U.S. patent application number 13/423884 was filed with the patent office on 2012-11-01 for composition comprising plant and/or fish oils and non-oxidizable fatty acid entities.
This patent application is currently assigned to LIFE SCIENCE NUTRITION AS. Invention is credited to Rolf Berge.
Application Number | 20120276212 13/423884 |
Document ID | / |
Family ID | 35149662 |
Filed Date | 2012-11-01 |
United States Patent
Application |
20120276212 |
Kind Code |
A1 |
Berge; Rolf |
November 1, 2012 |
COMPOSITION COMPRISING PLANT AND/OR FISH OILS AND NON-OXIDIZABLE
FATTY ACID ENTITIES
Abstract
The present invention concerns a composition prepared from a
combination of plant oil and/or fish oil and a compound comprising
non. .beta.-oxidizable fatty acid analogues, and the use of said
composition for the preparation of a pharmaceutical or nutritional
composition for the prevention and/or treatment of insulin
resistance, obesity, diabetes, fatty liver, hypercholesterolemia,
dyslipidemia, atherosclerosis, coronary heart disease, thrombosis,
stenosis, secondary stenosis, myocardial infarction, stroke,
elevated blood pressure, endothelial dysfunction, procoagulant
state, polycystic ovary syndrome, the metabolic syndrome, cancer,
inflammatory disorders and proliferate skin disorders. The present
invention also concerns an animal feed prepared from a combination
of plant oil and/or fish oil and a compound comprising non
.beta.-oxidizable fatty acid analogues, the use of said feed for
improving the body composition of an animal, and a product produced
from said animal.
Inventors: |
Berge; Rolf; (Bones,
NO) |
Assignee: |
LIFE SCIENCE NUTRITION AS
Hovdebygda
NO
|
Family ID: |
35149662 |
Appl. No.: |
13/423884 |
Filed: |
March 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10550129 |
May 9, 2006 |
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PCT/NO2005/000271 |
Jul 19, 2005 |
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13423884 |
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Current U.S.
Class: |
424/523 ;
514/560 |
Current CPC
Class: |
A61P 15/00 20180101;
A61P 43/00 20180101; A61P 25/28 20180101; A61P 3/04 20180101; A61P
19/00 20180101; A61P 29/00 20180101; A23L 33/115 20160801; A61P
5/48 20180101; A61P 25/00 20180101; A61P 37/00 20180101; A61P 21/00
20180101; A61P 3/00 20180101; A61K 31/232 20130101; A23V 2002/00
20130101; A23K 10/12 20160501; A61P 5/14 20180101; A61P 7/10
20180101; A61P 3/06 20180101; A61P 3/10 20180101; A61P 9/12
20180101; A61K 31/685 20130101; A61P 7/00 20180101; A61P 37/06
20180101; A61P 17/00 20180101; A61K 31/10 20130101; A61P 5/30
20180101; A61P 7/02 20180101; A61P 9/00 20180101; A61P 1/16
20180101; A61K 31/19 20130101; A23K 50/30 20160501; A23L 33/12
20160801; A61K 45/06 20130101; A61P 5/50 20180101; A61P 9/10
20180101; A61P 31/04 20180101; A61P 35/00 20180101; A23K 50/75
20160501; A61P 9/08 20180101; A61K 31/231 20130101; A61K 38/01
20130101; A61K 31/10 20130101; A61K 2300/00 20130101; A61K 31/19
20130101; A61K 2300/00 20130101; A61K 31/231 20130101; A61K 2300/00
20130101; A61K 31/232 20130101; A61K 2300/00 20130101; A61K 31/685
20130101; A61K 2300/00 20130101; A61K 38/01 20130101; A61K 2300/00
20130101; A23V 2002/00 20130101; A23V 2250/164 20130101; A23V
2250/70 20130101; A23V 2250/18 20130101 |
Class at
Publication: |
424/523 ;
514/560 |
International
Class: |
A61K 35/60 20060101
A61K035/60; A61P 5/48 20060101 A61P005/48; A61P 3/04 20060101
A61P003/04; A61P 3/10 20060101 A61P003/10; A61P 1/16 20060101
A61P001/16; A61P 3/06 20060101 A61P003/06; A61P 9/10 20060101
A61P009/10; A61P 9/00 20060101 A61P009/00; A61P 7/02 20060101
A61P007/02; A61P 25/00 20060101 A61P025/00; A61P 9/12 20060101
A61P009/12; A61P 15/00 20060101 A61P015/00; A61P 3/00 20060101
A61P003/00; A61P 35/00 20060101 A61P035/00; A61P 29/00 20060101
A61P029/00; A61K 31/202 20060101 A61K031/202 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 19, 2004 |
NO |
20043091 |
Jul 19, 2004 |
NO |
20043093 |
Dec 17, 2004 |
NO |
20045544 |
Claims
1.-47. (canceled)
48. A method of improving the body composition of a subject
comprising the administration of a pharmaceutical or nutritional
composition comprising a combination of tetradecylthioacetic acid
or a salt thereof and fish oil under conditions that the body
composition of said subject is improved.
49. Method according to claim 48, where the compositions comprise a
daily dosage of said tetradecylthioacetic acid or a salt thereof of
from about 1-2000 mg.
50. Method of claim 48, wherein said tetradecylthioacetic acid is
provided as a phospholipid.
51. Method of claim 48, wherein said tetradecylthioacetic acid is
provided as a triglyceride.
52. Method of claim 48, wherein said fish oil comprises omega-3
fatty acids or derivatives thereof.
Description
FIELD OF INVENTION
[0001] The use of a combination of compounds comprising non
.beta.-oxidizable fatty acid entities and plant oil or fish oil has
shown surprising synergistic effects. The present invention
concerns a composition prepared from a combination of compounds
comprising non .beta.-oxidizable fatty acid entities and plant oil
and/or fish oil. Said composition may be used for the preparation
of a pharmaceutical or nutritional composition for the prevention
and/or treatment of insulin resistance, obesity, diabetes, fatty
liver, hypercholesterolemia, dyslipidemia, atherosclerosis,
coronary heart disease, thrombosis, stenosis, secondary stenosis,
myocardial infarction, stroke, elevated blood pressure, endothelial
dysfunction, procoagulant state, polycystic ovary syndrome, the
metabolic syndrome, cancer, inflammatory disorders and proliferate
skin disorders. Said composition may also be used as an additive to
animal fodder for routine feeding of animals in order to affect
their body composition in general and fatty acid composition
specifically.
BACKGROUND OF THE INVENTION
[0002] In earlier patent applications, the inventors have described
beneficial applications of non .beta.-oxidizable fatty acid
analogues of the present invention in the treatment and prevention
of obesity (NO 2000 5461), diabetes (NO 2000 5462), primary and
secondary stenosis (NO 2000 5463), cancer (NO 2002 5930),
proliferate skin disorders (NO 2003 1080), inflammatory and
autoimmune disorders (NO 2003 2054).
[0003] Surprisingly, the present inventors have now shown that the
use of a combination of compounds comprising non .beta.-oxidizable
fatty acid entities with plant oil or fish oil has synergistic
beneficial biological effects. The inventors show that the
combination of non .beta.-oxidizable fatty acid entities with plant
oil or fish oil lowers the concentration of plasma cholesterol,
triglycerides and phospholipids, and increase fatty acyl CoA
oxidase activity. In addition, the inventors describe how non
.beta.-oxidizable fatty acid entities and plant or fish oils can be
directly added to animal feed. The feed is digestible, and has
shown surprising effects on the fatty acid composition of the
animals. Based on these unexpected findings, it is anticipated that
the combination of compounds comprising non .beta.-oxidizable fatty
acid entities and plant oil and/or fish oil will have an increased
preventive and/or therapeutic effect on all the diseases the
compounds comprising non .beta.-oxidizable fatty acid entities are
effective against, compared to that of the compounds comprising the
fatty acid analogues alone.
[0004] Lipid is a cheaper energy source than protein, and farmers
want the animals to obtain as much energy as possible from lipid in
the diet (by increased lipid oxidation) and as little as possible
from protein. Animals also tend to grow more rapidly when their
feed contains a large proportion of fats, which is generally a
desirable feature. The invention is here exemplified by reference
to fish farming and fish, specifically Atlantic salmon.
[0005] More of the protein can be used in this way for muscle
growth. It has previously been shown that non .beta.-oxidizable
fatty acid analogues such as the 3-thia fatty acid
tetradecylthioacetic acid (TTA) can lead to increased hepatic and
muscle mitochondrial and peroxisomal fatty acid oxidation in
mammals (Berge et at, 1989b, Aarsland et al., 1989, Asiedu et al.,
1993, Skrede et al., 1993, Asiedu et al., 1995) and reduced body
fat in rats (Spiegelman 1998, Madsen et al, 2002).
DETAILED DESCRIPTION OF THE INVENTION
[0006] The present invention relates to the use of a preparation
comprising a combination of:
1) plant oil and/or fish oil; and 2) one or more compounds
comprising non .beta.-oxidizable fatty acid entities represented by
(a) the general formula R''--COO--(CH.sub.2).sub.2n+1--X--R',
wherein X is a sulphur atom, a selenium atom, an oxygen atom, a
CH.sub.2 group, a SO group or a SO.sub.2 group; n is an integer of
0 to 11; and R' is a linear or branched alkyl group, saturated or
unsaturated, optionally substituted, wherein the main chain of said
R' contains from 13 to 23 carbon atoms and optionally one or more
heterogroups selected from the group comprising an oxygen atom, a
sulphur atom, a selenium atom, an oxygen atom, a CH.sub.2 group, a
SO group and a SO.sub.2 group; and R'' is a hydrogen atom or an
alkyl group containing from 1 to 4 carbon atoms; and/or (b) the
general formula (I),
##STR00001##
wherein R1, R2, and R3 represent [0007] i) a hydrogen atom; or
[0008] ii) a group having the formula CO--R in which R is a linear
or branched alkyl group, saturated or unsaturated, optionally
substituted, and the main chain of said R contains from 1 to 25
carbon atoms; or [0009] iii) a group having the formula
CO--(CH.sub.2).sub.2n+1--X--R', wherein X is a sulphur atom, a
selenium atom, an oxygen atom, a CH.sub.2 group, a SO group or a
SO.sub.2 group; n is an integer of 0 to 11; and R' is a linear or
branched alkyl group, saturated or unsaturated, optionally
substituted, wherein the main chain of said R' contains from 13 to
23 carbon atoms and optionally one or more heterogroups selected
from the group comprising an oxygen atom, a sulphur atom, a
selenium atom, an oxygen atom, a CH.sub.2 group, a SO group and a
SO.sub.2 group; [0010] iv) an entity selected from the group
comprising --PO.sub.3CH.sub.2CHNH.sub.3COOH (serine),
PO.sub.3CH.sub.2CH.sub.2NH.sub.3 (ethanolamine),
PO.sub.3CH.sub.2CH.sub.2N(CH.sub.3).sub.3 (choline),
PO.sub.3CH.sub.2CHOHCH.sub.2OH (glycerol) and PO.sub.3(CHOH).sub.6
(inositol); wherein R1, R2, and R3 are chosen independently from
i), ii), or iv), but at least one of R1, R2, or R3 is defined by
iii); and/or (c) the general formula (II),
##STR00002##
[0010] wherein A1, A2 and A3 are chosen independently and represent
an oxygen atom, a sulphur atom or an N--R4 group in which R4 is a
hydrogen atom or a linear or branched alkyl group, saturated or
unsaturated, optionally substituted, containing from 1 to 5 carbon
atoms; wherein R1, R2, and R3 represent [0011] i) a hydrogen atom
or a linear or branched alkyl group, saturated or unsaturated,
optionally substituted, containing from 1 to 23 carbon atoms; or
[0012] ii) a group having the formula CO--R in which R is a linear
or branched alkyl group, saturated or unsaturated, optionally
substituted, and the main chain of said R contains from 1 to 25
carbon atoms; or [0013] iii) a group having the formula
CO--(CH.sub.2).sub.2n+1--X--R', wherein X is a sulphur atom, a
selenium atom, an oxygen atom, a CH.sub.2 group, a SO group or a
SO.sub.2 group; n is an integer of 0 to 11; and R' is a linear or
branched alkyl group, saturated or unsaturated, optionally
substituted, wherein the main chain of said R' contains from 13 to
23 carbon atoms and optionally one or more heterogroups selected
from the group comprising an oxygen atom, a sulphur atom, a
selenium atom, an oxygen atom, a CH.sub.2 group, a SO group and a
SO.sub.2 group; [0014] iv) an entity selected from the group
comprising --PO.sub.3CH.sub.2CHNH.sub.3COOH (serine),
PO.sub.3CH.sub.2CH.sub.2NH.sub.3 (ethanolamine),
PO.sub.3CH.sub.2CH.sub.2N(CH.sub.3).sub.3 (choline),
PO.sub.3CH.sub.2CHOHCH.sub.2OH (glycerol) and PO.sub.3(CHOH).sub.6
(inositol); wherein R1, R2, and R3 are chosen independently from
i), iii), or iv), but at least one of R1, R2, or R3 is defined by
iii); and/or a salt, prodrug or complex of the compounds according
to (a)-(c).
[0015] In a preferred embodiment of a compound according to the
invention at least one of R1, R2 or R3 is an alkyl.
[0016] In a preferred embodiment of a compound according to the
invention at least one of R1, R2 or R3 is an alkene.
[0017] In a preferred embodiment of a compound according to the
invention at least one of R1, R2 or R3 is an alkyne.
