated fatty
CA 02444088 2003-10-21
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acid, linoleic acid (18:2), is cholesterol-lowering, both independent of other
fatty acids, but
especially when exchanged for dietary saturated fatty acids. However, I8:2
also lowers HDL-C
at high intakes (>20% energy, Mattson et al., 26 J. Laid Res.l94, 1985).
Applicant has found that, in normocholesterolemic individuals and when dietary
cholesterol is less than 300 mg/day, 18:2 and 14:0 are the two key fatty acids
that affect the
plasma lipid profile. The response exists across species and the effect of
18:2 is nonlinear, i.e.,
there is a "threshold" level of 18:2 above which a further effect on plasma
cholesterol is
minimal. Thus, in the absence of dietary cholesterol and in subjects with
normal LDL receptor
activity, 14:0 appears to be the only fatty acid that raises (in a linear
fashion) the plasma
cholesterol, whereas 18:2 (because of the nonlinear response to 18:2) lowers
it up to a certain
"threshold", level of dietary 18:2. This is most graphically des<;ribed by the
dietary ratio of the
percentage of dietary calories consumed as 18:2 vs. 14:0 °,~~ energy.
However, accurate
prediction requires the use of a multiple regression equation involving the
two fatty acids.
Palmitic and oleic acids appear to be neutral in these situations.
Example 2: Optimized Fat Blends
Referring to Figs. 11-15, these data demonstrate the plasma cholesterol
response to
variations in dietary 18:2/14:0 ratios. These data were generated using animal
fat with a reduced
cholesterol concentration (as described in Marschner et al., supra) and show
that the ratio of
18:2/14:0 is a useful predictor of the effect of a chosen blend on the
cholesterol response in
animals (including humans).
Young adult male gerbils were fed purified diets, containing 20% (w/w) fat
(40% of
dietary calories) for 4 weeks at which time plasma cholesterol and
triglyeeride concentrations
were determined. The choice of fat ranged from coconut oil (86% saturated
fatty acids) to
safflower oil (9% saturated fatty acids). In addition, stock supplies of three
animal fats (butter,
2S lard, and tallow) or these three fats reduced in cholesterol were fed to
the animals. A detailed
description of the basic diet and feeding protocol is published in 122 J.
Nutr. 374, 1992. Dietary
cholesterol was also added back to the cholesterol-reduced product and fed to
a separate group of
animals. In the case of tallow, special fractions of stearin and olefin were
fed as well.
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The data clearly show the beneficial effect of removing cholesterol from the
animal fats
as evidenced by the reduction in plasma cholesterol concentration with butter
(Figure 12), lard
(Figure 13), and tallow (Figure 14).
In Figure 11 when graded amounts of 18:2 (supplied as safflower oil) were
blended with
either cholesterol-reduced butter or cholesterol-reduced tallow it was
discovered that weight
proportions of 1:4 (safflower oil/tallow) and 1:1.5 (safflower/butaer) were
necessary to lower the
plasma cholesterol to basal values for gerbils (i.e., to 75 mg/dl) equivalent
to 120-130mg/dl for
humans. Based upon the known 18:2 and 14:0 content of these separate fats, and
consequently
these fat blends, these weight proportions indicate that an 18:2/14:0 ratio
between 2-9 (and
particularly between 4 and 8, or even 6 and 8) is adequate to neutralize and
actually lower the
hypercholesterolemic effect normally associated with these two saturated
animal fats (tallow and
butter). Moreover, the data show that blending the fats within: this range of
18:2/14:0 ratios
actually rendered the fats maximally hypocholesterolemic.
A single 18:2/14:0 ratio applicable to all fat blends is not practical because
(as the
regression equation discussed below indicates) the cholesterol-lowering impact
of increasing
dietary consumption of 18:2 on serum cholesterol is logarithmic. To determine
precisely what
the ratio should be to neutralize or lower the plasma cholesterol, one must
apply a full multiple
regression equation as expressed for a particular animal species (see Table
1). This will indicate
the exact percentage of dietary energy as calories (% energy) added as 18:2
which is required to
counteract any given intake of 14:0. Since the regression equations for cebus,
gerbils and
humans are so similar, information obtained from cebus and gerbils is highly
predictive of the
humans response. This has allowed the formulation of optimum fat blends for
humans.
