U.S. patent application number 10/758719 was filed with the patent office on 2004-08-12 for method for supplying bioavailable methionine to a cow.
Invention is credited to Bennett, Robert, Gros, Georges, Robert, Jean-Claude.
Application Number | 20040154549 10/758719 |
Document ID | / |
Family ID | 26234645 |
Filed Date | 2004-08-12 |
United States Patent
Application |
20040154549 |
Kind Code |
A1 |
Robert, Jean-Claude ; et
al. |
August 12, 2004 |
Method for supplying bioavailable methionine to a cow
Abstract
The present invention relates to a method for supplying
bioavailable methionine to a cow which comprises supplying to the
cow an ester of methionine or methionine amide and/or an ester of
the hydroxy analogue of methionine or a salt thereof.
Inventors: |
Robert, Jean-Claude;
(Antony, FR) ; Bennett, Robert; (Antony, FR)
; Gros, Georges; (Antony, FR) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Family ID: |
26234645 |
Appl. No.: |
10/758719 |
Filed: |
January 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10758719 |
Jan 16, 2004 |
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10336912 |
Jan 6, 2003 |
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10336912 |
Jan 6, 2003 |
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10060327 |
Feb 1, 2002 |
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6528541 |
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10060327 |
Feb 1, 2002 |
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09794347 |
Feb 28, 2001 |
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6372788 |
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09794347 |
Feb 28, 2001 |
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09438521 |
Nov 12, 1999 |
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6221909 |
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Current U.S.
Class: |
119/51.01 |
Current CPC
Class: |
A23K 20/142 20160501;
A23K 20/105 20160501; A61P 15/00 20180101; A61P 1/00 20180101; A61K
31/223 20130101; A23K 50/10 20160501 |
Class at
Publication: |
119/051.01 |
International
Class: |
A01K 001/10; A01K
005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 13, 1998 |
FR |
FR 98 14249 |
Jul 29, 1999 |
FR |
FR 99 10050 |
Claims
We claim:
1. A method for supplying bioavailable methionine to a cow, which
comprises administering to the cow at least one of an ester of
methionine, an ester of methionine amide, and an ester of the
hydroxy analogue of methionine, or which comprises administering to
the cow a salt of at least one of the esters.
2. A method as claimed in claim 1, in which the at least one ester
or its salt is administered to the cow by feeding to the cow a feed
containing the at least one ester or its salt as a supplement.
3. A method as claimed in claim 1, in which the at least one ester
is an alkyl ester having 1 to 12 carbon atoms.
4. A method as claimed in claim 3, in which the alkyl ester has 1
to 10 carbon atoms.
5. A method as claimed in claim 1, in which the at least one ester
is an alkyl ester and is methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, sec-butyl, tertriary-butyl, n-pentyl, isopentyl,
n-hexyl or isohexyl.
6. A method as claimed in claim 4, in which the alkyl ester has 1
to 4 carbon atoms.
7. A method as claimed in claim 6, in which the alkyl ester is
branched.
8. A method as claimed in claim 7, in which the alkyl ester is the
isopropyl ester.
9. A method as claimed in claim 8, in which the alkyl ester is the
isopropyl ester of the hydroxy analogue of methionine.
10. A method as claimed in claim 7, in which the alkyl ester is the
tertiary-butyl ester.
11. A method as claimed in claim 10, in which the alkyl ester is
the tertiary-butyl ester of methionine.
12. A method of supplying at least 50% bioavailable methionine to a
cow, which comprises administering to the cow the tertiary butyl
ester of methionine or the isopropyl ester of the hydroxy analogue
of methionine.
13. A method of improving milk obtained from a dairy cow, which
comprises supplying to the cow at least one of an ester of
methionine, an ester of methionine amide, and an ester of the
hydroxy analogue of methionine, or which comprises administering to
the cow a salt of at least one of the esters.
14. A method as claimed in claim 13, wherein the improvement in the
milk comprises increased protein content in the milk.
15. A method as claimed in claim 13, wherein the improvement in the
milk comprises increased fat content in the milk.
16. A ration comprising a grain portion, a concentrate portion and
a supplement, said supplement comprising at least one of an ester
of methionine, an ester of methionine amide, and an ester of the
hydroxy analogue of methionine, or said supplement comprising a
salt of at least one of the esters.
17. A ration as claimed in claim 16, in which the supplement
comprises an amount of the at least one ester calculated as
methionine equivalent of up to 75 g.
18. A ration as claimed in claim 17, comprising an amount of the at
least one ester calculated as methionine equivalent of 10 to 30
g.
19. A ration as claimed in claim 16, in which the at least one
ester is the isopropyl ester of the hydroxy analogue of
methionine.