[0018] In a preferred embodiment of a compound according to the
invention at least one of R1, R2 or R3 is tetradecylthioacetic
acid.
[0019] In a preferred embodiment of a compound according to the
invention at least one of R1, R2 or R3 is tetradecylselenoacetic
acid.
[0020] Preferred embodiments of the compounds according to the
invention are non .beta.-oxidizable fatty acids.
[0021] In a preferred embodiment of a compound according to the
invention X is a sulphur atom or a selenium atom.
[0022] Preferred embodiments of the compounds according to the
invention are tetradecylthioacetic acid (TTA),
tetradecylselenoacetic acid and 3-Thia-15-heptadecyne.
[0023] In a preferred embodiment of a compound according to the
invention n is 0 or 1.
[0024] In a preferred embodiment of a compound according to the
invention said compound is a phospholipid, wherein said
phospholipid is selected from the group comprising phosphatidyl
serine, phosphatidyl choline, phosphatidyl ethanolamine,
phosphatidyl inositol, phosphatidyl glycerol, diphosphatidyl
glycerol.
[0025] In a preferred embodiment of a compound according to the
invention said compound is a triacylglycerol.
[0026] In a preferred embodiment of a compound according to the
invention said compound is a diacylglycerol.
[0027] In a preferred embodiment of a compound according to the
invention said compound is a monoacylglycerol.
[0028] In a preferred embodiment of a compound according to the
invention said compound is the phosphatidylcholine (PC) derivative
1,2-ditetradecylthioacetoyl-sn-glycero-3-phosphocholine.
[0029] In a preferred embodiment of a compound according to the
invention said compound is the phosphatidylethanolamine (PE)
derivative
1,2-ditetradecylthioacetoyl-sn-glycero-3-phosphoethanolamine.
[0030] Preferred embodiments of the compounds according to the
invention are mono-, di- or tri-acylglycerides.
[0031] Preferred embodiments of the compounds according to the
invention are tri-acylglycerides comprising tetradecylthioacetic
acid (TTA).
[0032] In a preferred embodiment of a compound according to formula
(II) A1 and A3 both represent an oxygen atom, while A2 represent a
sulphur atom or an N--R4 group in which R4 is a hydrogen atom or a
linear or branched alkyl group, saturated or unsaturated,
optionally substituted, containing from 1 to 5 carbon atoms.
[0033] The compounds according to the invention are analogues of
naturally occurring compounds, and as such are recognized by the
same systems which process the natural compounds, including the
enzymes that .beta.- and in some cases co-oxidize natural long
chain fatty acids. The analogues differ from their naturally
occurring counterparts in that they cannot be completely oxidized
in this manner.
[0034] The compounds according to the invention may be non
.beta.-oxidizable fatty acid analogues, as represented by the
formula R''CCO--(CH.sub.2).sub.2n+1--X--R'. However, said compounds
may also be more complex structures derived from one or more of
said non .beta.-oxidizable fatty acid analogues, as represented by
the general formulas (I) or (II). These compounds are analogues of
naturally occurring mono-, di-, and triacylglycerols, or
phospholipids including phosphatidyl serine, phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl
glycerol, and diphosphatidyl glycerol. Said compounds may also
comprise a substitution in the glycerol backbone, as shown in
formula (II). Said substitution of the oxygen(s) is achieved by
replacing the oxygen(s) with sulphur or a nitrogen containing
group. This may block hydrolysis before uptake by the intestines,
thus increasing the bioavailability of the compounds.
[0035] The above complex structures derived from one or more of
said non .beta.-oxidizable fatty acid entities have their effect
because the fatty acid analogues they comprise are not capable of
being fully .beta.-oxidized. Said complex structures may have an
effect as complete structures, and as naturally resulting
degradation products comprising the fatty acid analogues. Because
the compounds are not able to be fully .beta.-oxidized, they will
build up, and this triggers an increase in the .beta.-oxidation of
naturally occurring fatty acids. Many of the effects of the
compounds according to the invention are due to this increase in
.beta.-oxidation.
[0036] During .beta.-oxidation, a fatty acid is enzymatically
oxidized cleaved between carbons 2 and 3 (when counting from the
carboxylic end of the fatty acid), resulting in the removal of the
two carbon atoms on either side of the oxidation site as acetic
acid. This step is then repeated on the now two carbons shorter
fatty acid, and repeated again until the fatty acid is fully
oxidized. .beta.-oxidation is the usual way in which the majority
of fatty acids are catabolized in vivo. The .beta.-oxidation
blocking by the compounds according to the invention is achieved by
the insertion of a non-oxidizable group in the X position in the
formula of the present invention. Because the mechanism for
.beta.-oxidation is well known, X is defined as S, O, SO, SO.sub.2,
CH.sub.2 or Se. Anyone skilled in the art would assume, without an
inventive step, that these compounds would all block
.beta.-oxidation in the same manner.
[0037] In addition, the compounds may contain more than one block,
i.e. in addition to X, R' may optionally comprise one or more
heterogroups selected from the group comprising an oxygen atom, a
sulphur atom, a selenium atom, an oxygen atom, a CH.sub.2 group, a
SO group and a SO.sub.2 group. As an example, one may insert two or
three sulphurs as X to induce a change in the degradation of the
fatty acid and thus a modulated effect. Multiple sulphur atoms
would also modulate the polarity and stability somewhat. From a
pharmacological viewpoint it is generally desirable to be able to
present a spectrum of compounds rather than just one single
compound to avoid or counteract problems with resistance.
[0038] In addition to the identity of X, its position is also an
issue. The distance of X from the carboxylic end of the fatty acid
is defined by how many CH.sub.2 groups are positioned between X and
the carboxylic end of the fatty acid, which is defined by
(CH.sub.2).sub.2n+1, where n is an integer of 0 to 11. Thus these
are an odd number of CH.sub.2 groups, that is; the position of X
relative to the carboxyl group is such that X eventually blocks
.beta.-oxidation. The range of n is chosen to include all
variations of the fatty acid analogue which has the desired
biological effect. Since .beta.-oxidation in theory can work on
infinitely long molecules, a could be infinite, but in practice
this is not so. The fatty acids which normally undergo
.beta.-oxidation are usually 14 to 24 carbon atoms long, and this
length is therefore most ideal for undergoing enzymatic
.beta.-oxidation. The ranges of n and R' are thus given so that the
fatty acid analogues will cover this range. (Likewise, option ii)
of formulas (I) and (II) and define R to have 1 to 25 carbon
groups, and option i) of formula (U) define the alkyl group to
contain from 1 to 23 carbon atoms, to be analogous to naturally
occurring compounds.) The total number of carbon atoms in the fatty
acid backbone is preferably between 8 and 30, most preferably
between 12 and 26. This size range is also desirable for the uptake
and transport through cell membranes of the fatty acid analogues of
the present invention.
[0039] Although all fatty acid anagoges with an odd positioning of
the .beta.-oxidation blocker X away from the carboxylic end block
.beta.-oxidation, the extent of their biological effect may be
variable. This is due to the difference in biological degradation
time of the various compounds. The inventors have done experiments
to show the effect of moving X further from the carboxylic fatty
acid end. In these experiments the activity (in
nmol/min/mg/protein) of mitochondrial .beta.-oxidation in the liver
of fatty acid analogues was measured with sulphur in the 3, 5 and 7
positions relative to the carboxyl end. The activities were 0.81
for sulphur in the 3.sup.rd position, 0.61 for sulphur in the
5.sup.th position, 0.58 for sulphur in the 7.sup.th position, and
0.47 for palmitic acid, the non .beta.-oxidation blocking control.
This shows, as expected, that .beta.-oxidation is indeed blocked by
fatty acid analogues with varying positioning of the block, and
that the effect thereof is lessened the further away from the
carboxylic end the block is positioned at, because it takes the
.beta.-oxidation longer to reach the block so more of the fatty
acid analogue is degraded by then. However, as the decline is great
for going from the 3.sup.rd to 5.sup.th position, but small going
from the 5.sup.th to 7.sup.th position, it is reasonable to assume
that this decline will continue to be less as one moves out the
chain, and thus that it will be very far out indeed before no
effect (compared to the control) is seen at all.
[0040] Thus, it is reasonable to include as compounds of the
present invention, fatty acid analogues and other compounds
represented by the general formulas (I) and (U); (which comprise
said fatty acid analogue(s),) which block n-oxidation at different
distances from the carboxylic end of the analogues, as the
compounds of the present invention all do indeed block
.beta.-oxidation, even if the effect thereof can be modulated. This
modulation will after all differ under wearying conditions; in
different tissues, with wearying dosages, and by changing the fatty
acid analogue so that it is not so easily broken down, as will be
described next. Thus it is reasonable to include in the formula all
distances of the .beta.-oxidation blocker from the carboxylic end
of the fatty acid analogue which are biologically relevant.
[0041] Although fatty acid analogues as described with a block in
the X position cannot undergo .beta.-oxidation, they may still
undergo .omega.-oxidation. This is a much less common and slower
biological process, which oxidizes the fatty acid not from the
carboxylic end, but rather from the methyl/hydrophobic head group,
here termed R'. In this pathway the carbon atom at the .omega.-end
of the fatty acid is hydroxylated by a member of the cytochrome
P450 enzyme family. This hydroxylated fatty acid is then converted
into an aldehyde by an alcohol dehydrogenase, and subsequently this
aldehyde is converted into a carboxyl group by an aldehyde
dehydrogenase. As a consequence, the final product of the pathway
is a dicarboxylic fatty acid, which can be degraded further by
.omega.-oxidation from the .omega.-end.
[0042] .omega.-oxidation is believed to be the main pathway for
degradation of the fatty acid analogues as described with a block
in the X position. Experiments were thus performed where R' was
changed to block .omega.-oxidation, by introducing a triple bond at
the methyl end of the fatty acid analogue. This resulted in the
fatty acid analogue 3-thia-15-heptadecyn, which when tested showed
the expected result: a substantially increased degradation time in
vivo. This is important for the use of the fatty acid analogues in
pharmaceutical preparation, as it may potentiate the effects of the
.beta.-oxidizable fatty acid analogues by further slowing down
their breakdown.
[0043] Again, as with the blocking of .beta.-oxidation, it is
routine to find other fatty acid analogues witch would block
.omega.-oxidation in exactly the same manner, based upon knowledge
of how .omega.-oxidation occurs. A double bond will for instance
have the exact same effect as the triple bond did, and it is
therefore included in the definition of the methyl/hydrophobic head
group end of the molecule, here termed R', that it may be saturated
or unsaturated. A branch may also block oxidation, so R' is defined
as linear or branched.
[0044] In order to block .omega.-oxidation by the insertion of a
substitute in R', said R' may be substituted in one or several
positions with heterogroups selected from the group comprising an
oxygen atom, a sulphur atom, a selenium atom, an oxygen atom, a
CH.sub.2 group, a SO group and a SO.sub.2 group. R' may also be
substituted with one or more compounds selected from the group
comprising fluoride, chloride, hydroxy, C.sub.1-C.sub.4 alkoxy,
C.sub.1-C.sub.4 alkylthio, C.sub.2-C.sub.5 acyloxy or
C.sub.1-C.sub.4 alkyl.
[0045] Thus the compounds according to the present invention are
either fatty acids analogous to naturally occurring fatty acids,
which are not capable of being .beta.-oxidized, or naturally
occurring lipids comprising said fatty acid analogues. In vivo, the
fatty acid analogues show a strong preference for being
incorporated into phospholipids. In some cases it is indeed
advantageous to mimic nature and incorporate the fatty acid
analogues in naturally occurring lipids, such as mono-, di-, and
triglycerides and phospholipids. This changes the absorption of the
compounds (when comparing fatty acids to fatty acids incorporated
in larger lipid structures) and may increase the bioavailability or
stability.
[0046] As an example, one could make a complex by including a fatty
acid(s) which are not capable of being .beta.-oxidized into a
triacylglycerol. Such compounds are encompassed by formulas (I) and
(U). If such a triacylglycerol was taken orally, for instance in an
animal feed product, it would probably be transported like any
triacylglycerol, from the small intestine in chylomicrons and from
the liver in the blood in lipoproteins to be stored in the adipose
tissue or used by muscles, heart or the liver, by hydrolyzes of the
triacylglycerol into glycerol and 3 free fatty acids. The free
fatty acids would at this point be the parent compound of the
present invention, and not a complex anymore.
[0047] Yet other possible glycerophospholipid derivatives of the
fatty acids of the present invention includes, but are not limited
to, phosphatidyl cholines, phosphatidyl ethanolamines, phosphatidyl
inositols, phosphatidyl serines and phosphatidyl glycerols.
[0048] Another esterification of fatty acids found in vivo which
could be easily used to make a complex for a compound of the
present invention would be to make the alcohol or polyalcohol
corresponding to the fatty acid, for example one could make a
sphingolipid derivative such as ceramide or sphingomyelin by making
the corresponding amino alcohol. Like the glycerophospholipid
complexes, such complexes would be very water insoluble and less
hydrophilic. These kinds of hydrophobic complexes of the present
invention would pass easier through biological membranes.
[0049] Other possibilities of polar complexes of the present
invention may be, but are not limited to, lysophospholipids,
phosphatidic ads, alkoxy compounds, glycerocarbohydrates,
gangliosiedes, and cerebrosides.