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Table 1
Re,~ession Equations For Cholesterol Response to Dietary
FAs: 4 Species Comparison
Gerbil PC1~ - 126 + 8 Ej4;o - 40 logEl8;2 r2=0.91 (25 diets)
Cebus PC - 192 + 10 El4:o - 48 logElB:z r2=0.92 (16 duets)
Human PC - 229 + 8 Et4:o - 36 logEls:a r2=0.85 (17 eliets)
Hamster PC - 160 + 5 Ela:o -~ 26 log Et8:2 r2=0.74 (13 diets)
In summary, plasma cholesterol response to the dietary intake e~f 18:2 does
not vary on a linear
basis (i.e., it is logarithmic). Thus, a single 18:2/14:0 ratio (which implies
a linear relationship)
to provide the benefits of the present invention would be unrealistically
limiting and an
oversimplification of the data. However, the beneficial hypocholesterolemic
response in man
achieved through this invention is obtained by preparing animal and vegetable
oil blends whose
18:2/14:0 ratio based on weight percentage composition fall between 2 and 9.
This range would
represent dietary consumption between 10 and 25 grams per day 18:2 for an
average human
male. More precisely, ratios between 2 and 5 will neutralize (prevent the
usual cholesterolemic
effect caused by) the cholesterol-reduced saturated fat consumed alone. Ratios
between 5 and 9
allow the fat blend to actually lower the plasma cholesterol concentration
equal to the same
extent as that induced by a polyunsaturated vegetable oil.
Referring specifically to Fig. 1 S, the synergistic affect of the ratio
discussed above with
use of cholesterol-reduced animal fat is demonstrated (column 5). The level of
plasma
cholesterol in hamsters is significantly less than expected from either
feeding of cholesterol-
reduced milk fat alone or feeding of the appropriate proportion of oil
(polyunsaturated fatty
acids) to the stock animal fat.
Primate Analyses
The above data on predictive values of fatty acid ratios were obtained with
hamsters and
gerbils. Below is provided an analysis of data obtained with monkeys and
humans which
demonstrates the universal applicability of the claimed ratio, and in
particular its utility in
'/ PC represents the predicted plasma cholesterol concentration. The constant
term (eg, 138 for gerbils)
represents the reference plasma cholestero:~ value for a given species
independent of any specific fatty acid impact.
E,4;o is the % energy as 14:0 in the diet, while LogE~B;~ is log % energy as
18:2 in the diet, 1z represents the percent
of total variation in plasma cholesterol explained by the regression equation
(~, 95% for gerbil equation).
CA 02444088 2003-10-21
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humans. The following demonstrates that it is the ratio of dietary consumption
of 18:2 and 14:0
that is critical to predicting the cholesterol response in normal individuals.
Results obtained in cebus monkeys (see below) indicate that dietary myristic
(14:0) and
palmitic (16:0) acids exert disparate effects on cholesterol metabolism, while
the ability of
S linoleic acid (18:2) to decrease total plasma cholesterol displays an upper
limit or threshold.
Reanalysis of published data (see below) suggests a similar situation pertains
in humans.14:0
appears to be the principal saturated fatty acid that raises plasma
cholesterol whereas 18:2 lowers
it. Oleic acid (18:1) appears neutral. The effect of 16:0 may vary. In
normocholesterolemic
subjects consuming diets containing < 300 mg/day of cholesterol, 16:0 appears
to be without
effect on plasma cholesterol. However, in hypercholesterolemic subjects (>22S
mg/dL) and
especially those consuming diets providing cholesterol intakes of > 400
mg/day, dietary 16:0
may expand the plasma cholesterol pool.
Example 3: Cebus Monkeys
The cebus was utilized because its plasma cholesterol is extremely sensitive
to variations
in dietary fat saturation. Although more sensitive than humans in the
magnitude of their
response, cebus respond in the same manner.