20. A ration as claimed in claim 19, wherein the isopropyl ester is
present in an amount of from 7 to 65 g per cow per day.
21. A ration as claimed in claim 16, in which the at least one
ester is the tert-butyl ester of methionine.
22. A ration as claimed in claim 21, in which the tert-butyl ester
is present in an amount of from 7 to 65 g per cow per day.
23. A unit dosage form, comprising an amount of at least one of an
ester of methionine, an ester of methionine amide, and an ester of
the hydroxy analogue of methionine, or comprising a salt of at
least one of the esters, suitable for dosage for one cow for one
day.
24. A method of improving the condition of a cow, which comprises
supplying to the cow at least one of an ester of methionine, an
ester of methionine amide, and an ester of the hydroxy analogue of
methionine, or which comprises administering to the cow a salt of
at least one of the esters.
25. A method as claimed in claim 24, in which the at least one
ester is an alkyl ester having 1 to 12 carbon atoms.
26. A method as claimed in claim 24, wherein the improvement in
condition of the cow comprises improved fertility.
27. A method as claimed in claim 24, wherein the improvement in
condition of the cow comprises improved liver function.
28. A method as claimed in claim 24, wherein the improvement in
condition of the cow comprises an increase in energy.
Description
[0001] This application claims the benefit of foreign priority to
French patent application no. 98 14249, filed Nov. 13, 1998, and
French patent application no. 99 10050, filed Jul. 29, 1999. Both
of these foreign priority documents are incorporated by reference
herein.
[0002] The present invention relates to a method for supplying
bioavailable methionine to a cow which comprises administering to
the cow an ester of methionine or methionine amide and/or an ester
of the hydroxy analogue of methionine or a salt thereof. The
present invention also relates to a method of improving milk
obtained from dairy cows and in particular to a method which
comprises supplying to the dairy cow an ester of methionine or
methionine amide and/or an ester of the hydroxy analogue of
methionine or a salt thereof.
[0003] Protein is one of the major nutrients in the diets of
lactating cows. The cows however do not actually require proteins
but instead they require the specific amino acids, which are the
building blocks that make up their own protein.
[0004] It is known that methionine is a limiting amino acid and in
particular for milk production it is believed that a well balanced
level of methionine will result in effective levels of milk
production. It is also believed that an increase in methionine
levels can result in increased milk production.
[0005] It is therefore desirable to maintain or even enhance the
level of methionine. Methionine can be added directly to the cow's
diet. However, the free form of this amino acid is rapidly degraded
by bacteria in the rumen and consequently only a small portion of
the methionine enters the bloodstream. There have been many
attempts to overcome this problem and in general the methionine is
introduced into the diet in a protected or modified form,
permitting the compound to pass through the rumen unaffected. The
methionine released from the protected or modified form then enters
the small intestine and is absorbed into the bloodstream. One of
the most widely studied compounds for this particular purpose is
the hydroxy analogue of methionine, namely 2-hydroxy4-(methylthio)
butanoic acid, generally referred to as HMB.
[0006] WO 99/04647, published on 4th Feb. 1999, discloses a method
of introducing methionine into the rumen by supplementing the feed
with the hydroxy analogue of methionine. In this patent
application, it is claimed that the hydroxy analogue is
substantially unaffected by rumen degradation, passing through the
rumen and consequently providing at least 20%, preferably at least
40% of the hydroxy analogue for absorption into the bloodstream
through the intestine. The patent application refers to the hydroxy
analogue, its salts, esters, amides and oligomers as being `rumen
by-pass` and claims an improved efficient means of introducing
methionine into the bloodstream of the cow. The claimed advantage
of the disclosed compounds in this documents is that the compounds
by-pass the rumen and are absorbed in the intestine.
[0007] There are also many publications on the effect of the
hydroxy analogue of methionine and a publication by Charles Schwab,
from a presentation given at a conference in May 1998, reviews all
of the publications and concludes that the hydroxy analogue of
methionine is thought to by-pass the rumen for intestinal
absorption but will only do so if it is administered at a dose
above 60 g per animal per day, preferably above 90 g per animal per
day. At lower doses, it would appear, according to the author, that
the hydroxy analogue of methionine is to a large extent, consumed
by the micro organism in the rumen.
[0008] The best determination of the absorption of the hydroxy
analogue of methionine is the determination of the bioavailability
in the blood. The bioavailability is characterised by the level of
appearance of methionine in the blood compared with the amount of
methionine equivalent of compound introduced into the feed ration.