[0050] Although there can be large structural differences between
different compounds comprising non .beta.-oxidizable fatty acid
entities of the present invention, the biological functions of all
the compounds are expected to be very similar because they all
block .beta.-oxidation in the same trimmer. This inability of the
fatty acid analogues to be .beta.-oxidized (and in some cases,
.omega.-oxidized,) causes the analogues to build up in the
mitochondria, which triggers the .beta.-oxidation of the in vivo
naturally occurring fatty acids, which in turn leads to many of the
biological effects of the compounds comprising fatty acid analogues
of the present invention. (Berge R K et al. (2002) Curr Opin
Lipidol 13(3):295-304)
[0051] The fatty acid .beta.-oxidation pathway is the main pathway
for the metabolism of fats. The initial and rate limiting reaction
is carried out in the peroxisomes of the liver by acyl-CoA oxidase.
Acyl-CoA oxidase catalyze the dehydrogenation of acyl-CoA
thioesters to the corresponding trans-2-enoyl CoA. A fatty acid
analogue according to formula (I); tetradecylthioacetic acid (TTA),
has been used previously by the present inventors to test the
various biological effects of the fatty acids. In the current
invention, its effect on acyl-CoA oxidase was tested, as well as
the effect of various plant oils and fish oil, alone or in
concurrence. TTA alone showed effected a large increase in this
enzyme activity compared to the negative control. The plant and
fish oils alone exhibited a very small increase in acyl-CoA oxidase
activity compared to the negative control. Sunflower oil did not
increase the activity of TTA when administered together. This is
what one would expect, that the acyl-CoA oxidase activity of TTA
with oils would stay the same as without added oils. Fish and olive
oil showed a slight potentiation of the increase in acyl-CoA
oxidase activity by TTA. Soy oil without TTA had negligible effects
on -CoA oxidase activity, but combined with TTA it exhibited a 60%
increase when compared to the effects of TTA alone. This
potentiating of TTA as an acyl-CoA oxidase activator by soy oil is
quite unexpected.
[0052] In the present invention, the effect of non
.beta.-oxidizable fatty acid entities on phospholipids levels were
also tested, as well as the effect of various plant oils and fish
oil, alone or in concurrence with TTA. Sunflower and fish oil
reduced the phospholipids level, and potentiated TTAs ability to do
so beyond the phospholipids lowering abilities of either TTA or the
oils alone. Soy oil and olive oil actually increased the
phospholipids levels, but, unexpectedly, these oils substantially
potentiated TTAs ability to decrease phospholipids levels. The
effect of soy oil is especially noteworthy, on its own it increased
the phospholipids levels by 10% compared to the control, but when
given with TTA it lowered the phospholipids level by an additional
40% compared to TTA alone.
[0053] In the present invention, the effect of non
.beta.-oxidizable fatty add entities on cholesterol levels were
also tested, as well as the effect of various plant oils and fish
oil, alone or in concurrence with TTA. TTA lowered the cholesterol
level more than any of the plant or fish oils alone. Sunflower oil
or fish oil without TTA lowered the cholesterol levels somewhat,
with TTA the cholesterol levels were lowered beyond that of TTA
alone. Olive and soy oil actually increased the cholesterol levels
on their own, but, quite unexpectedly, when added to TTA these oils
improved on TTAs ability to lower cholesterol. This TTA
potentiating effect was greatest with soy oil, which reduced the
cholesterol level with 60% when compared with TTA alone.
[0054] TTA has been shown to reduce the plasma triacylglycerol
level by increasing the number of mitochondria and stimulating
mitochondrial .beta.-oxidation of normal saturated and unsaturated
fatty acids to ketone bodies (Froyland Let al. (1997) J Lipid Res
38:1851-1858). In the present invention, it was found that this
effect was further unexpectedly potentiated by the addition of
plant and fish oils. Olive, sunflower and fish oil all lowered the
triacylglycerol levels on their own, sunflower and fish oil even
more so than TTA alone, and further potentiated the cholesterol
lowering effect of TTA beyond that seen for either the oils or TTA
on its own. Soy oil showed the most spectacular results; on it's
own it actually increased cholesterol levels by 15% compared to the
control, but quite unexpectedly it potentiated TTAs cholesterol
lowering effect by 130%.
[0055] In the present invention, the effect of feeding Atlantic
salmon a feed comprising non (1-oxidizable fatty acid analogues,
oil, common feed components and optionally a fermented soy protein
material was tested. In example 2.1, fish feed was composed from
coating common feed pellets with fish oil including TTA. This feed
was then used in example 22 as the food supply for Atlantic salmon,
and the presence of TTA had beneficial effects on the thus produced
compared to fish fed equivalent feed without TTA (examples 2.3 and
2.4).
[0056] The common feed pellets used comprised mostly fish meal,
some wheat and a vitamin and mineral additive. The oil used for the
coating of the pellets was of marine origin, from capelin, and had
various amounts of TTA mixed in. Table 1 describes the formulation
and chemical composition of the diets. The origin of the protein
(fish meal) or carbohydrates (wheat) is in itself not so important,
the important part is that this is a common feed, well suited for
the test species (in this example Atlantic salmon), which upon
addition of TTA exhibits beneficial effects.
[0057] Although the source of the protein itself is not important,
it has been shown in a co-pending application (NO20043093) that TTA
administered together with protein has an added beneficial effect
as compared to TTA alone. The source of fat chosen for the diet
might be more important, as TTA herein has been shown to have
surprising synergistic effects with oils, especially of marine
origin. Thus, the fact that this common feed is high in fats and
protein and low in carbohydrates probably increased the beneficial
effects of TTA over TTA being administered alone, or in a diet with
more carbohydrates.
[0058] In example 2.4, the effects of a specific protein material,
a fermented soy protein material, was ascertained. The fermented
soy protein material is resulting from a fermentation of soy beans.
It comprises modified and un-modified soy proteins and isoflavones,
as well as other soy constituents. A preferred embodiment of the
invention uses the fermented soy protein material
Gendaxin.RTM..
[0059] Table 2 describes the fatty acid composition of the diets.
There were only minor differences in the fatty acid composition of
the diets (all contained nearly 100% fish oil), the percentage of
n-3 fatty acids (FA) was almost equal. Diets supplemented with TTA,
however, led to substantial changes in the percentage of n-3 fatty
acid composition of the phospholipids (PL), triacylglycerols (TAG)
and free fatty acids (FFA) of gills, heart and liver of Atlantic
salmon. Administration of TTA during the 8 weeks also resulted in a
decreased percentage of saturated FAs in almost all the lipids
fractions. The percentage of the n-3 FAs, especially DHA, increased
hi the gills and heart, as can be seen in example 2.3.
[0060] Atlantic salmon fed diets containing TTA grew at a slower
rate than fish fed the control diet. The body lipid level in fish
fed the diets supplemented with TTA was significantly lower than it
was in fish fed the control diet.
[0061] There are health benefits to the fish itself by being fed a
feed according to the invention. Old fish may experience arterial
sclerosis and resulting health problems just like humans, and a
lowering of lipids will have a beneficial effect on this.
[0062] In general, lean meat, as obtained by the method of the
present invention, is considered beneficial in most animal species
reared for consumption. Thus the effect of lowering the total lipid
levels is in itself advantageous. In addition, the specific changes
in fatty acid composition are particularly positive. It is widely
recognized that consuming less saturated fatty acids is healthy,
and an increased consumption of n-3 has been associated with a
whole host of health benefits, from reducing the chance of heart
diseases to anti-inflammatory effects and even smarter babies.
[0063] Other animal products obtained from animals fed the feed of
the present invention may also have beneficial effects. As an
example would fish oil thus obtained have an advantageous
nutritional composition when compared to oil from fish fed
commercial diets. Other products, such as fish skins, may also have
beneficial effects seeing as the whole body composition is
improved.
[0064] The level of fatty acids in the blood is normally determined
by the relative rates of lipolysis and esterification in adipose
tissue, and the uptake of fatty acids in the muscles. In the
muscles, fatty acids inhibit glucose uptake and oxidation.
Increased levels of fatty acids and triacylglycerol in the blood
and muscles therefore correlate with obesity and insulin
resistance, as well as a reduced ability to metabolize glucose
(Olefsky J M (2000) J Clin Invest 106:467-472; Guerre-Millo M et
al. (2000) J Biol Chem 275:16638-16642). Stimulation of fatty acid
oxidation and decreased plasma fatty acid concentration by non
.beta.-oxidizable fatty acid entities and plant and/or fish oils
can therefore prevent and treat insulin resistance and diseases
caused thereby (Shulman G I (2000) J Clin Invest 106(2):171-176).
TTA has been found to completely prevent high-fat diet induced
insulin resistance and adiposity, and reduce adiposity,
hyperglycaemia and insulin sensitivity in obese rats (Madsen M et
al. (2002) J Lipid Res 43(5):742-50). Due to the unexpected
synergetic results found by the inventors using both TTA and plant
and/or fish oils, without being bound to any specific theory of why
the results are as shown, we now expect that this combination will
be even more effective in the treatment of these conditions. We
also expect TTA to be potentiated by fish and plant oils in
treating related diseases and disorders including elevated blood
pressure, increased lipid and cholesterol levels, endothelial
dysfunction, procoagulant state, polycystic ovary syndrome and the
metabolic syndrome.
[0065] The peroxisome proliferator-activated receptor (PPAR) family
are pleiotropic regulators of cellular functions such as cellular
proliferation, differentiation and lipid homeostasis (Ye J M et al.
(2001) Diabetes 50:411-417). The PPAR family is comprised of three
subtypes; PPAR.alpha., PPAR.beta., and PPAR.gamma.. TTA is a potent
ligand of PPAR.alpha. (Forman B M, Chen J, Evans R M (1997) Proc
Natl Aced Sci 94:4312-4317; Gottlicher M et al. (1993) Biochem
Pharmacol 46:2177-2184; Berge R K et al. (1999) Biochem J
343(1):191-197), and activate PPAR.beta. and PPAR.gamma. as well
(Raspe E et al. (1999) J Lipid Res 40:2099-2110). As a PPAR.alpha.
activator TTA stimulate the catabolism of fatty acids by increasing
their cellular uptake. Lowering the plasma triglyceride levels with
TTA caused a shift in liver cellular metabolism, towards
PPAR.alpha. regulated fatty acid catabolism in mitochondria. (Gray
H J et al. (2003) J Biol Chem 278(33):30525-33) While the effect of
TTA on plasma triacylglycerol is direct by PPAR.alpha. activation,
which is demonstrated by the abolishment of this effect in
PPAR.alpha. knockout mice, fish oil does reduce plasma
triacylglycerol even in knockout mice (Dallongeville J et al.
(2001) J Biol Chem 276:4634-4639).
[0066] Supplement with dietary n-3 poly unsaturated fatty acids
like those found in fish oil stimulate hepatic peroxisomal acyl-CoA
oxidase activity and thus fatty acid oxidation in the liver and to
a smaller extent in skeletal muscle (Ukropec J et al. (2003) Lipids
38(10):1023-9). A fish oil rich diet has been shown to increase
both the activity and mRNA levels of hepatic mitochondrial and
peroxisomal fatty acids oxidation enzymes (Hong D D et al. (2003)
Biochim Biophys Acta: Mol Cell Biol Lipids 1635 (1):29-36). Fish
oil induced an increase in abundance of peroxisomal acyl-CoA
oxidase in the liver but not muscles of rats, and the authors
hypothesise that this is due to n-3 fatty acids protect against
fat-induced insulin resistance by serving a PPAR.alpha. ligands,
inducing hepatic (not intramuscular) peroxisome proliferation.
PPAR.alpha. gene expression did not change. (Neschen S et al.
(2002) Am J Physiol Endocrinol Metab 282:E395-E401)
[0067] As can be seen in the above paragraphs, the biochemical
details of exactly how TTA and fish oil influence fat metabolism
are not known in detail. Even less is known of how plant oils
positively influence fat metabolism as described in the present
invention (Rustan A C, Christiansen E N, Drevon C A (1992) Biochem
J 283(2)333-339). The effects may or may not be through the same
paths, both TTA and oils may for instance act as PPAR.alpha.
ligands, or act independently of PPAR.alpha.. If they work through
the same paths, one would not expect TTA to be potentiated by the
oils, because TTA is a strong PPAR.alpha. activator which one would
expect would fully saturate the PPAR.alpha. activation. To even get
an additive effect of the TTA effect plus the plant or fish oil
effect when combining the two would then be unexpected. To get a
synergistic effect way above the additive effect, as is seen
especially for TTA and soy oil in all tests of the present
invention, but also to a lesser extent with TTA and fish or
sunflower oil in lowering triacylglycerol levels, and olive and
sunflower oil in lowering cholesterol levels, is even more
surprising. .beta.-oxidizable fatty acid analogues have many
effects, and we do not know how they are all brought about, but
based upon the unexpected results of the present invention we
expect them all to be potentiated by plant and fish oils without
being bound to any specific theory.
[0068] PPAR ligands affect proliferation of various cancer cell
lines. TTA in particular has been found to reduce proliferation of
many cancer cell lines (Berge K et al. (2001) Carcinogenesis
22:1747-1755; Abdi-Dezfuli F et al. (1997) Breast Cancer Res Treat
45:229-239; Tronstad K J et al. (2001) Biochem Pharmacol
61:639-649; Tronstad K J et al. (2001) Lipids 36:305-313). This
reduction is related to reduction in triacylglycerol levels
(Tronstad K J et al. (2001) Biochem Pharmacol 61:639-649), and is
mediated by both PPAR dependent and independent pathways (Berge K
et al. (2001) Carcinogenesis 2.2:1747-1755). Since oils improve
TTA's ability to lower triacylglycerol levels and is a PPAR ligand,
it is therefore highly likely that it will improve the
anti-proliferative effects of TTA as well, making this an
improvement upon TTA's cancer prevention and treatment abilities.