Applicant analyzed data accumulated from 16 dietary fat: feeding trials in
rebus monkeys
over six years. Utilizing the approach originally used by Hegsted et al. (17
Am. J. Clin. Nutri.
281, 1965) to quantitate the effects of dietary fat on serum cholesterol in
man, the data was
subjected to multiple regression analysis to ascertain the ability of specific
dietary fatty acids to
predict the plasma cholesterol concentration.
The data base used for the analyses described herein represent a summary of
results from
various feeding studies over the six years in which rebus monkeys were fed 16
cholesterol free
purified diets as described in Table 2, Diets 1 and 2, from Pronczuk et al.,
26 Lipids 213, 1991
2S diets, 3-7 from Hayes et al., S3 Am. J. Clin. Nutr. 491, 1991, diets 8-10
from Khosla et al., SS
Am. J. Clin. Nutr. 51, 1992, diets 11-13 from Hegsted, supra, and diets 14-16
from Khosla et al.,
6 FASEB. J. (Abstract 1992).
CA 02444088 2003-10-21
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Table 2
Diets, percentage energy from dietary fatty acids, and the observed plasma
cholesterol
______________________________________________ Dietary fatty acids
_____________________________________ p1 asma Cholesterol'
Dieta(n)12:0 14:0 16:0 18:0 18:1 18:2 18:3
1(4) 0.00 0.06 3.69 0.68 7.78 18.57 0.19 152110
2(4) 14.73 6.88 4.00 1.27 3.35 0.78 0.00 26325
3(8) 14.82 5.83 3.32 1.02 2.91 2.64 0.28 24617
4(8) 7.38 2.98 2.67 0.93 11.74 4.96 0.37 1918
5(8) 4.15 1.80 7.78 1.12 11.53 4.12 0.25 186113
6(8) 0.06 0.31 12.49 1.27 11.47 4.77 0.31 16111
7(8) O.I2 0.22 7.25 1.2I 12.74 8.43 0.84 15I~9
8(9) 0.64 0.52 2.08 1.00 29.64 5.76 0.08 1426
9(9) 0.92 0.56 16.28 1.92 15.64 3.92 0.16 1456
~
10(9) 0.60 0.52 2.52 0.12 5.48 29.12 0.16 1185
'~
11(10) 19.12 7.52 4.28 1.32 3.76 3.40 0.36 23310
12(10) 0.08 0.40 16.12 1.64 14.80 6.16 040 1558
13(10) 0.16 0.28 9.36 1.56 16.44 10.88 1.08 1458
14(6) 0.00 0.47 16.96 1.52 8.84 3.22 0.00 183 11
15(I2) 0.00 0.31 11.19 1.27 13.80 3.44 0.00 1775
16(6) 0.00 0.25 7.41 1.21 17.95 3.63 0.00 176 10
~ ( ~
Diets were fed with fat contributing either 31% energy (#s 1-7 & 14-16), or
40% energy (#s $-
13). All diets were cholesterol-free. The fatty acid composition of each diet
was determined by GLC.
Dietary fats were formulated (using either a single oil or blends of oils) as
follows: 1, Corn Oil; 2,
Coconut Oil; 3 & 11, 90% Coconut Oil/ 10% Soybean Oil; 4, 4.'i% Coconut Oil/
40% High Oleic
Safflower Oil/ 15% Soybean Oil; 5, 45% Palm Oil/ 22% Coconut Oil/ 20% High-
Oleic Safflower Oil/
13% Soybean OiI; 6 & 12, 90% Palm Oil/ IO% Soybean Oil; 7 & 13, 45% Palm Oil/
40% Soybean Oil/
15% High-Oleic Safflower Oil; 8, High-Oleic Safflower Oil; 9, Palrn Oil; 10,
High Linoleic Safflower
Oil; 14, 95% Palm Stearin/ 5% High-Linoleic Safflower Oil; 15, 54°'o
Palm Stearin/ 43% Olive Oil/ 3%
High-Linoleic Safflower Oil; 16, 24% Palm Stearin/ 75% Olive Oil/ 1% High-
Linoleic Safflower Oil.