This determination takes into account the passage of the hydroxy
analogue through the rumen, its degree of absorption irrespective
of the place of absorption during the digestive transit and the
degree enzymatic conversion of the hydroxy analogue into
methionine. At a dose of methionine equivalent to 50 g per day per
cow, it is described in this article that methionine protected
against degradation in the rumen with a polymer, in particular the
product sold under the trade name Smartamine.TM., has a rumen
by-pass of 90%; the hydroxy analogue gives a bioavailability of
only 3%.
[0009] A paper in J Dairy Science 1988, 71, pp3292 to 3301
discloses the introduction of the methyl ester or the ethyl ester
of the hydroxy analogue of methionine to the diet of a cow in an
attempt to increase the level of milk production. The results from
the study indicate that these esters are rapidly converted to the
hydroxy analogue of methionine and subsequently degraded in the
rumen of the animal. Specifically, after incubation for six hours
in rumen juices, only 1.8% and 3% of the methyl and ethyl ester of
the hydroxy analogue respectively, remains. This is compared with
34% and 85% of methionine and the hydroxy analogue of
methionine.
[0010] We have now found, contrary to the teachings of the
aforementioned prior art, that certain esters of methionine or a
methionine amide and/or the hydroxy analogue of methionine have a
favourable effect in cows. We have surprisingly found that certain
compounds introduce methionine into the bloodstream of the rumen
more effectively and more rapidly than the known prior art. We have
also found that these particular compounds do not enter the
bloodstream through rumen by-pass and intestinal absorption but by
absorption through the rumen wall. We have also found that
introducing the specific ester compound into the diet of dairy cows
through the feed ration results in desired improvement in milk
production.
[0011] Accordingly the present invention provides a method for
supplying bioavailable methionine to a cow which comprises
administering to the cow an ester of methionine or methionine amide
and/or an ester of the hydroxy analogue of methionine or a salt
thereof.
[0012] For the purposes of the present invention, by cow is meant
cattle, namely beef cows and dairy cows.
[0013] In particular the present invention provides a method for
supplying bioavailable. methionine to a cow which comprises
administering to the cow a branched alkyl ester of methionine or
methionine amide and/or a branched alkyl ester of the hydroxy
analogue of methionine.
[0014] The use of the claimed esters provides the advantage over
the prior art in that it provides a greater amount of methionine
into the bloodstream of the cow than the methionine derivatives of
the prior art. Furthermore, we have surprisingly found that the use
of the particular esters results in very rapid absorption of
methionine into the bloodstream. The ester derivatives according to
the present invention appear not only to avoid rumeri degradation
but surprisingly introduce methionine into bloodstream by
absorption through the rumen wall. This is contrary to the
aforementioned prior art wherein the hydroxy analogue compounds of
methionine are known to either degrade in the rumen or by-pass the
rumen and absorb through the intestine.
[0015] As is evident from the prior art in this area, studies to
introduce methionine into the bloodstream of the ruminant have
concentrated on the use of rumen by-pass compounds as the quickest
and most effective means of introducing methionine into the
bloodstream. We have found that the addition of the esters of the
present invention to the diet of the cow can result, in some cases,
in more than 50% of methionine equivalent being absorbed directly
through the rumen wall. Not only do these esters have a high
bioavailability level but they allow methionine or biologically
equivalent compounds to enter the blood steam very quickly after
intake by the cow through rumen absorption. This result is
surprising and quite unexpected because until now, it has actually
been believed that only compound such as volatile fatty acids,
ammonia and dioxycarbons are absorbed through the rumen wall.
[0016] The present invention also seeks to provide an improvement
in the condition of the cow and the use of the specific esters of
the present invention can result in an improvement in the weight
gain, an improvement in the fertility, an increase in energy as
well as an improvement in the function of the liver.
[0017] The effect on the liver function as a result of the
administration of the ester is an important benefit. This effect
may be characterised by a reduction in metabolic problems through
an improvement in the very low density lipoproteins. Also thought
likely, is a reduction in blood ketosis and a limitation of hepatic
steatosis.
[0018] The administration of the ester can also have a beneficial
effect on reproduction. The interval between calving and
reproduction may be shortened. This effect is also characterised by
an increase in the percentage fertilisation during
insemination.
[0019] It also appears that the use of the specific esters may
result in a stimulation of rumen fermentation, thus resulting in
more digestible organic matter and therefore more energy.
[0020] We have also found that when the esters of the present
invention are given to dairy cows, there is an improvement in the
milk obtained thereof.
[0021] According to another aspect of the present invention, there
is provided a method of improving milk from a dairy cow which
comprises administering to the cow an ester of methionine or
methionine amide and/or an ester of the hydroxy analogue of
methionine or a salt thereof.
[0022] In particular, the present invention provides a method of
improving milk from a dairy cow which comprises administering the
cow a branched alkyl ester of methionine or a branched alkyl ester
of the hydroxy analogue of methionine.