TTA may be used for the prevention and/or treatment of cancer
including inhibition of: primary and secondary neoplasms, the
growth of tumours, invasion of a primary tumour into connective
tissue, and formation of secondary tumours (NO 2002 5930).
[0069] In general, PPAR agonists and poly unsaturated fatty acids
modulate the inflammatory response. TTA modulate inflammatory
response by depressing the release of inflammatory cytokine
interleukin-2 and suppressing PHA stimulated proliferation of
peripheral mononuclear cells (Aukrust P et al. (2003) Eur J Clin
Invest 33(5):426-33). The modulation of cytokine by TTA may be PPAR
mediated or through altered prostaglandin levels or by modification
of lipid mediated signal transduction, the latter which also is the
proposed mechanism of action for poly unsaturated fatty acids, as
those found in plant and fish oils. Now that the inventors have
found the unexpected results of the present invention, they
therefore expect that plant oil and/or fish oil in combination with
non .beta.-oxidizable fatty acid entities will potentiate the
effect of the fatty acid analogues on inflammatory disorders
including immune mediated disorders such as rheumatoid arthritis,
systemic vasculitis, systemic lupus erythematosus, systemic
sclerosis, dermatomyositis, polymyositis, various autoimmune
endocrine disorders (e.g. thyroiditis and adrenalitis), various
immune mediated neurological disorders (e.g. multiple sclerosis and
myastenia gravis), various cardiovascular disorders (e.g.
myocarditis, congestive heart failure, arteriosclerosis and stable
and unstable angina, and Wegener's granulomatosis), inflammatory
bowel diseases, Chron's disease, non specific colitis,
pancreatitis, nephritis, cholestatis/fibrosis of the liver, and
acute and chronic allograft rejection after organ transplantation,
as well as proliferate skin disorders like psoriasis, atopic
dermatitis, non-specific dermatitis, primary irritant
contact-dermatitis, allergic contact-dermatitis, lamellar
ichthyosis, epidermolytic hyperkeratoses, pre-malign sun-induced
keratoses, and seborrhoea, and diseases that have an inflammatory
component such as e.g. Alzheimer's disease or impaired/improvable
cognitive function.
FIGURE LEGENDS
[0070] FIG. 1 shows that the increase in fatty acyl-CoA activity by
TTA is potentated by soy oil, olive oil and fish oil.
[0071] FIG. 2 shows that the phospholipids lowering effect of TTA
is potentated by sunflower oil, soy oil and fish oil.
[0072] FIG. 3 shows that the cholesterol lowering effect of TTA is
potentated by olive oil, sunflower oil, soy oil and fish oil.
[0073] FIG. 4 shows that the triacylglycerol lowering effects of
TTA is potentated by olive oil, sunflower oil, soy oil and fish
oil.
DEFINITIONS USED IN THE APPLICATION
Animals
[0074] In this context the term "animals" include mammals such as
humans and farm (agricultural) animals, especially the animals of
economic importance such as gallinaceous birds, bovine, ovine
caprine and porcine mammals, especially those that produce products
suitable for the human consumption, such as meat, eggs and milk.
Further, the term is intended to include fish and shellfish, such
as salmon, cod, Tilapia, clams, oysters, lobster or crabs. The term
also includes domestic animals such as dogs and cats.
Animal Feed
[0075] The term animal feed refers to food for animals (as defined
above). Animal feed usually comprise appropriate amounts of fats,
proteins, carbohydrates, vitamins and minerals necessary for the
sustenance of the intended animal recipient, and may comprise
additional components for the improvement of taste, texture,
colour, smell, stability, storage life etc, or antibiotics or other
components added for the benefit of the health of the animal. The
animal feed is preferably but not necessary dry matter, most
preferably a pellet material. The term animal feed is also intended
to include nutritional Compositions, veterinary compositions,
and/or functional food products for animal consumption.
Meat
[0076] The word meat refers to flesh from any animal as defined
above. Thus, the protein containing flesh from mammals, birds, fish
and shellfish is all referred to as meat The term "meat product"
refers to any product produced from meat as defined above.
Plant and/or Fish Oils
[0077] These include all oils of plant or marine origin, including
but not limited to fatty or fixed oils as well as essential or
volatile oils, and any combination thereof. They do not necessarily
need to be in liquid form. Sunflower oil, which was used in the
present invention, is really oil from the sunflower seed, not the
flower itself.
Fish Oil
[0078] This term include all oils of a marine origin.
Nutritional Composition
[0079] This term is meant to include any ingestible material,
including but not restricted to nutritional supplements, functional
foods, herbal supplements etc. for human or animal consumption. The
term also includes food products for human consumption and animal
fodder, wherein the composition of the present invention is an
additive, and not the main ingredient. This especially concerns
animal fodder, where any fodder can be supplemented with the
composition of the present invention, to attain the biological
effects thereof.
Treatment
[0080] In relation to the pharmaceutical applications of the
invention the term "treatment" refers to a reduction of the
severity of the disease.
Prevention
[0081] The term "prevention" refers to the preventing of a given
disease, i.e. a compound of the present invention is administered
prior to the onset of the condition. This means that the compounds
of the present invention can be used as prophylactic agents or as
ingredients in a nutritional composition in order to prevent the
risk or onset of a given disease.
Nutritional Composition
[0082] This term is meant to include any ingestible material,
including but not restricted to nutritional supplements, functional
foods, herbal supplements etc. for human or animal consumption. The
term also includes food products for human consumption and animal
fodder, wherein the composition of the present invention is an
additive, and not the main ingredient. This especially concerns
animal fodder, where any fodder can be supplemented with the
composition of the present invention, to attain the biological
effects thereof.
Administration of the Compounds of the Present Invention
[0083] As a pharmaceutical medicament the compounds of the present
invention may be administered directly to the animal by any
suitable technique, including parenterally, intranasally, orally,
or by absorption through the skin. They can be administered locally
or systemically. The specific route of administration of each agent
will depend, e.g., on the medical history of the recipient human or
animal.
[0084] Examples of parenteral administration include subcutaneous,
intramuscular, intravenous, intra-arterial, and intra-peritoneal
administration
[0085] As a general proposition, the total pharmaceutically
effective amount of each of the non oxidizable fatty acid entities
administered parenterally per dose will preferably be in the range
of about 1 mg/kg/day to 200 mg/kg/day of patient body weight for
humans, although, as noted above, this will be subject to a great
deal of therapeutic discretion. A dose of 5-50 mg/kg/day is most
preferable. A dose of 1-300 mg/kg/day of oil is preferable, and a
dose of 10-150 mg/kg/day is most preferable.
[0086] If given continuously, the compounds of the present
invention are each typically administered by 1-4 injections per day
or by continuous subcutaneous infusions, for example, using a
mini-pump. An intravenous bag solution may also be employed. The
key factor in selecting an appropriate dose is the result obtained,
as measured by decreases in total body weight or ratio of fat to
lean mass, or by other criteria for measuring control or prevention
of obesity or prevention of obesity-related conditions, as are
deemed appropriate by the practitioner.
[0087] For parenteral administration, in one embodiment, the
compounds of the present invention are formulated generally by
mixing each at the desired degree of purity, in a unit dosage
injectable form (solution, suspension, or emulsion), with a
pharmaceutically acceptable carrier, i.e., one that is non-toxic to
recipients at the dosages and concentrations employed and is
compatible with other ingredients of the formulation.
[0088] Generally, the formulations are prepared by contacting the
compounds of the present invention each uniformly and intimately
with liquid carriers or finely divided solid carriers or both.
Then, if necessary, the product is shaped into the desired
formulation. Preferably the carrier is a parenteral carrier, more
preferably a solution that is isotonic with the blood of the
recipient Examples of such carrier vehicles include water, saline,
Ringer's solution, and dextrose solution. Non-aqueous vehicles such
as fixed oils and ethyl oleate are also useful herein, as well as
liposomes.
[0089] The carrier may suitably contain minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids,
such as glycine, glutamic acid, aspartic acid, or arginine;
monosaccharides, disaccharides, and other carbohydrates including
cellulose or its derivatives, glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; counterions such as sodium; and/or non-ionic surfactants
such as polysorbates, poloxamers, or PEG.
[0090] For oral pharmacological compositions such carrier material
as, for example, water, gelatine, gums, lactose, starches,
magnesium-stearate, talc, oils, polyalkene glycol, petroleum jelly
and the like may be used. Such pharmaceutical preparation may be in
unit dosage form and may additionally contain other therapeutically
valuable substances or conventional pharmaceutical adjuvants such
as preservatives, stabilising agents, emulsifiers, buffers and the
like. The pharmaceutical preparations may be in conventional liquid
forms such as tablets, capsules, dragees, ampoules and the like, in
conventional dosage forms, such as dry ampoules, and as
suppositories and the like.
[0091] In addition the compounds of the present invention, i.e. the
compounds comprising .beta.-oxidizable fatty acid analogue and
plant and/or fish oils, may be used in nutritional preparations, as
defined earlier, in which case the dosage of non .beta.-oxidizable
fatty acid analogue preferable is as described pharmaceuticals or
less, while the amounts of plant and/or fish oil preferable are
suitable for the preparation of food and feed materials. As a part
of a nutritional composition, and especially animal fodder, the
plant and/or fish oils can be a substantial part of the fodder, and
thus have a nutritional value as well as potentiating the non
.beta.-oxidizable fatty acid analogues. Oil of the present
invention can comprise up to all of the fat in a nutritional
composition. In animal fodder, the amount of non .beta.-oxidizable
fatty acid analogue can be up to 10 times that in products for
human consumption, that is, up to 2 g/kg/day of animal body weight.
Such animal fodder may be used for routine feeding of Animals. An
animal feed composition may also comprise fermented soy protein
material. Fermented soy protein material is especially useful as a
functional protein in food products, particularly when used as a
substitute for natural plasma in animal feeds and in pet foods. An
Animal feed composition may also comprise additional ingredients
such as fats, sugars, salt, flavourings, minerals, etc. The product
may then be formed into chunks resembling natural meat chunks in
appearance and texture. The product of the invention has the
further advantages that this is readily formulated to contain
necessary nutrients, is easily digested by the animals and is
palatable to the animals.
EXPERIMENTAL SECTION
[0092] The preparation of non .beta.-oxidizable fatty acid entities
according to the present invention is disclosed in detail in the
applicant's earlier Norwegian patent applications no. 20005461,
20005462, 20005463 and 20024114. These documents also describe
toxicity studies of TTA. Preparation of mono-, di-, and
triglycerides and nitrogen comprising lipids according to the
invention is disclosed in detail in U.S. patent application Ser.
No. 10/484,350. The preparation of phospholipids including serine,
ethanolamine, choline, glycerol, and inositol according to the
invention is disclosed in detail in the applicant's earlier
Norwegian patent application no. 20045562.
Example 1
Biological Effects in Rats of the Composition According to the
Invention
1.1 Experimental Setup
Chemicals
[0093] Chemicals were obtained from common commercial sources and
were of reagent grade. The fish oil was obtained from Hordafor,
while the plant oils were obtained from Mills.
Carboxymethylcellulose (CMC) was used as a control (negative).
Experimental Animals
[0094] Male Wistar rats weighing from 250 to 358 g, were bought
from AnLab Ltd. (Prahg, The Check Republic), and were kept in wire
cages in a temperature of 22+/-1.degree. C. and light controlled
(light from 7 am to 7 pm) room. There were no restrictions put on
food and water intake. Three rats were kept in each cage. Increase
in weight and food intake was monitored daily.
Experimental Diets
[0095] The rats were fed a standard Chow ST1 diet (from Vela;
Prahg, The Check Republic).
Treatments
[0096] Male Wistar rats were allowed to acclimatize to the new
surroundings before initiation of the experiment. They were then
treated daily for 10 days by gavage. CMC was used as a carrier and
negative control. Each treatment group numbered 4 rats. The groups
that were treated with TTA were given 150 mg/kg body weight/day
dissolved in CMC or oils. The groups that were treated with oils
(sunflower, soy, olive or fish) were given 3 mL (ca 2.5 g)/kg body
weight/day. CMC was used as a carrier and negative control. The day
after the last treatment the rats were sacrificed.
Sacrifice and Tissue Retrieval
[0097] The rats were anaesthetized with a 1:1 mixture of
Hypnorm.TM. (fentanyl citrate 0.315 mg/ml and fluanisone 10 mg/ml,
Janssen Animal Health) and Dormicum.RTM. (midazolam 5 mg/ml, F.
Hoffmann-La Roche) injected subcutaneously. Blood was drawn
directly from the heart using a heparin rinsed syringe. The liver
was immediately removed, weighed and divided into two parts, which
were immediately chilled on ice or frozen in liquid nitrogen,
respectively. Plasma and tissues were stored at -80.degree. until
analysis. The protocol was approved by the Norwegian State Board of
Biological Experiments with Living Animals.
Preparation of Hepatic Subcellular Fractions
[0098] Livers from the rats were homogenised individually in
ice-cold sucrose-solution (0.25 mol/L sucrose in 10 mmol/L HEPES
buffer pH 7.4 and 1 mmol/L EDTA) using a Potter-Elvehjem
homogeniser. Subcellular fractionation of the livers was performed
as previously described (Berge R K at al. (1984) Eur J Biochem 141:
637-44). The procedure was performed at 0-4.degree. C., and the
fractions were stored at -80.degree. C. Protein was assayed with
the BioRad protein assay kit using bovine serum albumin as the
standard.