6 Number of monkeys. ° mg/dl plasma, Mean f SEM.
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The diet provides either 31°/~ or 40% of the energy as fat with the
range in % energy from the
most predominant fatty acids as follows: 12:0 (0-19%); I4:0 (0-7.5%); I6:0 (2-
17%); 18:0 (0.7-
1.9%); 18:1 (3-30%); 18:2 (1-29%); 18:3 (0-1.1%). In all cases total
cholesterol was determined
enzymatically on fasting plasma samples.
The final data-set includes I29 cholesterol values generated from a group of
16 monkeys
fed a total of 16 different diets. The composition of the diets has been
detailed previously
(Hayes et al., 53 Am. J. Clin. Nutr. 49I, 1991; I~hosla et al., '.>5 Am. J.
Clin. Nutr. 51, 1992;
Pronczuk et al., 26 Lipids 213, 1991, all hereby incorporated by reference
herein). The dietary
protein source was either lactalbumin (Diet #s 1-7, 11-16) or l:actalbumin and
casein (Diet #s
8-10). The fat source fed was either a single oil (Diet #s 1, 2 and 8-10) or
blend of oils (Diet #s
3-7 and II-16) designed to isolate specific fatty acid effects. To ensure that
the diets were
essentially cholesterol-free, only vegetable oils were employed. These
included coconut oil,
corn oil, soybean oil, hi-oleic safflower oil, hi-linoleic safflower oil, palm
oil, and olive oil.
With the exception of two diets (each fed to four different anu:r~als) all
diets were fed to 6-12
monkeys for 6-12 week periods. For all diets the fatty acid composition was
determined by GLC
(Haves et al., 53 Am. J. Clin. Nutr. 491, 1991).
In an attempt to define the plasma cholesterol (PC) response in terms of its
dietary fatty
acid descriptor(s), the observed plasma cholesterol (mean for a given diet)
was regressed against
the dietary energy (% of total) contributed by a specific fatty acids) to
generate the appropriate
multiple regression equations. kith seven dietary variables (the 7 major fatty
acids) a total of
127 possible regression equations resulted. Calculations were carried out on a
Macintosh Plus°
computer (Apple Systems Inc., Cupertino, CA) using the Statview 512+~ (Brain
Power Inc.,
Calabasca, CA) and Cricket Graphs (Cricket Software Inc;., Philadelphia PA)
statistical
packages.
The 129 individual cebus plasma cholesterol (PC) responses to all 16
cholesterol-free
diets averaged 1744 mg/dL (mean ~ SE) with a range of 96-355 mg/dL, indicating
that the
inherent cholesterol level for this group of monkeys was essentially normal.
On the basis of
individual fatty acids (Table 3), myristic acid alone explained 80% of the
variation in plasma
cholesterol (Eqn C8) while linoleic acid accounted for 66% of the observed
variation (Eqn C12).
No significant relationship was observed when palmitic, stearic or linolenic
acids were
considered alone.
CA 02444088 2003-10-21
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PC = 151 + 14E14:o r2 - 0.80 (C8)
PC = 240 - 90.6 log Elg:lz r2 - 0.66 (C12)
Table 3
Coefficients for Individual fatty acid regression in equations for cebus
monkeys fed 16
dietary fats.
.......... .. ...... .. ....... .. ....... ........~7ariable(s)...
.................. .. .......... .....
Ea 12:0 14:0 16.0 18:0 18:1 I8:2 18:3 Ib t2 SE
C? 5.46 - - - - - - 255 0.?52 20.10
C8 - 14.02 - - - -- - 151 0.800 18.60
C9 - - 1.94 - - - - 192 0.064 40.40
C10 - - - 5.81 - - - 1?0 0.004 41.67
Cl1 - - - - 3.39 - - 216 0.324 34.32
C12 - - - - - 90.60'r - 240 0.655 24.51
CI3 - - - - - - 2f.93 185 0.050 40.68
a Equation, Regressions C7, C8, Cll, C12 were significant at p<0.001.