[0023] Where the esters of the present invention are supplied to
dairy cows we have found that by supplementing the normal daily
feed of the dairy cow with an ester of methionine or methionine
amide and/or an ester of the hydroxy analogue of methionine or a
salt thereof, there is a surprising improvement in the quality of
the milk obtained from the dairy cow. In particular, we have found
that the introduction of the specific esters into the diet of the
dairy cow results in an increase in the protein content of the
milk.
[0024] Furthermore, in addition to the protein level, it has been
found that the administration of the specific esters of methionine
or methionine amide and/or esters of the hydroxy analogue of
methionine or a salt thereof can result in improvements in the
volume of milk produced and the fat content of the milk.
[0025] The increase in protein content as a result of the
administration of the ester can be evaluated as being generally
between 0.5 and 4 g of protein per litre of milk. The proteins
which are generally increased are alpha, beta and kappa, especially
the beta and kappa proteins which have a favourable effect on the
cheese making properties of the milk produced.
[0026] The foregoing objects may be obtained in whole or in
part.
[0027] The present invention is directed to a method of supplying
bioavailable methionine to the cow which comprises administering to
the cow an ester of methionine or methionine amide and/or ester of
the hydroxy analogue of methionine or a salt thereof. Suitable
esters are alkyl esters. The alkyl group may be linear, branched or
cyclic having 1 to 12 carbon atoms, preferably 1 to 10, most
preferably 1 to 4 carbon atoms.
[0028] Suitable esters of methionine and the hydroxy analogue of
methionine include the methyl ester, ethyl ester, n-propyl ester,
isopropyl ester, butyl esters, namely n-butyl ester, sec butyl
ester, isobutyl ester and tertiary butyl ester, pentyl esters and
hexyl esters, especially n-pentyl, isopentyl, n-hexyl and isohexyl
esters. Suitable amides of methionine included the alkyl ester of
N-acyl methioninates for example alkyl N-acetyl methioninates.
[0029] Preferably, the ester is a branched or linear alkyl ester,
especially a branched alkyl ester, for example the isopropyl ester
and the tertiary butyl ester. As regards the ester of methionine,
the most preferred is the isopropyl ester and tertiary butyl ester.
As regards the hydroxy analogue of methionine, the most preferred
is the tertiary butyl and the isopropyl ester.
[0030] In particular, it has been found that the use of the
isopropyl ester of the hydroxy. analogue of methionine is
particularly effective, being capable of providing at least 50% of
methionine equivalent to the bloodstream by absorption across the
rumen wall. The isopropyl ester of the hydroxy analogue of
methionine has been found to display a bioavailability of
methionine of more than 50%.
[0031] Furthermore it has been found that with the isopropyl ester
of the hydroxy analogue, the bioavailability peak appears in the
blood relatively quickly following the administration indicating,
that the ester is absorbed directly through the rumen wall thus
indicating that the ester is not rumen by-pass.
[0032] It has also been found that the tertiary butyl ester of
methionine is capable of providing approximately 80% methionine
equivalent to the cow by rumen absorption. This specific ester also
appears to enter the blood stream very quickly, providing
methionine within less than one hour of intake.
[0033] The ester may be supplied to the cow in any suitable way.
Preferably, the ester is supplied as a feed supplement and may be
supplied to the cow through the normal daily feed. Cows are fed a
ration which comprises a concentrate portion and a forage portion.
According to another aspect of the present invention there is
provided a ration comprising a forage portion, a concentrate
portion and a supplement, said supplement comprising an ester of
methionine or methionine amide and/or an ester of the hydroxy
analogue of methionine or a salt thereof.
[0034] Suitable esters in the ration are esters as hereinbefore
described. A preferred ration comprises a forage portion, a
concentrate portion and the isopropyl ester of the hydroxy analogue
of methionine.
[0035] The amount of ester introduced into the feed of the cow may
vary from the breed of cow and from the stage of the milk producing
cycle. Suitably, the supplement comprises an amount of ester
calculated as methionine equivalent of up to 75 g, preferably from
5 to 50 g, especially from 10 to 30 g per animal per day.
[0036] The amount of ester required may be calculated using any
suitable means familiar to the person skilled in the art. Suitably,
the amount may be determined through the use of a computer
model.
[0037] Where the ration contains the tertiary butyl ester or the
isopropyl ester of the hydroxy analogue of methionine, the ester
may be present in a concentration of from 7 to 65 g per animal per
day, most preferably from 10 to 30 g per animal per day of ester.
Where the ration contains the isopropyl ester or the tertiary butyl
ester of methionine, the ration suitably comprises from 7 to 65 g,
most preferably from 10 to 30 g of the ester.