1.2 Biological Effects in Rats of the Composition According to the
Invention
[0099] Enzyme Assay for fatty acyl-CoA Oxidase
[0100] Fatty acyl-CoA oxidase activity was measured in the
peroxisomal liver fraction as previously described (Small G M,
Burdett K, Connock M J (1985) Biochem J 227: 205-10). The results
were given as fatty acyl-CoA oxidase activity per total protein,
baseline activity (activity of control) was subtracted, and the
data which is presented in FIG. 1 were normalized to the activity
of TTA.
Lipid Analysis
[0101] Plasma and liver lipids were measured enzymatically on the
Technicon Axon system (Miles, Tarrytown, N.Y.) using the
Triglyceride kit from Bayer, Total cholesterol (Bayer, Tarrytown,
N.Y.), and the PAP150 kit for choline containing phospholipids from
bioMerieux. The results were given per total protein, and the data
which is presented in FIGS. 2-4 were normalized to the activity of
the positive control (no added TTA or oils; i.e. "normal"
levels).
Example 2
Biological Effects in Atlantic Salmon of the Composition According
to the Invention
2.1 Experimental Setup Including Preparation of the Fish Feed
[0102] The experimental fishmeal-based diets were provided by EWOS
and contained 0.01% Y.sub.2O.sub.3 as an inert marker for
digestibility determination (3 mm pellets). Table 1 shows the
formulations and chemical compositions of the three diets. All the
three diets were produced from one feed mix. The different diets
were obtained by coating the common feed pellet with the different
oils and mixtures. The diets contained either fish oil (capelin
oil) (Control), fish all supplemented with 0.5% TTA (0.5% TTA) or
fish oil supplemented with 1.5% TTA (1.5% TTA).
TABLE-US-00001 TABLE 1 Formulation and chemical composition of the
diets Control 0.5% TTA 1.5% TTA Formulation (% of total) Fish meal,
LT 67.8 67.8 67.8 Capelin oil.sup.a 21.3 21 20.7 TTA 0.1 0.3 Wheat
10.4 10.4 10.4 Astax.sup.b-Cantax.sup.c 0.06 0.06 0.06
Mineral/Vitamin premix 0.49 0.49 0.49 Yttrium oxide 0.01 0.01 0.01
Chemical composition Dry matter (%) 97.1 96.1 93.8 Protein (%) 51.9
51.4 49.7 Fat (%) 26.9 26.7 26.7 Ash (%) 10.8 10.4 10 Energy
(MJ/kg) 23.8 23.7 23.2 Diets: fish oil (Control), fish oil added
0.5% TTA (0.5% TTA), fish oil added 1.5% TTA (1.5% TTA).
.sup.aCapelin oil, Norsildmel, Norway. .sup.bAsta, BASF, lucanthin
red. .sup.cCanta, lucanthin pink.
[0103] The fatty acid composition of the diets clearly reflected
that of the fish oil used (capelin oil) (Table 2). The capelin oil
contained relatively high levels of the monounsaturated FM and was
also rich in the long-chain n-3 FAs, 20:5 n-3 (EPA) and 22:6 n-3
(DHA). The feed, however, contained a significant amount of fish
meal, which contained n-3 FAs, ensuring that the levels of these FA
in the diet where higher than those in the added oil.
[0104] In addition to the above diets, identical diets but with
0.5% Gendaxin and 0% or 0.9% TTA (based on the total dry weight of
the feed) were prepared.
TABLE-US-00002 TABLE 2 Fatty acid composition of the diets Fatty
acids Control 0.5% TTA 1.5% TTA 12:0 0.1 0.1 0.1 14:0 6.7 6.6 6.6
15:0 0.3 0.3 0.3 16:0 11.8 11.6 11.5 16:1 n - 7 7.1 7 6.9 16:1 n -
9 0.4 0.3 0.3 16:2 n - 7 0.4 0.4 0.4 17:0 0.1 0.1 0.1 18:0 1.4 1.3
1.3 18:1 n - 6 0.4 0.4 0.4 18:1 n - 7 3.1 3 3 18:1 n - 9 11.5 11.6
11.3 18:2 n - 6 2.7 2.7 2.6 18:3 n - 6 0.1 0.1 0.1 18:3 n - 3 0.7
0.7 0.7 18:4 n - 3 2.1 2.1 2.1 TTA 0.5 1.5 20:0 0.1 0.1 0.1 20:1 n
- 9 0.5 0.5 0.5 20:1 n - 11 17.9 17.9 17.7 20:2 n - 6 0.2 0.2 0.2
20:3 n - 3 nd nd nd 20:4 n - 3 0.4 0.4 0.4 20:4 n - 6 0.3 0.3 0.3
20:5 n - 3 5.9 5.9 5.8 22:0 0.2 nd nd 22:1 n - 9 1.9 1.9 1.8 22:1 n
- 11 14.1 14 14 22:2 n - 6 0.1 0.2 0.2 22:5 n - 3 0.5 0.4 0.5 22:6
n - 3 6.4 6.2 6.2 .SIGMA. Saturates 20.7 20.1 20 .SIGMA. n - 3 15.9
15.7 14.1 .SIGMA.n - 6 3.5 3.5 3.4 Control: fish oil, 05% TTA: fish
oil added 0.5% TTA, 1.5% TTA: fish oil added 1.5% TTA. The quantity
of each fatty acid is given as a percentage of the total fatty
acids.
2.2: Rearing Atlantic Salmon on Feed Comprising TTA
Fish, Facilities and Experimental Design
[0105] The trial was conducted at AKVAFORSK Research Station,
Sunndalsera, Norway. Atlantic salmon (Salmon salar) with a mean
initial weight of approximately 86 g were placed into 15
cylinder-conical tanks (0.85 m diameter), 40 fish per tank. The
tanks were supplied with seawater with a constant temperature of
12.degree. C. The fish were acclimatised to the temperature and fed
a commercial feed for two weeks before the start of the trial. The
growth trial consisted of one period of 8 weeks.
[0106] The diets were as described above in table 2, containing
either fish oil (capelin oil) (Control), fish oil supplemented with
0.5% TTA (0.5% TTA) or fish oil supplemented with 1.5% TTA (1.5%
TTA). The tree diets were randomly assigned to triplicate tanks.
The feed was distributed by electrically driven disc-feeders
(Akvaprodukter A S, Sunndalsora). The tanks were designed such that
waste feed was collected from the effluent water in wire mesh
boxes. Wasted feed was collected, and this allowed the weight of
feed consumed to be calculated.
[0107] The Gendaxin containing diets were used in a separate
experiment, but the design thereof was the same as described
above.
Initial and Final Sampling
[0108] The fish were fasted for 2 days before the initial sampling.
Six fish from each tank were anaesthetised in MS-222 at the
beginning and at the end of the experiment, and the mean weight and
mean length were determined. These six fish were killed by a blow
to the head and the abdomen out open. Samples of liver, heart,
gills and kidney were immediately frozen in liquid nitrogen and
stored at -80.degree. C. These samples were subsequently used for
the analysis of fatty acid composition. A further five fish per
tank were anaesthetised and killed. These fish were used for
determination of the composition of the whole body.
[0109] The fish were not fasted before the final sampling. Five
fish from each tank were stripped to collect faecal samples
following the procedure described by Austreng (Aquaculture, 1978
13:265-272). Faecal samples from each tank were pooled. The samples
were stored at -20.degree. C. prior to analyses.
[0110] The second gill arch was removed from anaesthetised fish and
rinsed in ice-cold SEI buffer (150 mM sucrose, 10 nM EDTA, 50 mM
imidazole, pH 7.3) and immediately frozen in liquid nitrogen. Gill
tissues were stored at -80.degree. C. Livers were homogenized in
ice-cold sucrose medium.
Growth
[0111] The mean weight of the fish nearly tripled during the trial,
in al dietary groups, from an initial value of 86 g to a final
value of approximately 250 g. The SGRs decreased with increasing
dietary doses of ITA, from and SGR of 1.8 in the control group to
an SGR of 1.7 in the 0.5% TTA group and an SGR of 1.5 in the 1.5%
TTA group (Table 3). There were no significant differences in the
condition factor between the dietary groups (Table 3).
TABLE-US-00003 TABLE 3 Effect of dietary inclusion of TTA and oil
on feed intake and growth on Atlantic salmon Control 0.5% TTA 1.5%
TTA Initial weight 85 .+-. 2 88 .+-. 1 86 .+-. 2 Final weight 278
.+-. 7 267 .+-. 3 233 .+-. 1 CF 1.2 .+-. 0.02 1.1 .+-. 0.02 1.2
.+-. 0.02 TGR 2.6 .+-. 0.44.sup.c .sup. 2.5 .+-. 0.05.sup.b 2.0
.+-. 0.06.sup.a SGR 1.8 .+-. 0.02.sup.c .sup. 1.7 .+-. 0.03.sup.b
1.5 .+-. 0.05.sup.a Total FER 1.2 .+-. 0.05 1.2 .+-. 0.04 1.1 .+-.
0.12 Values are means .+-. SEM (n = 3) CF (%): Condition factor,
SGR: Specific growth rate, TGC: Thermal growth coefficient, FER:
Feed efficiency ratio (wet gain/dry feed intake).
.sup.abcDifferences between mean values within a given row are
significant (p .ltoreq. 0.05), as indicated by different
superscript letters.
Feed Intake and Nutrient Digestibility
[0112] There were only small differences in digestibility in this
trial (Table 4). The digestibilities of FAs in all dietary groups
were high, being greater than 96% for the sum of all FAs for the
fish fed the control diet and the 0.5% TTA diets, and greater than
90% for the fish fed the 1.5% TTA diet. The digestibilities of the
saturated FAs were, in general, lower than those for the other
FAs.
TABLE-US-00004 TABLE 4 Nutrient digestibility in Atlantic salmon
Control 0.5% TTA 1.5% TTA Energy 89.5 .+-. 0.08 89.0 .+-. 0.58 87.1
.+-. 1.22 Protein 87.7 .+-. 0.15 87.9 .+-. 0.31 87.5 .+-. 0.44 Fat
97.3 .+-. 0.58 96.5 .+-. 1.27 94.9 .+-. 2.12 .SIGMA.Saturated .sup.
93.2 .+-. 1.54.sup.b .sup. 81.4 .+-. 1.95.sup.a .sup. 81.5 .+-.
2.18.sup.a .SIGMA.Monounsaturated 87.8 .+-. 5.66 90.4 .+-. 5.56
95.4 .+-. 1.87 .SIGMA.Polyunsaturated 90.9 .+-. 7.77 94.0 .+-. 4.45
92.5 .+-. 5.08 TTA 98.6 .+-. 0.45 97.0 .+-. 1.59 of protein, fat,
energy content and selected fatty acid in Atlantic salmon fed diets
containing Control: fish oil, 0.5% TTA: fish oil added 0.5% TTA,
1.5% TTA: fish oil added 1.5% Data are % means .+-. SEM Values
within the same row with different superscripts are significantly
different; nd = not detectable.
2.3 Biological Effects of the Composition According to the
Invention
Chemicals
[0113] Acetic acid, chloroform, petroleum ether and methanol were
all obtained from Merck (Darmstadt, Germany) Benzene was obtained
from Rathburn Chemicals Ltd. (Walkerburn, Scotland) and
2',7'-clichlorofluorescein from Sigma Chemical Co. (St. Louis, Mo.,
USA). Methanolic HCl and 2,2-dimethoxypropane was purchased from
Supelco Inc. (Bellfonte, Pa., USA). Glass-baked silica gel K6
plates were obtained from Whatman International Ltd. (Maidstone,
England).
Chemical Analysis
[0114] Fish sampled at the beginning and at the end of the
experiment were analysed for dry matter, fat, protein, ash and
energy content All diets and faecal samples were analysed for dry
matter (by drying at 105.degree. C. to constant weight), fat (by
ethyl-acetate extraction as described in NS 9402, 1994), protein
(by a Kjeltec Autoanalyser-N*625), starch, ash (by heating to
550.degree. C. until constant weight), energy and yttrium oxide
(using ICP-AES after wet asking the samples). The energy contents
of the diets, faeces and whole fish samples were determined by
adiabatic bomb calorimetry, using a Parr 1271 Bomb calorimeter.
Lipid Extraction and Fatty Acid Analysis
[0115] Total lipids were extracted from homogenised gills, liver
and heart using the method described by Folch (J Biol Chem 1957
226:497-509). The chloroform-methanol phases from the gills were
dried under nitrogen and dissolved in hexane. Phospholipids (PL),
triacylglycerol (TAG) and free fatty acids (FFA) were separated by
thin-layer chromatography (TLC) using a mixture of petroleum ether,
diethyl ether and acetic acid (113:20:2 by volume) as the mobile
phase. The lipids were visualised by spraying the TLC plates with
0.2% (w/v) 2',7'-dichlorofluorescein in methanol and they were
identified by comparison with known standards under UV-fight.
[0116] The spots corresponding to PL, FFA, and TAG were scraped off
into glass tubes and were then trans-methylated overnight with
2,2-dimethoxypropane, methanolic-HCL and benzene at room
temperature as described by Mason and Waller (Anal Chem 1964
36:583). The methyl esters were separated on a non-polar fused
capillary column by gas chromatography basically as described by
Rosjoa (Fish Physiol Biochem 1994 13:119-132). The methyl esters of
FAs were separated in a gas chromatograph (Perkin-Elmer Auto system
GC equipped with an injector, programmable split/splitless
injector) with a CP wax 52 column (with length 25 m, internal
diameter 0.25 mm and thickness of the film 0.2 .mu.m), flame
ionisation detector and 1022 data system. The carrier gas was He,
and the injector and detector temperatures were 280.degree. C. The
oven temperature was raised from 50.degree. C. to 180.degree. C. at
the rate of 10.degree. C. min.sup.-1, and then raised to
240.degree. C. at the rate of 0.7.degree. C. min.sup.-1. The
relative quantity of each fatty acid present was determined by
measuring the area under the peak corresponding to that fatty
acid.