Intercept of the regression equation r2 is a measure of the total variance
explained by the regression
equation, SE is the standard error around the regression line.
''r Indicates a log function
Myristic and linoleic acids had opposite effects, i-e., cholesterol-raising
and cholesterol-
lowering, respectively. Fig. 16(a) shows the effect on myristic acid on
cholesterol plasma levels.
In addition, the logarithmic nature of the response to 18:2 indicated a
nonlinear relationship
existed between increasing 18:2 intake and the observed pla;>ma cholesterol
(see Fig. I6b).
Although not a true dose-response curve for 18:2 (because increased ~/o energy
from 18:2 was
simultaneously coupled with decreased % energy from other fatay acids), Fig.
16b nevertheless
serves to illustrate the physiological impact of 18:2 on the nonlinear
relationship described by
Equation C8. It is apparent from Fig. 16b that increments of 18:2 reach a
"threshold" beyond
which further increases exert minimal impact on the plasma cholf;sterol level.
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The simplest, most inclusive multiple regression equation obtained by
including two or
more fatty acids (Eqn C14) revealed a regression coefficient of 0.92, and was
based on the
energy derived solely from myristic and linoleic acids which explained 92% of
the variation (r2)
in plasma cholesterol. The standard error about the regression was 12.6 mg/dL.
The constant
term (192) represents the "baseline" rebus plasma cholesterol value
independent of any dietary
fat effect.
PC =192 + 10 Elaao - 48 log Elg;2 r2=0.92 (C14)
The observed plasma cholesterol values plotted against the plasma cholesterol
predicted by Eqn
C14 are depicted in Figure 17.
Inclusion of one or two additional fatty acids, as well as 14:0 and 18:2
failed to improve
the predictability. Therefore, Eqn C 14 is predictive of cholesterol levels.
The logarithmic term
in Eqn C14 indicates a nonlinear response attributable to 18:2 intake.
Fig. 18 provides the simplest graphic illustration of the dietary fatty acid-
plasma
cholesterol relationship described by Eqns C14 and f11 (see below), by
plotting the ratio of the
I S energy from 18:2114:0 against the observed plasma cholesterol. The
decrease in plasma
cholesterol with an increasing 18:2/14:0 ratio exhibits a plateau (or
threshold) effect. ~nce this
threshold is reached, no further change in cholesterol is achieved. Numerous
other practical
combinations of fatty acids were regressed against the observed cholesterol
value but none
improved upon this simple ratio.
Example 4: Human Data
The above analysis of data from rebus fed cholesterol-free diets suggested
that a) all
saturated fatty acids (12C-18C) are not the same, b) the principal cholesterol-
raising and
cholesterol-lowering fatty acids are myristic and linoleic, respectively, and
that c) palmitic, oleic,
and stearic acids are neutral. In the most comprehensive study to date in
human subjects (36
diets) where individual dietary fatty acids are reported, Hegsted and
coworkers (17 Am. J. Clin.
Nutr. 17, 281, 1965) derived a relationship (Eqn H1).
DeltaSC=8.45 Delta E14:0 + 2.12 Delta E16:0-1.87 Delta E18:2 + 0,056 Delta C -
6.24 (HI)
This equation, with a multiple regression coefficient of 0.951, explained
about 95% of the
observed variation in serum cholesterol. Similar to the above rebus data,
changes in myristic
acid alone explained much of the total variation (69%) in the human serum
cholesterol response.
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In addition to myristic acid, equation Hl assigned a modest cholesterol-
raising role to both
palmitic acid and dietary cholesterol.
In contrast to the rebus diets, the human diets also contained dietary
cholesterol (range
106-686 mg per day), which is known to down-regulate LDL rf;ceptors. The human
data were
reanalyzed for a possible dietary cholesterol X dietary fatty ;acid
interaction. This analysis
revealed that the greatest deviation between observed and predicted serum
cholesterol (based on
Eqn H1) was attributable to diets with >400 mg cholesterol. Accordingly, the
human data were
split into two dietary groups, those receiving either <400 mg (ra.nge 1 I6-306
mg;n=19) or >400
mg (range 437-686 mg; n=I7) cholesterol per day. The cholesterol intake of the
former group
IO would presumably have minimal impact on LDL receptor status whereas some
degree of down-
regulation might be attributed to dietary cholesterol in the latter group.