[0038] According to another aspect of the present invention there
is provided a unit dosage form comprising an amount of ester as
herein before described suitable for dosage for one cow for one
day.
[0039] The forage portion may typically comprise corn silage, grass
silage, alfalfa silage and/or hay silage. The concentrate portion
may typically comprise grains such as corn, wheat, barley in
addition to sources of protein such as meal, rape seed, soyabean,
corn gluten and by products such as fish meal, blood meal, brewers
grain and the like.
[0040] The supplement comprising the ester may be mixed with the
forage portion and the grain portion at any suitable time. The
ester is a liquid and may be introduced by mixing in with the
forage portion and the concentrate portion prior to the formation
of the food pellets. Alternatively, the ester may be added to the
pellet ration by the farmer prior to feeding to the cow.
[0041] The ester when incorporated into the feed pellet either
before or after formation of the pellet is stable. In particular,
it has been found that the isbpropyl ester of the hydroxy analogue
is stable in the resulting pellet, retaining over 95% stability
over a long period. Thus, the use of the esters of the present
invention as a food supplement provides a stable source of
methionine.
[0042] The present invention will now be described in detail with
reference to the following examples wherein
EXAMPLE 1
Esters of the Hydroxy Analogue of Methionine
[0043] (a) PREPARATION OF THE ESTERS:
[0044] (1) isopropyl ester of the hydroxy analogue of
methionine
[0045] 314.4 g (1.88 mol) of 2 hydroxy-4 methylthio-butyronitrile
was placed in a stirred jacket reactor fitted with chicanes. 201.3
g (1.951 mol) of 95% sulphuric acid was added slowly whilst
maintaining the temperature below 50.degree. C. After the
introduction of the acid, the reaction temperature was maintained
at 45.degree. C. for 15 minutes. 227.3 g of isopropanol was added
to the reactor contents. The temperature of the reactor was then
increased at a rate of 5.degree. C. per minute until the
temperature at the bottom of the reactor reached 116.degree. C. and
the temperature at the top reached 75.degree. C. These reactor
conditions were maintained for 5 hours. Some of the distillate was
removed during that period and replaced with fresh isopropanol.
[0046] The reaction mixture was then neutralized with 161.2 g of
32% aqueous ammonia (2.72 mol of ammonia). Two phases were
obtained. 780 g of water and 449.7 g of dichloromethane were added.
The two resulting phases were separated to yield 939.1 g of organic
phase and 1247.4 g of aqueous phase.
[0047] The light fractions of the organic product were removed by
distillation. The temperature of the evaporating bath was increased
and the pressure reduced to approximately a few milibars. 263.5 g
of distillate was recovered. The titre of isopropyl ester of
methionine was found to be greater that 99%. The yield was 72%.
[0048] (2) methyl, ethyl, -butyl and cyclohexyl esters of the
hydroy analogue of methionine
[0049] These esters were prepared as detailed above but using the
appropriate alcohol.
[0050] (b) BIOAVAILABILITY
[0051] Spot doses of the following amounts of the esters prepared
as detailed above, equating to 50 g of methionine equivalent, were
given to 2 cows in the manner described in Example 2(b1) above.
[0052] methyl ester of HMB: 64.8 g
[0053] ethyl ester of HMB: 74.8 g
[0054] isopropyl ester of HMB: 80.5 g
[0055] n-butyl ester of HMB: 96 g
[0056] cyclohexyl ester of HMB: 97.5 g
[0057] see butyl ester of HMB: 79 g
[0058] The concentration of methionine and HMB was measured over a
period of 27 hours. The measurements were plotted and the areas
under the curve calculated to provide the bioavailability
results.