Calculations
[0117] Apparent digestibility coefficients (ADC) were calculated as
described by Austreng (Aquaculture, 1978 13:265-272). Condition
factor (CF), hepatosomatic index (HSI), specific growth factor
(SGR) and thermal unit growth coefficient (TGC) were calculated as
follows, based on individual recordings of weights and lengths:
[0118] SGR=(e.sup.(ln W.sup.j.sup.-ln
W.sup.0.sup.)/days)-1)*100
[0119] TGC=(W.sub.1.sup.1/3-W.sub.0.sup.1/3)*1000/(days*.degree.
C.)
where W.sub.0 is initial weight, W.sub.1 is final weight, and t
clay degrees.
[0120] CF=100*W*(fork length).sup.-3
[0121] HSI=100*liver weight*W.sup.-1
Statistical Analysis
[0122] All data was subjected to one-way analysis of variance
(ANOVA) and differences were ranked by Duncan's multiple range
test. The significance level was set at 5%.
Body and Liver Composition
[0123] Fish fed the 1.5% TTA diet had a lower body lipid level
(9.6%) than fish fed the control diet (10.6%) (Table 5). No
statistically significant differences were found in the total liver
lipid content between fish fed the control diet and fish fed the
TTA diets (Table 6). The hepatosomatic index was significantly
higher in fish fed the 1.5% TTA diet (1.2%) than in fish fed the
control diet (1.1%) diet (Table 6).
TABLE-US-00005 TABLE 5 Chemical composition of the carcass in % of
wet weight basis Start Control 0.5% TTA 1.5% TTA Crude lipid (%)
8.3 10.6 .+-. 0.01.sup.b 11.0 .+-. 0.42.sup.b 9.6 .+-. 0.09.sup.a
Crude protein(%) 16.8 18.1 .+-. 0.06.sup. 17.8 .+-. 0.19.sup. 18.0
.+-. 0.22.sup. Dry matter (%) 27.6 31.1 .+-. 0.12.sup.b 31.0 .+-.
0.15.sup.b 29.9 .+-. 0.10.sup.a Ash (%) 2.3 .sup. 2.0 .+-. 0.05
.sup. 2.0 .+-. 0.02 2.0 .+-. 0.06 Energy (MJ/Kg) 7.1 8.5 .+-.
0.03.sup.b 8.5 .+-. 0.10.sup.b 8.0 .+-. 0.05.sup.a Control: fish
oil, 0.5% TTA: fish oil added 0.5% TTA, 1.5% TTA: fish oil added
1.5% TTA. .sup.abDifferences between mean values within a given row
are significant (p .ltoreq. 0.05), as indicated by different
superscript letters.
TABLE-US-00006 TABLE 6 Effect of dietary inclusion of TTA and oils
on hepatosomatic index (HSI) and liver lipid content Control 0.5%
TTA 1.5% TTA HIS 1.1 .+-. 0.01.sup.a 1.1 .+-. 0.02.sup.a 1.2 .+-.
0.03.sup.b Liver lipid content (%) 4.9 .+-. 0.27.sup. 5.1 .+-.
0.12.sup. 5.7 .+-. 0.69.sup. Results are means .+-. SEM (n = 3).
Values within the same row with different superscript are
significantly different.
Fatty Acid Compositions of Liver, Gills and Heart
[0124] The fatty acid compositions of PL, TAG and FFA of gills,
liver and heart are shown in Tables 7, 8 and 9. TTA was
incorporated into the PL fraction of the gills (0.8%) and heart
(0.7%) of the Atlantic salmon fed the 1.5% TTA diet TTA was also
incorporated into the TO and the FFA fractions of the gills (Table
7). Traces of TTA and its .DELTA..sup.9 desaturase products were
incorporated into the liver lipids, while no .DELTA..sup.9
desaturase products from TTA were recovered in lipids from heart
and gills.
[0125] The percentage of n-3 FAs in the liver, gills and heart also
depended on the diet given to the fish. The percentage of EPA+DHA
was significantly higher in fish fed the 1.5% TTA diet than in
control fish, in all the lipid fractions of the gills and heart. In
the liver, on the other hand, TTA led to only a moderate increase
in the percentage of DHA and a slightly reduced percentage of EPA.
The percentage of palmitic acid (16:0) and the sum of all saturated
FAs were significantly lower in the PL fraction of the gills, heart
and liver of fish fed the 1.5% TTA diet than they were in fish fed
the control diet (Tables 7, 8, 9). The sum of monounsaturated FA
was significant lower in the TG and FFA fractions of gills in fish
fed the 1.5% TTA diet than in fish fed the control diet (Table 8).
In contrast, the percentage of the sum of monounsaturated FAs in PL
and TAG fractions of the liver was higher in fish fed increasing
doses of TTA (Table 9).
TABLE-US-00007 TABLE 7 Fatty acid composition of the gills
Phospholipids Triacylglycerol Fatty acids FO 0.5% TTA 1.5% TTA FO
0.5% TTA 1.5% TTA 14:0 3.1 .+-. 0.17 2.9 .+-. 0.15 2.6 .+-. 0.05
5.8 .+-. 0.09 5.6 .+-. 0.35 5.7 .+-. 0.18 16:0 25.2 .+-. 0.89.sup.b
21.7 .+-. 0.61.sup.a 20.9 .+-. 0.09.sup.a 15 .+-. 0.24 15.3 .+-.
0.04 15.4 .+-. 0.17 16:1n-7 2.8 .+-. 0.14 2.8 .+-. 0.61 2.9 .+-.
0.02 6.8 .+-. 0.44 6.1 .+-. 0.27 6.2 .+-. 0.19 16:3n-4 0.9 .+-.
0.22 0.8 .+-. 0.03 0.8 .+-. 0.08 nd nd nd 18:0 5.9 .+-. 0.41 5.1
.+-. 0.31 5.1 .+-. 0.18 2.9 .+-. 0.15 2.9 .+-. 0.12 3 .+-. 0.08
18:1n-6 0.3 .+-. 0.07 0.4 .+-. 0.02 0.4 .+-. 0.01 0.4 .+-. 0.01 0.4
.+-. 0.01 0.4 .+-. 0.01 18:1n-7 3.0 .+-. 0.07 3.2 .+-. 0.15 3.4
.+-. 0.21 3.6 .+-. 0.05 3.6 .+-. 0.04 3.6 .+-. 0.01 18:1n-9 14.0
.+-. 1.42 12.6 .+-. 0.47 12.7 .+-. 0.15 15.5 .+-. 0.45 15.7 .+-.
0.67 14.7 .+-. 0.22 18:2n-6 1.4 .+-. 0.75 1.01 .+-. 0.07 0.8 .+-.
0.18 2.5 .+-. 0.07 2.6 .+-. 0.15 2.4 .+-. 0.07 18:3n-3 0.2 .+-.
0.19 0.1 .+-. 0.03 nd 0.6 .+-. 0.02 0.6 .+-. 0.05 0.5 .+-. 0.02
18:3n-6 0.1 .+-. 0.05 0.1 .+-. 0.06 nd nd .sup. 1.1 .+-. 0.08.sup.b
.sup. 1.4 .+-. 0.13.sup.b 18:4n-3 nd nd nd 1.2 .+-. 0.06 nd 0.4
.+-. 0.38 TTA nd 0.8 .+-. 0.17 nd 0.7 .+-. 0.37 20:1n-9 0.3 .+-.
0.07 0.2 .+-. 0.01 0.3 .+-. 0.09 0.5 .+-. 0.03 0.5 .+-. 0.02 0.5
.+-. 0.04 20:1n-11 3.5 .+-. 0.11 3.8 .+-. 0.43 3.5 .+-. 0.18 14.7
.+-. 0.64 15.0 .+-. 0.80 13.6 .+-. 0.58 20:2n-6 nd nd nd 0.2 .+-.
0.11 0.2 .+-. 0.10 0.1 .+-. 0.10 20:3n-6 nd 0.1 .+-. 0.01 nd nd nd
0.1 .+-. 0.07 20:4n-3 0.27 .+-. 0.08 0.2 .+-. 0.002 0.3 .+-. 0.07
0.7 .+-. 0.02 0.6 .+-. 0.03 0.7 .+-. 0.01 20:4n-6 2.5 .+-. 0.10 2.5
.+-. 0.12 2.9 .+-. 0.21 0.6 .+-. 0.05 0.5 .+-. 0.05 0.7 .+-. 0.12
20:5n-3 6.2 .+-. 0.52 6.5 .+-. 0.23 6.9 .+-. 0.62 .sup. 3.9 .+-.
0.25.sup.ab .sup. 3.5 .+-. 0.10.sup.a .sup. 4 .+-. 0.09.sup.b
22:1n-9 nd nd nd nd nd nd 22:1n-11 0.8 .+-. 0.41 1.4 .+-. 0.44 1.03
.+-. 0.02 .sup. 10.4 .+-. 0.30.sup.ab 11.7 .+-. 0.51.sup.b 10.1
.+-. 0.50.sup.a 22:5n-3 0.8 .+-. 0.17 1.1 .+-. 0.003 1.2 .+-. 0.09
1.2 .+-. 0.05 1.0 .+-. 0.01 1.2 .+-. 0.02 22:6n-3 22.0 .+-.
1.27.sup.a 24.7 .+-. 0.71.sup.a 27.8 .+-. 0.57.sup.b 9.3 .+-. 0.52
9.2 .+-. 0.54 11.6 .+-. 0.42 Others.sup. 1.9 .+-. 0.29 5.0 .+-.
1.72 2.9 .+-. 0.34 1.5 .+-. 0.56 0.7 .+-. 0.09 0.8 .+-. 0.12
.SIGMA.saturated 36.2 .+-. 1.03.sup.b 30.9 .+-. 0.62.sup.a 29.1
.+-. 0.32.sup.a 24.0 .+-. 0.40 24.1 .+-. 0.19 24.3 .+-. 0.13
.SIGMA.Monounsaturated 25.8 .+-. 1.97 25.5 .+-. 2.04 25.6 .+-. 0.53
.sup. 52.1 .+-. 0.92.sup.ab 53.1 .+-. 0.93.sup.b 49.1 .+-.
1.12.sup.a .SIGMA.n-6 4.3 .+-. 1.10 3.7 .+-. 0.07 3.9 .+-. 0.18 3.4
.+-. 0.14 4.5 .+-. 0.07 4.6 .+-. 0.11 .SIGMA.n-3 29.7 .+-.
1.45.sup.a 33.0 .+-. 0.86.sup.a 36.9 .+-. 0.95.sup.b .sup. 16.8
.+-. 0.87.sup.ab 14.9 .+-. 0.44.sup.a 18.4 .+-. 0.58.sup.b Free
fatty acids Fatty acids FO 0.5% TTA 1.5% TTA 14:0 .sup. 2.5 .+-.
0.04.sup.b .sup. 2.4 .+-. 0.14.sup.b .sup. 2.0 .+-. 0.08.sup.a 16:0
20.4 .+-. 0.12.sup.c 17.8 .+-. 0.38.sup.b 15.7 .+-. 0.43.sup.a
16:1n-7 .sup. 4.1 .+-. 0.02.sup.b .sup. 4.0 .+-. 0.24.sup.b .sup.
3.5 .+-. 0.12.sup.a 16:3n-4 1.0 .+-. 0.05 0.9 .+-. 0.024 0.9 .+-.
0.05 18:0 5.6 .+-. 0.07 5.1 .+-. 0.04 5.1 .+-. 0.62 18:1n-6 0.5
.+-. 0.02 0.4 .+-. 0.01 0.4 .+-. 0.003 18:1n-7 .sup. 4.3 .+-.
0.12.sup.b .sup. 4.0 .+-. 0.05.sup.ab .sup. 3.8 .+-. 0.15.sup.a
18:1n-9 15.6 .+-. 0.47 14.7 .+-. 0.47 14.2 .+-. 0.36 18:2n-6 1.6
.+-. 0.01 1.7 .+-. 0.08 1.4 .+-. 0.07 18:3n-3 0.2 .+-. 0.01 0.2
.+-. 0.03 0.2 .+-. 0.01 18:3n-6 nd nd nd 18:4n-3 .sup. 0.3 .+-.
0.01.sup.b .sup. 0.1 .+-. 0.7.sup.ab .sup. 0.2 .+-. 0.03.sup.ab TTA
nd 0.7 .+-. 0.36 20:1n-9 .sup. 4.5 .+-. 0.20.sup.b .sup. 3.8 .+-.
0.08.sup.a .sup. 3.4 .+-. 0.07.sup.a 20:1n-11 .sup. 0.5 .+-.
0.01.sup.a .sup. 0.4 .+-. 0.02.sup.b .sup. 0.4 .+-. 0.04.sup.b
20:2n-6 0.3 .+-. 0.004 0.3 .+-. 0.02 0.3 .+-. 0.03 20:3n-6 0.3 .+-.
0.01 0.3 .+-. 0.01 0.3 .+-. 0.02 20:4n-3 0.4 .+-. 0.07 0.5 .+-.