When contributions from both dietary cholesterol and pa:lmitic acid were
ignored, 85.4%
of the variation in serum cholesterol could be explained solely by the intake
of 14:0 and 18:2
(Eqn HS). Thus, with cholesterol intake <400 mg (and presumably LDL receptor
activity not
compromised), inclusion of 16:0 and/or dietary cholesterol failed to improve
the predictability
after 14:0 and 18:2 had been considered, a finding comparable to the rebus
data.
Delta SC=9.10 Delta El4:o-I.78 Delta ElB:z-I0.1 ~ (HS).
Therefore, the simplest equatian for this group of humans with limited dietary
cholesterol
intake (Eqn HS) is very similar to that for rebus monkeys (Eqn C",14). Fig. 19
compares a plot of
the observed serum cholesterol for the 19 diets from the low-cholesterol group
(<400 mg per
day) with that predicted on the basis of Eqns HS and H1. The fit of the data
based on Equation
HS (using only I4:0 and 18:2) was comparable to that based on Eqn Hl (using
14:0, 16:0, 18:2
and dietary cholesterol).
When dietary cholesterol intake exceeded 400 mg per da:y, the regression
equation based
solely on 14:0 and I8:2 intake (Equation H6) accounted for 83.5% of the
observed variation in
serum cholesterol (r=0.914).
DeltaSC = 7.50 DeltaEta:o - 2.45 DeltaEl8:.2 + 10.89 (H6)
By contrast, the original regression equation (Eqn HI) that included 14:0,
16:0, 18:2 and dietary
cholesterol accounted for 93% of the variation and is superior for predicting
the serum
cholesterol response in this group with a high cholesterol intake. Thus,
inclusion of 16:0 in the
regression at the higher intake of dietary cholesterol appears to improve
predictability.
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Referring to Figs. 16 and 18, the response in plasma cholesterol to changes in
dietary
18:2 appeared nonlinear. Although these figures do not represent classic dose-
response curves
because changes in dietary energy from a particular fatty acid .are
necessarily accompanied by
changes in other fatty acids as well, the association with dietary I8:2 was
the only nonlinear
relationship observed in both monkeys and humans. Thus, the ''threshold"
concept seems
reasonable, i.e., the "threshold" (% energy) of 18:2 needed to counteract a
specific saturated fatty
acid (I4:0) varies depending on the % energy of 14:0 present.
This threshold concept is more appropriate than prior f;quations which
describe linear
relationships and imply that the plasma cholesterol can increase or decrease
indefinitely.
Empirical observation and physiological constraints suggest that the latter
situation is not the
case, since the observed plasma cholesterol in primates typically ranges
between fixed limits
(I I0-350 mg/dL). By the same token, an upper threshold in the response to
myristic acid at high
intakes is predicted. fIowever, in practical terms with real fats, it is not
possible to exceed 6-7%
energy from dietary myristate (as coconut oil).
Fortunately, essentially all practical human diets supply 14:0 and 18:2 within
the range of
the most dynamic portion of the curie in Fig. 18. Thus, the opportunity exists
to maximize the
dietary fat-associated reduction in plasma cholesterol by manipulating the
intake of these two
fatty acids. Fig. 16b implies that once the plateau of the curve is reached (5-
6% energy as 18:2
in most cases, but as low as 2-3% energy in the absence of dietary 14:0 and
cholesterol), the
composition of additional dietary fatty acids may not matter. Tlais would
explain why 18:1 and
even 16:0 appear to substitute for 18:2 once the threshold for 18:2 is
exceeded. Furthermore, in
practical dietary situations (i.e., those with modest intake of I4:0) in
normocholesterolemic
individuals (<200mg/dL) consumption of I8:2 in excess of 5-6% energy would be
without
appreciable beneficial effect and could have detrimental effects (i.e., would
be superfluous). The
cebus data, like the human data further suggest that 16:0 and 18::1 exert
minimal influence on the
plasma cholesterol concentration, emphasizing the neutrality of these two
fatty acids (the main
fatty acids in human diets) in normal individuals (man and animals). In this
case, "normal"
refers to an up-regulated LDLr activity reflected by a plasma cholesterol
concentration less than
200 mg/dL (LDL < 130 mg/dL).