[0059] Bioavailability was determined with reference to
Smartamine.TM.. The bioavailability results of the esters are given
in Table 1
1TABLE 1 BIOAVAILABILITY RESULTS ESTERS OF THE HYDROXY ANALOGUE OF
METHIONINE (HMB) Time after administration Ester (hours) 0 1 2 3 5
7 27 Bioavailability Isopropyl [met]* 0.27 1.53 1.96 2.49 2.93 2.93
1.00 59% ester of HMB [HMB]* 0 1.90 1.00 0.99 0.38 0.30 0 Methyl
ester [met]* 0.42 1.26 1.58 1.64 1.66 1.86 0.53 39% of HMB [HMB]* 0
1.35 0.60 0.65 0.30 0.33 0 ethyl ester of [met]* 0.44 1.61 1.87
1.94 1.97 2.13 0.66 35% HMB [HMB]* 0.00 2.39 0.92 0.50 0.27 0.32 0
n-butyl ester [met]* 0.33 0.67 0.70 0.74 0.91 1.07 0.49 17% of HMB
[HMB]* 0.00 0.23 0.12 0.15 0.26 0 0 Sec butyl [met]* 0.31 1.25 1.48
1.53 1.14 1.22 0.42 31% ester of HMB [HMB]* 0 2.15 1.06 0.59 0.42
0.52 0 cyclohexyl [met]* 0.36 0.55 0.87 1.06 1.07 1.09 0.47 20%
ester of HMB [HMB]* 0.00 0.36 0.21 0.23 0.26 0.24 0 *concentration
measured in mg/100 g of blood plasma; met = methionine
EXAMPLE 2
Esters of Methionine
[0060] (a) PREPARATION OF ESTERS
[0061] The esters of methionine were prepared according to the
following general procedure:
[0062] Methionine and 1.2 eq of sulphuric acid relative to the
methionine to be esterified were introduced into the alcohol
corresponding to the nature of the alkyl chain. The resulting
mixture was refluxed whilst removing water to shift the
equilibrium. The mixture was neutralised with ammonia to isolate
the ester. The alcohol was distilled off. The ester obtained was
extracted with dichloromethane and washed with water. The
dichloromethane was evaporated.
[0063] The esters of methionine prepared according to this process
are: methyl methioninate, -propyl methioninate, -butyl
methioninate, n-hexyl methioninate, -octadecyl methioninate, ethyl
N-acetyl methioninate, methionine methyl ester hydrochloride,
methionine ethyl ester hydrochloride, isopropyl methioninate,
tertiary butyl methioninate, cyclohexyl methioninate, sec butyl
methioninate and dodecyl methiohinate.
[0064] (b) BIOAVAILABILITY
[0065] The bioavailability of the esters was evaluated.
[0066] (1) methyl methioninate and n-propyl methioninate
[0067] 56 g of methyl methioninate and 72 g of n-propyl
methioninate (providing an equivalent 50 g of DL-methionine),
prepared as detailed above, were supplied to two cows as a spot
dose at 7.45 am just before the morning feed.
[0068] The ration given to the cows was distributed as two equal
meals at 08.00 hours and 16.00 hours comprised 7 kg of hay and 2 kg
concentrate.
2 WEEK COW 1 COW 2 1 methyl methioninate -propyl methioninate 2 --
-- 3 n-propyl methioninate methyl methioninate 4 -- --
[0069] Samples of blood were taken by jugular vein puncture 09.00,
10.00, 11.00, 13.00 and 15.00 hours on the day where the ester was
given to the animal and at 09.00, 12.00 and 15.00 hours on the day
before and two days after supply.
[0070] The plasma from each sample was isolated from the blood
samples by centrifuging the blood at 3000 revs per minute for 10
minutes. The samples were stored in a freezer. The assay for
methionine was carried out according to the standard procedure of
Moore and Stein.
[0071] The results were plotted using the conventional AUC method
wherein the areas under the curves are calculated to obtain the
bioavailability values for each ester.
[0072] The bioavailability results for the esters are given in
Table 2
[0073] (2) n-hexy methioninate, -butyl methioninate, n-octadecyl
methioninate ethyl N-acetyl methioninate
[0074] Spot doses of the following amounts of the esters prepared
as detailed above, equating to 50 g of methionine, were given to 2
cows in the manner described in (b1) above.
[0075] n-hexy methioninate: 79 g
[0076] n-butyl methioninate: 86 g
[0077] n-octadecyl methioninate: 227 g
[0078] ethyl N-acetyl methioninate: 75 g
[0079] The ration given to the cows was distributed as two equal
meals at 08.00 hours and 16.00 hours and comprised 7 kg of hay and
2 kg concentrate comprising 41% barley, 37% dehydrated beet pulp,
5% molasses, 2% urea and 15% soyabean 48.
[0080] The esters were given to the cow according to the following
schedule:
3 WEEK COW 1 COW 2 3 butyl methioninate octadecyl methioninate 5
octadecyl methioninate n-hexyl methioninate 7 ethyl N-acetyl
methioninate n-butyl methioninate 10 n-hexyl methioninate ethyl
N-acetyl methioninate
[0081] The bioavailability results for the esters are given in
Table 2
[0082] (3) methionine ethyl ester hydrochloride
[0083] The procedure of (b1) was repeated using 72 g of methionine
ethyl ester hydrochloride, prepared as detailed above
[0084] The daily ration given to the cows was as in (b2)
[0085] The ester was given to the cow according to the following
schedule:
4 WEEK COW 1 COW 2 1 methionine ethyl methionine ethyl ester
hydrochloride ester hydrochloride 2 -- --
[0086] Blood samples were taken at the same times as in (b1)
[0087] The bioavailability result for this ester is given in Table
2
[0088] (4) dodecyl methioninate and isopropyl methioninate
[0089] 106.5 g of dodecyl methioninate and 64.1 isopropyl
methioninate (providing an equivalent 50 g of DL-methionine),
prepared as detailed above, were supplied to two cows as detailed
in (b1) above.