0.02 0.5 .+-. 0.02 20:4n-6 5.6 .+-. 0.26 .sup. 6.0 .+-. 0.08.sup.ab
.sup. 6.2 .+-. 0.33.sup.ab 20:5n-3 9.2 .+-. 0.30 10.1 .+-. 0.23
10.1 .+-. 0.53 22:1n-9 .sup. 0.2 .+-. 0.02.sup.b nd.sup.a nd
22:1n-11 .sup. 1.2 .+-. 0.12.sup.b .sup. 0.8 .+-. 0.15.sup.ab .sup.
0.6 .+-. 0.08.sup.a 22:5n-3 .sup. 1.6 .+-. 0.04.sup.a .sup. 2.0
.+-. 0.14.sup.ab .sup. 2.3 .+-. 0.19.sup.b 22:6n-3 17.2 .+-.
0.09.sup.a 21.8 .+-. 1.5.sup.b 23.9 .+-. 0.67.sup.b Others.sup. 2.0
.+-. 0.17 2.0 .+-. 0.36 4.6 .+-. 1.01 .SIGMA.saturated 29.0 .+-.
0.13.sup.b 25.7 .+-. 0.54.sup.a 23.3 .+-. 1.30.sup.a
.SIGMA.Monounsaturated 31.2 .+-. 0.83.sup.b 28.4 .+-. 0.99.sup.a
25.6 .+-. 0.64.sup.a .SIGMA.n-6 8.2 .+-. 0.26 8.6 .+-. 0.12 8.6
.+-. 0.37 .SIGMA.n-3 28.6 .+-. 0.38.sup.a 34.3 .+-. 1.58.sup.b 36.6
.+-. 1.23.sup.b FO: fish oil, 0.5% TTA: fish oil added 0.5% TTA,
1.5% TTA: fish oil added 1.5% TTA. The quantity of each fatty acid
is given as percentage of the total fatty acids (FA). Data are
means .+-. SEM. Values within the same row with different
superscripts are significantly different, p < 0.05, n = 3; nd =
not detected. .sup. Includes nd. FA and some FA with percentages
less than 1. indicates data missing or illegible when filed
TABLE-US-00008 TABLE 8 Fatty acid composition of the heart.
Phospholipids Triacylglycerol Fatty acids FO 0.5% TTA 1.5% TTA FO
0.5% TTA 1.5% TTA 14:0 1.6 .+-. 0.20 1.6 .+-. 0.16 1.2 .+-. 0.22
6.2 .+-. 0.35 6.1 .+-. 0.20 5.9 .+-. 0.20 16:0 22.8 .+-. 0.12.sup.b
22.2 .+-. 0.5.sup.b 19.0 .+-. 0.79.sup.a 14.4 .+-. 1.2 13.9 .+-.
0.70 13.8 .+-. 0.19 16:1n-7 1.6 .+-. 0.01 1.8 .+-. 0.17 1.8 .+-.
0.09 6.7 .+-. 0.18 6.7 .+-. 0.34 6.6 .+-. 0.07 18:0 4.1 .+-. 0.19
3.9 .+-. 0.09 3.5 .+-. 0.22 2.3 .+-. 0.15 2.2 .+-. 0.07 2.3 .+-.
0.14 18:1n-6 0.3 .+-. 0.05 0.3 .+-. 0.05 0.3 .+-. 0.01 0.3 .+-.
0.13 0.4 .+-. 0.01 0.4 .+-. 0.01 18:1n-7 2.7 .+-. 0.15 2.9 .+-.
0.06 3.0 .+-. 0.1 3.8 .+-. 0.09 3.6 .+-. 0.11 3.6 .+-. 0.09 18:1n-9
7.2 .+-. 0.09 8.3 .+-. 0.20 8.0 .+-. 0.49 15.0 .+-. 0.14 14.5 .+-.
0.15 14.1 .+-. 0.22 18:2n-6 1.0 .+-. 0.20 1.0 .+-. 0.09 1.2 .+-.
0.05 2.4 .+-. 0.22 2.1 .+-. 0.46 2.5 .+-. 0.07 18:3n-3 nd nd 0.1
.+-. 0.08 0.4 .+-. 0.19 0.9 .+-. 0.33 0.6 .+-. 0.01 18:4n-3 .sup.
nd.sup.a .sup. nd.sup.ab .sup. 0.2 .+-. 0.01.sup.b nd nd nd TTA nd
0.7 .+-. 0.4 nd nd 20:1n-9 .sup. 3.8 .+-. 0.07.sup.a .sup. 4.3 .+-.
0.29.sup.ab .sup. 4.5 .+-. 0.13.sup.b 16.9 .+-. 0.69 16.2 .+-. 0.56
16.6 .+-. 0.81 20:1n-11 nd nd nd 0.4 .+-. 0.19 0.5 .+-. 0.04 0.5
.+-. 0.03 20:2n-6 nd nd 0.2 .+-. 0.003 0.2 .+-. 0.11 0.2 .+-. 0.1
0.3 .+-. 0.01 20:3n-6 nd nd 0.1 .+-. 0.07 .sup. nd.sup.a .sup. 0.1
.+-. 0.03.sup.b .sup. nd.sup.a 20:4n-3 0.8 .+-. 0.04 0.5 .+-. 0.07
0.7 .+-. 0.09 0.5 .+-. 0.27 0.7 .+-. 0.01 0.7 .+-. 0.052 20:4n-6
1.5 .+-. 0.03 1.4 .+-. 0.07 1.5 .+-. 0.08 0.1 .+-. 0.06 nd 0.2 .+-.
0.08 20:5n-3 10.7 .+-. 0.27.sup.b .sup. 9.0 .+-. 0.51.sup.b .sup.
8.4 .+-. 0.09.sup.a .sup. 3.3 .+-. 0.05.sup.a .sup. 3.4 .+-.
0.06.sup.a .sup. 3.7 .+-. 0.05.sup.b 22:1n-9 0.8 .+-. 0.09 1.0 .+-.
0.15 1.0 .+-. 0.09 1.2 .+-. 0.59 1.7 .+-. 0.02 1.7 .+-. 0.08
22:1n-11 nd nd nd 11.23 .+-. 0.24 11.6 .+-. 0.27 11.4 .+-. 0.32
22:5n-3 .sup. 1.8 .+-. 0.06.sup.a .sup. 2.0 .+-. 0.05.sup.a .sup.
2.4 .+-. 0.06.sup.b 0.8 .+-. 0.38 1.1 .+-. 0.06 1.2 .+-. 0.06
22:6n-3 35.5 .+-. 0.08 35.9 .+-. 1.39 38.5 .+-. 1.4 7.9 .+-. 0.44
7.6 .+-. 0.37 8.4 .+-. 0.43 others.sup. 1.4 .+-. 0.21 1.3 .+-. 0.22
1.8 .+-. 0.03 0.2 .+-. 0.09 0.4 .+-. 0.06 0.4 .+-. 0.10
.SIGMA.Saturated 32.2 .+-. 3.09.sup.b 28.1 .+-. 0.76 24.3 .+-.
1.00.sup.a 24.8 .+-. 2.57 22.8 .+-. 0.89 23.0 .+-. 0.42
.SIGMA.Monounsaturated 12.7 .+-. 3.02 17.8 .+-. 0.59 17.8 .+-. 0.76
55.6 .+-. 1.54 55.3 .+-. 1.22 55.5 .+-. 1.40 .SIGMA. n-6 2.3 .+-.
0.25 2.6 .+-. 0.17 3.0 .+-. 0.15 2.7 .+-. 0.34 2.4 .+-. 0.41 2.8
.+-. 0.07 .SIGMA.n-3 49.5 .+-. 0.60 47.5 .+-. 1.6 50.4 .+-. 1.4
13.7 .+-. 1.42 15.2 .+-. 0.83 16.1 .+-. 0.74 Free fatty acids Fatty
acids FO 0.5% TTA 1.5% TTA 14:0 1.2 .+-. 0.14 1.5 .+-. 0.09 1.5
.+-. 0.14 16:0 21.9 .+-. 0.93 21.1 .+-. 0.59 20.4 .+-. 1.16 16:1n-7
2.0 .+-. 0.11 1.4 .+-. 0.7 2.1 .+-. 0.1 18:0 5.2 .+-. 0.4.sup.b
.sup. 5.0 .+-. 0.26.sup.ab .sup. 4.2 .+-. 0.20.sup.a 18:1n-6 .sup.
nd.sup.a .sup. 0.4 .+-. 0.01.sup.b .sup. 0.3 .+-. 0.01.sup.b
18:1n-7 3.6 .+-. 0.09 3.5 .+-. 0.20 3.3 .+-. 0.15 18:1n-9 8.4 .+-.
0.10 8.6 .+-. 0.40 8.2 .+-. 0.47 18:2n-6 1.5 .+-. 0.14 1.4 .+-.
0.07 1.1 .+-. 0.14 18:3n-3 nd 0.2 .+-. 0.08 nd 18:4n-3 nd nd nd TTA
nd nd 20:1n-9 .sup. 5.9 .+-. 0.15.sup.b .sup. 5.9 .+-. 0.18.sup.b
.sup. 5.2 .+-. 0.13.sup.a 20:1n-11 nd nd nd 20:2n-6 nd nd nd
20:3n-6 nd nd nd 20:4n-3 0.5 .+-. 0.26 0.7 .+-. 0.14 0.6 .+-. 0.07
20:4n-6 20:5n-3 9.8 .+-. 0.65 8.9 .+-. 0.50 9.3 .+-. 0.42 22:1n-9
nd nd nd 22:1n-11 .sup. 2.1 .+-. 0.17.sup.b .sup. 2.1 .+-.
0.13.sup.b .sup. 1.4 .+-. 0.16.sup.a 22:5n-3 .sup. 1.8 .+-.
0.07.sup.a .sup. 2.0 .+-. 0.05.sup.b .sup. 2.3 .+-. 0.03.sup.c
22:6n-3 30.7 .+-. 0.82.sup.a 33.2 .+-. 1.07.sup.a 35.3 .+-.
1.5.sup.b others.sup. 1.3 .+-. 0.60 1.4 .+-. 0.45 1.4 .+-. 0.91
.SIGMA.Saturated 28.4 .+-. 0.83 27.7 .+-. 0.97 26.4 .+-. 1.19
.SIGMA.Monounsaturated 22.3 .+-. 0.47 21.9 .+-. 0.97 20.7 .+-. 0.87
.SIGMA. n-6 3.2 .+-. 0.21 3.5 .+-. 0.08 3.2 .+-. 0.08 .SIGMA.n-3
42.9 .+-. 0.5 45.0 .+-. 1.71 47.5 .+-. 1.36 FO: fish oil, 0.5% TTA:
fish oil added 0.5% TTA, 1.5% TTA: fish oil added 1.5% TTA. The
quantity of each fatty acid is given as percentage of the total
fatty acids (FA). Data are means .+-. SEM. Values within the same
row with different superscripts are significantly different, p <
0.05, n = 3; nd = not detected. .sup. Includes nd. FAs and some FAs
with percentages less than 1. indicates data missing or illegible
when filed
TABLE-US-00009 TABLE 9 Fatty acid composition of the liver.
Phospholipids Triacylglycerol Fatty acids FO 0.5% TTA 1.5% TTA FO
0.5% TTA 1.5% TTA 14:0 .sup. 2.6 .+-. 0.06.sup.b 2.3 .+-. 0.09
.sup. 2.2 .+-. 0.05.sup.a 3.5 .+-. 0.65 3.8 .+-. 0.36 2.6 .+-. 1.16
16:0 21.3 .+-. 0.90.sup.b 17.3 .+-. 0.36.sup.a 17.2 .+-. 0.41.sup.a
10.0 .+-. 0.58 9.7 .+-. 1.62 6.7 .+-. 3.34 16:1n-7 .sup. 1.7 .+-.
0.04.sup.b 1.7 .+-. 0.02 .sup. 1.6 .+-. 0.03.sup.a 6.1 .+-. 0.06
5.6 .+-. 0.05 4.8 .+-. 0.8 16:3n-4 0.5 .+-. 0.05 0.5 .+-. 0.02 0.5
.+-. 0.04 0.4 .+-. 0.03 0.4 .+-. 0.06 0.2 .+-. 0.11 18:0 2.9 .+-.
0.3.sup.a .sup. 3.6 .+-. 0.17.sup.b .sup. 3.5 .+-. 0.04.sup.b 2.5
.+-. 0.39 2.1 2.5 .+-. 0.19 18:1n-6 .sup. 0.4 .+-. 0.01.sup.a .sup.
0.4 .+-. 0.02.sup.b .sup. 0.4 .+-. 0.01.sup.ab 0.4 .+-. 0.004 0.3
.+-. 0.001 nd 18:1n-7 .sup. 1.9 .+-. 0.08.sup.a .sup. 2.3 .+-.
0.09.sup.b .sup. 2.1 .+-. 0.04.sup.ab 4.5 .+-. 0.21 4.6 .+-. 0.61
4.4 .+-. 0.19 18:1n-9 8.6 .+-. 0.19 9.3 .+-. 0.18 9.3 .+-. 0.27
25.2 .+-. 2.14 23.5 .+-. 2.8 25.4 .+-. 4.60 18:2n-6 1.1 .+-. 0.11
1.5 .+-. 0.12 1.4 .+-. 0.02 2.3 .+-. 0.22 2.7 .+-. 0.13 3.2 .+-.