The acute-slope regression ira Fig. 18 shows that small increments of dietary
14:0 are
extremely cholesterolemic when consumed by sensitive individuals (or species)
at low thresholds
CA 02444088 2003-10-21
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of 18:2. Although 14:0 is often discounted because of its relatively Iow
intake (i.e., typically 0-
2% total dietary energy), the rapid rise in total cholesterol over this range
of 14:0 at a fixed low
threshold of 18:2 suggests that 14:0 is the most potent fatty acid
contributing to the saturated fat
effect and that the 18:2 threshold would exert a counterbalancing effect only
as 18:2
consumption increased from 2-6 energy %, i.e., the range found in diets of
most populations.
These data thus indicate, as discussed above, that fatty acid intake should be
maintained
in a proper balance between 18:2 and 14:0 intake, i.e., within the ratio
described above for
normocholesterol individuals. Thus, blends of fats having such a ratio are
useful in aiding an
individual to maintain this ratio, and to consume food with high metabolic
value.
Example 5: Prototype Blends
Prototype blends by weight of selected vegetable oils with cholesterol-reduced
tallow
necessary to produce an 18:2/14:0 ratio of at least 5:1 that will assure
maximal lowering of
plasma cholesterol by the resulting shortening blend are shown in Table 4.
Ratios in parenthesis
represent blends that produce an 18:2/14:0 ratio of approximately 3:1,
allowing neutralization of
the typically cholesterolemic effect of cholesterol-reduced tallow when
consumed by humans. A
sample calculation is as follows: one part by weight sunflower oil containing
66% by weight
18:2 (see USDA Handbook No. 18-4, Composition of Foods-Fats and ~ils Raw
Processed
Prepared 1979) blended with eight parts cholesterol-reduced t;~.llow
(containing 4% by weight
18:2 and approximately 3.7% by weight 14:0) yields a blend having an 18:2/14:0
ratio as
follows: (1 x 66% + 8 x 4%) divided by 8 x 3.7% = 3.3:1).
Table 4
Blend Constituents Blend Proportions
Stripped tallow: soybean oil 3.5:I (7:1)
Stripped tallow: corn oil 3.5:1 (6:1)
Stripped tallow: canola oil (rapeseed)1.8:1 (4:1)
Stripped tallow: sunflower oil 4.0:1 (8:
I
)
Stripped tallow: palm olefin 1.0:I (2:1)
Stripped tallow: peanut oil 2.0:I (4:1)
Stripped tallow: safflower oil 4.5:1 (9:1
)
Stripped tallow: cottonseed oil 3.0:I (6:1)
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Table 5
Food Categories Individual Products
Spreads Margarine
Butter
Fats and Oils Shortening for baking
Cooking oil
French Fries
Fried Chicken
(or Chicken Nuggets)
Ready-to-Eat Baked
Goods Muffins
Breads
Rolls
All purpose baking
mixes
(e.g., Bisquick)
Ready-to-Eat Snacks Cookies
Crackers
Snack Chips Potato Chips
Corn Chips
Tortilla Chips
Popcorn with Topping
Food Categories Individual Products
Dressings & Sauces Salad Dressing
Dressings for Salads
Mayonnaise
Pasta Sauces
Dairy Products Ice cream
(May be more than one Ice cream novelties
category) (incl. sandwiches)
Cheeses
Process cheese
Milk ( 1 % and 2%)
Sour cream
Cream Cheese
Creams
Pizza
Ground beef
Sausage (All types)
Eggs
Desserts Puddings
Pies
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Cheese cake
'6Nhipped topping
Breakfast Items Cereals (esp. granola)
~7affles
Popcorn with Topping
Other embodiments are within the following claims.