[0090] The esters were given to the cow according to the following
schedule:
5 WEEK COW 1 COW 2 3 isopropyl isopropyl methioninate methioninate
5 isopropyl isopropyl methioninate methioninate 7 dodecyl dodecyl
methioninate methioninate
[0091] Blood samples were taken from each cow according to the
regime of (b1).
[0092] The bioavailability results for the esters are given in
Table 2
[0093] (5) cyclohexyl methioninate, methionine methyl ester
hydrochloride and sec butyl methioninate
[0094] Spot doses of the following esters prepared as detailed
above, equating to 50 g equivalent of methionine were given to 2
cows in the manner described in (b1) above:
[0095] cyclohexyl methioninate: 122 g
[0096] methionine methyl ester hydrochloride: 73 g
[0097] sec butyl methioninate: 72 g
[0098] The bioavailability results are given in Table 2
6TABLE 2 BIOAVAILABILITY RESULTS ESTERS OF METHIONINE Time after
administration ESTER (hours) 0 1 2 3 5 7 28 Bioavailability %
n-octadecyl [met]* 0.37 0.37 0.37 0.34 0.37 0.31 0.38 1
methioninate Dodecyl [met]* 0.33 0.38 0.37 0.37 0.44 0.45 0.28 3
methioninate n-butyl [met]* 0.32 1.10 0.74 0.63 0.62 0.61 0.27 8
methioninate n-hexyl [met]* 0.31 1.22 0.92 0.94 1.06 1.07 0.35 17
methioninate n-propyl [met]* 0.32 2.29 1.70 1.55 1.27 1.14 0.32 22
methioninate ethyl N-acetyl [met]* 0.26 1.84 1.97 1.53 1.12 0.86
0.36 20 methioninate Methionine [met]* 0.36 2.78 2.60 2.30 2.00
1.43 0.38 30 ethyl ester hydrochloride Methyl [met]* 0.34 4.08 3.87
3.25 2.57 2.51 0.41 51 methioninate Isopropyl [met]* 0.33 3.62 3.21
2.86 2.22 1.93 0.35 44 methioninate Tertiary butyl [met]* 0.37 4.84
4.87 4.98 4.39 4.19 0.76 80 methioninate Cyclohexyl [met]* 0.29
2.55 2.27 1.88 1.61 1.55 0.38 35 methioninate Methionine [met]*
0.41 1.96 1.43 1.27 1.18 1.22 0.43 25 methyl ester hydrochloride
sec butyl [met]* 0.35 2.75 2.49 2.39 1.75 1.56 0.44 28 methioninate
*concentration measured in mg/100 g of blood plasma; met =
methionine
EXAMPLE 3
Kinetics
[0099] The kinetics of availability of methionine and HMB in the
bloodstream were determined for the isopropyl ester of the hydroxy
analogue of methionine and compared with the hydroxy analogue of
methionine (a compound not according to the present invention).
[0100] The procedure of Example 2 was repeated wherein samples of
the isopropyl ester of the hydroxy analogue (69 g) and the hydroxy
analogue (Alimet.TM.-57 g) were given to four cows. The methionine
and HMB levels in the blood plasma taken from the cows were
analysed and the results are given in Tables 3 and 4 below.
[0101] It can be seen from the results that the isopropyl ester of
the hydroxy analogue of methionine provides methionine and HMB to
the bloodstream much quicker than HMB itself, thus indicating the
ester is absorbed through the rumen wall.