0.87 18:3n-3 nd nd nd 0.4 .+-. 0.08 0.5 .+-. 0.09 0.6 .+-. 0.23
18:3n-6 nd 0.1 .+-. 0.004 0.1 .+-. 0.004 nd nd nd 18:4n-3 .sup.
nd.sup.a .sup. 0.1 .+-. 0.03.sup.b .sup. 0.1 .+-. 0.00.sup.b 0.7
.+-. 0.10 0.8 .+-. 0.18 0.5 .+-. 0.27 .DELTA..sup.9-desaturased TTA
0.1 .+-. 0.01 0.1 .+-. 0.01 nd nd TTA 0.05 .+-. 0.02 nd nd nd
20:1n-9 .sup. 0.1 .+-. 0.07.sup.a .sup. 0.2 .+-. 0.01.sup.b .sup.
0.2 .+-. 0.01.sup.b 0.5 .+-. 0.01 0.3 .+-. 0.01 0.5 .+-. 0.001
20:1n-11 .sup. 4.6 .+-. 0.24.sup.a .sup. 5.2 .+-. 0.25.sup.b .sup.
5.2 .+-. 0.05.sup.a 17.3 .+-. 0.89 16.7 .+-. 0.65 16.6 .+-. 0.28
20:2n-6 0.4 .+-. 0.03 0.4 .+-. 0.02 0.4 .+-. 0.01 0.4 .+-. 0.01 0.5
.+-. 0.07 0.4 .+-. 0.26 20:3n-6 0.4 .+-. 0.02 0.4 .+-. 0.02 0.4
.+-. 0.02 nd nd nd 20:4n-3 0.7 .+-. 0.05 0.8 .+-. 0.02 0.8 .+-.
0.02 0.7 .+-. 0.06 0.8 .+-. 0.08 0.4 .+-. 0.21 20:4n-6 .sup. 1.8
.+-. 0.16.sup.ab .sup. 1.6 .+-. 0.06.sup.a .sup. 2.0 .+-.
0.06.sup.b nd nd nd 20:5n-3 10.3 .+-. 0.78 9.7 .+-. 0.17 9.3 .+-.
0.30 2.5 .+-. 0.13 2.7 .+-. 0.19 1.9 .+-. 0.27 22:1n-9 nd nd nd 1.3
.+-. 0.03 1.3 .+-. 0.19 0.9 .+-. 0.43 22:1n-11 0.5 .+-. 0.09 0.4
.+-. 0.20 0.6 .+-. 0.02 8.2 .+-. 0.53 8.7 .+-. 1.45 8.7 .+-. 0.95
22:5n-3 1.6 .+-. 0.81 2.6 .+-. 0.08 2.6 .+-. 0.11 1.1 .+-. 0.004
1.3 .+-. 0.03 0.7 .+-. 0.35 22:6n-3 35.6 .+-. 0.51 36.2 .+-. 0.28
36.7 .+-. 0.49 5.2 .+-. 0.49 7.3 .+-. 1.28 6.3 .+-. 0.65
others.sup. 0.7 .+-. 0.16 1.5 .+-. 0.23 1.3 .+-. 0.06 2.9 .+-. 1.06
1.4 .+-. 0.87 4.5 .+-. 3.08 .SIGMA.saturated 27.5 .+-. 0.49.sup.b
23.8 .+-. 0.49.sup.a 23.6 .+-. 0.40.sup.a 18.4 .+-. 0.57 18.9 .+-.
1.93 20.5 .+-. 3.66 .SIGMA.Monounsaturated 18.1 .+-. 0.29.sup.a
19.7 .+-. 0.26.sup.b 19.7 .+-. 0.33.sup.b 63.6 .+-. 2.70.sup.a 61.3
.+-. 2.25.sup.b 58.7 .+-. 0.79.sup.b .SIGMA.n-6 3.7 .+-. 0.38 4.1
.+-. 0.10 4.3 .+-. 0.10 2.8 .+-. 0.34 3.1 .+-. 0.05 2.5 .+-. 0.004
.SIGMA.n-3 49.9 .+-. 1.03 51.3 .+-. 0.18 51.4 .+-. 0.14 11.7 .+-.
0.56 14.4 .+-. 1.06 12.8 .+-. 0.20 Free fatty acids Fatty acids FO
0.5% TTA 1.5% TTA 14:0 3.9 .+-. 0.57 4.0 .+-. 0.04 3.5 .+-. 0.16
16:0 17.7 .+-. 1.7 16.6 .+-. 0.58 16.1 .+-. 0.35 16:1n-7 5.0 .+-.
0.93 5.0 .+-. 0.13 5.0 .+-. 0.09 16:3n-4 0.8 .+-. 0.07 0.9 .+-.
0.01 0.8 .+-. 0.06 18:0 2.8 .+-. 0.14 2.2 .+-. 0.31 2.4 .+-. 0.08
18:1n-6 0.5 .+-. 0.07 0.5 .+-. 0.002 0.5 .+-. 0.03 18:1n-7 3.9 .+-.
0.13 4.4 .+-. 0.09 4.4 .+-. 0.15 18:1n-9 24.9 .+-. 3.60 21.7 .+-.
0.67 23.0 .+-. 0.72 18:2n-6 4.0 .+-. 1.7 2.3 .+-. 0.11 2.2 .+-.
0.14 18:3n-3 1.0 .+-. 0.63 0.4 .+-. 0.05 0.4 .+-. 0.05 18:3n-6 nd
.sup. 0.1 .+-. 0.06.sup.b .sup. nd.sup.a 18:4n-3 0.4 .+-. 0.12 0.3
.+-. 0.08 0.4 .+-. 0.09 .DELTA..sup.9-desaturased TTA nd nd TTA nd
nd 20:1n-9 0.3 .+-. 0.02 0.3 .+-. 0.01 0.2 .+-. 0.11 20:1n-11 6.7
.+-. 0.70 .sup. 7.8 .+-. 0.30.sup.ab .sup. 9.2 .+-. 0.54.sup.b
20:2n-6 0.6 .+-. 0.25 0.4 .+-. 0.01 0.4 .+-. 0.02 20:3n-6 .sup. 0.3
.+-. 0.12.sup.b .sup. 0.2 .+-. 0.01.sup.ab .sup. nd.sup.a 20:4n-3
1.0 .+-. 0.10 1.2 .+-. 0.04 1.3 .+-. 0.08 20:4n-6 0.8 .+-. 0.07 0.8
.+-. 0.04 0.7 .+-. 0.02 20:5n-3 5.9 .+-. 0.77 5.9 .+-. 0.46 5.4
.+-. 0.43 22:1n-9 0.5 .+-. 0.07 0.6 .+-. 0.05 0.4 .+-. 0.21
22:1n-11 1.9 .+-. 0.3 2.5 .+-. 0.28 2.4 .+-. 0.36 22:5n-3 1.5 .+-.
0.23 1.7 .+-. 0.10 1.1 .+-. 0.54 22:6n-3 11.3 .+-. 0.69 15.1 .+-.
0.74 14.9 .+-. 0.40 others.sup. .sup. 2.4 .+-. 0.57.sup.a .sup. 3.0
.+-. 0.11.sup.ab .sup. 3.2 .+-. 0.16.sup.b .SIGMA.saturated 25.8
.+-. 2.10 24.2 .+-. 0.75 23.4 .+-. 0.43 .SIGMA.Monounsaturated 43.8
.+-. 1.65 43.0 .+-. 1.39 45.3 .+-. 1.55 .SIGMA.n-6 5.8 .+-. 2.08
3.8 .+-. 0.11 3.4 .+-. 0.13 .SIGMA.n-3 21.7 .+-. 1.09 25.2 .+-.
1.27 23.9 .+-. 1.05 FO: fish oil, 0.5% TTA: fish oil added 0.5%
TTA, 1.5% TTA: fish oil added 1.5% TTA. The quantity of each fatty
acid is given as percentage of the total fatty acids (FA). Data are
means .+-. SEM. Values within the same row with different
superscripts are significantly different, p < 0.05, n = 3; nd =
not detected. .sup. Includes nd. FAs and some FAs with percentages
less than 1. indicates data missing or illegible when filed
2.4 Biological Effects of the Composition According to the
Invention and a Fermented Protein Material
Chemicals
[0126] Gendaxin was obtained from Aximed, Bergen, Norway. One
capsule of Gendaxine contains 35 mg isoflavones, inter alia 10 mg
Genistein and 15 mg Daidzein.
Lipid Analysis
[0127] Plasma lipids were measured enzymatically on the Technicon
Axon system (Miles, Tarrytown, N.Y.) using the Triglyceride kit
from Bayer, Total cholesterol (Bayer, Tarrytown, N.Y.), and the
PAP150 kit for choline containing phospholipids from bioMerieux.
The results were given in mmol/l, and the data is presented in
table 10 below.
TABLE-US-00010 TABLE 10 Total cholesterol, triglycerides and
phospholipids of the plasma. Cholesterol Triglycerides
Phospholipids Control 10.02 2.95 11.98 0.25% Gendaxin 9.14 2.71
11.19 0.5% Gendaxin + 9.10 2.12 10.66 0.9% TTA
[0128] It is evident from the above data that addition of Gendaxin
to the fish feed has a positive effect on the fatty acid
composition of the plasma of the salmon. The cholesterol,
triglyceride and phospholipids levels all decreased with 0.25%
Gendaxin added to the fish feed when compared to the
control-Further addition of Gendaxin and TTA improved the fatty
acid composition of the plasma additionally.
Enzyme Assay
[0129] Fatty acyl-CoA oxidase activity was measured in the
peroxisomal liver fraction as previously described (Small G M,
Burdett K, Connock M J (1985) Biochem J 227: 205-10). The results
were given as fatty acyl-CoA oxidase activity per total protein,
and are shown in table 11 below.
TABLE-US-00011 TABLE 11 Hepatic .beta.-oxidation. Beta oxidation
Control 0.940 0.5% Gendaxin + 0.9% TTA 1.501
[0130] It is evident from the above data that addition of Gendaxin
and TTA to the fish feed has a positive effect on .beta.-oxidation,
as the .beta.-oxidation is highly increased.
Example 3
[0131] In line with the experimental setup given in example 1, we
have conducted a feeding experiment on Male Wistar rats (see Table
12) with the following feed components: [0132] 30% fat [0133] 20%
protein [0134] 5% fiber [0135] 10% sucrose [0136] 3.5% AIN93G
mineral mix [0137] 1.0% AIN-93 vitamin mix [0138] The remainding:
Starch
[0139] The fat component is 30% lard, or 2.5-5% of the lard is
exchanged with fish oil or 0.15 of the lard is exchanged with TTA.
The protein material is 20% milk protein (casein), or half of it is
exchanged with fish protein or "Bioprotein".
TABLE-US-00012 TABLE 12 HDL Ch Tg HDL Ch FFA PL Ch/Ch Treatment
mmol/L mmol/L mmol/L mmol/L mmol/L ratio FPH, 10% 2.03 1.16 1.63
0.34 1.48 0.80 Fish oil, 2.5% 1.77 1.27 1.42 0.40 1.47 0.80 Fish
oil, 5% 1.79 1.13 1.44 0.40 1.41 0.81 Fish oil 2.5% + FPH 10% 1.26
0.93 1.00 0.35 1.07 0.79 Fish oil 5% + FPH 10% 1.21 0.92 0.97 0.37
0.98 0.80 FPH 10% + TTA 0.15% 1.27 0.65 1.07 0.28 1.18 0.83 Fish
oil 2.5% + TTA 0.15% 1.21 0.43 1.00 0.25 1.01 0.83 Fish oil 5% +
TTA 0.15% 1.26 0.61 1.02 0.23 1.14 0.81 Fish oil 2.5% + FPH 10% +
TTA 0.15% 0.98 0.46 0.81 0.31 1.00 0.83 Fish oil 5% + FPH 10% + TTA
0.15% 1.02 0.72 0.84 0.32 1.00 0.84 TTA 0.15% 1.56 0.39 1.31 0.28
1.17 0.84 Bioprotein High 20% 1.23 0.80 0.95 0.41 1.00 0.77
Bioprotein Low 20% 1.28 0.61 1.04 0.34 1.01 0.81 Kontroll (Casein
20%) 1.90 1.04 1.52 0.38 1.41 0.80 Weight WAT WAT Liver/ WAT WAT of
rat Liver epi ret bw epi/bw ret/bw gram gram gram gram ratio ratio
ratio FPH, 10% 460.83 10.04 7.99 9.90 2.17 1.69 2.12 Fish oil, 2.5%
427.83 9.44 7.00 8.67 2.21 1.63 2.01 Fish oil, 5% 445.17 9.87 7.59
9.79 2.21 1.70 2.20 Fish oil 2.5% + FPH 10% 438.83 10.01 6.33 8.80
2.28 1.42 2.02 Fish oil 5% + FPH 10% 432.50 10.18 7.31 8.86 2.35
1.67 2.05 FPH 10% + TTA 0.15% 436.17 15.55 6.80 8.25 3.56 1.54 1.88
Fish oil 2.5% + TTA 0.15% 405.00 13.25 4.59 7.09 3.26 1.13 1.75
Fish oil 5% + TTA 0.15% 442.83 14.80 5.48 7.95 3.34 1.22 1.77 Fish
oil 2.5% + FPH 10% + TTA 0.15% 438.50 15.66 6.03 8.20 3.57 1.36
1.85 Fish oil 5% + FPH 10% + TTA 0.15% 449.67 16.29 7.18 9.19 3.62
1.58 2.02 TTA 0.15% 404.00 13.13 4.25 6.28 3.26 1.05 1.55
Bioprotein High 20% 413.33 9.00 5.02 6.99 2.18 1.21 1.69 Bioprotein
Low 20% 420.67 8.97 4.63 6.65 2.13 1.09 1.58 Kontroll (Casein 20%)
417.36 8.91 5.94 8.46 2.13 1.42 2.03
* * * * *