7TABLE 3 BLOOD PLASMA METHIONINE CONCENTRATIONS (mg/100 g of
plasma) Time after 10 20 30 40 50 60 75 90 120 240 COMPOUND
administration 0 mins mins mins mins mins mins mins mins mins mins
HMB COW 1 0.32 0.31 0.34 0.29 0.27 0.27 0.28 0.29 0.30 0.26 0.48
HMB COW 2 0.39 0.32 0.35 0.35 0.34 0.34 0.35 0.29 0.31 0.39 0.67
HMB COW 3 0.40 0.35 0.36 0.36 0.34 0.34 0.34 0.31 0.33 0.42 0.59
HMB COW 4 0.30 0.33 0.34 0.36 0.31 0.26 0.24 0.25 0.28 0.32 0.46
Isopropyl COW 1 0.33 0.72 1.00 1.13 1.30 1.46 1.60 1.69 1.74 1.96
2.12 ester of HMB Isopropyl COW 2 0.33 0.45 0.67 0.71 0.75 0.77
0.86 1.11 1.43 1.75 1.98 ester of HMB Isopropyl COW 3 0.37 0.37
0.50 0.76 0.89 1.05 1.10 1.21 1.44 1.68 2.39 ester of HMB Isopropyl
COW 4 0.37 0.26 0.45 0.70 0.82 0.92 1.19 1.40 1.54 1.79 2.00 ester
of HMB
[0102]
8TABLE 4 BLOOD PLASMA HMB CONCENTRATIONS (mg/100 g of plasma) Time
after 10 20 30 40 50 60 75 90 120 240 COMPOUND administration 0
mins mins mins mins mins mins mins mins mins mins HMB COW 1 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.03 0.06 HMB COW 2 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.10 0.45 HMB COW 3 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.51 HMB COW 4 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.24 Isopropyl COW 1
0.00 4.02 4.19 3.64 3.40 3.11 2.71 2.35 2.23 1.78 0.58 ester of HMB
Isopropyl COW 2 0.00 1.05 1.24 1.31 1.39 1.46 1.53 3.11 2.38 2.54
0.82 ester of HMB Isopropyl COW 3 0.00 0.28 0.93 1.45 1.96 1.99
2.37 2.94 3.49 3.11 1.09 ester of HMB Isopropyl COW 4 0.00 0.17
0.57 1.22 1.36 1.84 3.31 3.73 2.37 2.31 0.92 ester of HMB
EXAMPLE 4
Milk Production
[0103] Example (a) isopropyl ester of the hydroxy analogue of
methionine and the isopropyl ester of methionine
[0104] The isopropyl ester of the hydroxy analogue of methionine
was given to 16 cows over a period of 8 weeks. Each cow was given
daily corn silage and a supplement to cover 100% of requirement and
a 115% PDIE (protein digestible in the intestine) requirement. The
daily supplement consisted of 4.3 kg of a high energy concentrate
which consists of 19.8% barley, 21.1% wheat, 37.5% beet pulp, 2.3%
animal fat, 1.1% salts, 0.6% calcium carbonate and 1.1% sodium
bicarbonate; 2.2 kg of tanned soya cake, 1 kg of normal soya cake,
240 g of urea and 300 g of vitamin and mineral supplements.
[0105] The method according to the present invention was carried
out by splitting the cows into three groups and giving the
following supplement to the normal diet to provide 12.5 g of
bioavailable methionine per animal per day.
[0106] Treatment 1: 1 kg of soya cake
[0107] Treatment 2: 1 kg of soya cake 20 g of polymer coated
methionine (comparative example)
[0108] Treatment 3: 1 kg of soya cake supplemented with 3%
isopropyl ester of HMBI containing 57% equivalent methionine
[0109] Treatment 4: 1 kg of soya cake supplemented with 2.5%
isopropyl ester of methionine containing 76% equivalent
methionine.
[0110] The supplements were given to the cows according to the
following schedule:
9 PERIOD Group* D1 to D15 D15 to D30 D31 to D45 D46 to D60 1
Control isopropyl ester isopropyl Polymer- without of HMB ester of
protected additive methionine methionine 2 isopropyl Control
without Polymer- isopropyl ester ester of additive protected of HMB
methionine methionine 3 isopropyl Polymer- Control without
isopropyl ester ester of protected additive of methionine HMB
methionine 4 Polymer- isopropyl ester isopropyl Control without
protected of methionine ester of additive methionine HMB *4 cows
per group
[0111] The results from the analyses of the milk produced are given
below in Table 5
10TABLE 5 RESULTS ON MILK PRODUCTION Daily amount Butter Content
Protein content of of milk of Milk Milk COMPOUND (kg/cow) g/kg g/kg
Control 31.4 39.1 30.1 isopropyl ester of 32.7 43.3 30.6 methionine
isopropyl ester of 32.3 44.3 30.8 HMB COMPARATIVE: 31.4 40.3 30.9
Polymer-protected methionine
[0112] It can be seen from the results that the addition of the
isopropyl esters of methionine and the isopropyl ester of the
hydroxy analogue of methionine to the diet of the cow results in
milk with higher fat content and higher protein content,
EXAMPLE 5
Liver and Fertility
[0113] The procedure of Example 4 was repeated and observations on
the liver function and fertility of the cows were made. Substantial
improvements were observed in the cows receiving the esters.
EXAMPLE 6
Milk Production
[0114] The procedure of Example 4 was repeated using rations
containing other esters the choice of ester was made in
confirmation with the results reported in examples 1 and 2 which
show that the bioavailability of methionine in the blood of dairy
cows is improved when using the esters of the present invention and
rations containing these esters. Improved milk is also
obtained.
* * * * *