U.S. patent application number 10/182854 was filed with the patent office on 2003-08-21 for method for maintaining or improving the synthesis of mucins.
Invention is credited to Ballevre, Olivier, Breuille, Denis, Finot, Paul-Andre.
Application Number | 20030157186 10/182854 |
Document ID | / |
Family ID | 23982983 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157186 |
Kind Code |
A1 |
Ballevre, Olivier ; et
al. |
August 21, 2003 |
Method for maintaining or improving the synthesis of mucins
Abstract
Methods for maintaining, improving or increasing the synthesis
of mucins by administering a nutritional composition or supplement
that contains a therapeutically effective amount of threonine are
provided. The present invention further provides methods for
treating a variety of disease states characterized by alterations
to the mucin levels, such as, intestinal inflammatory and bacteria
infections or other like disease states.
Inventors: |
Ballevre, Olivier;
(Lausanne, CH) ; Finot, Paul-Andre; (St-Legier,
CH) ; Breuille, Denis; (Saint-Saturnin, FR) |
Correspondence
Address: |
BELL, BOYD & LLOYD LLC
P. O. BOX 1135
CHICAGO
IL
60690-1135
US
|
Family ID: |
23982983 |
Appl. No.: |
10/182854 |
Filed: |
February 21, 2003 |
PCT Filed: |
January 31, 2001 |
PCT NO: |
PCT/EP01/01013 |
Current U.S.
Class: |
424/535 ;
514/12.2; 514/2.4; 514/23; 514/5.6; 514/547 |
Current CPC
Class: |
A23L 33/12 20160801 |
Class at
Publication: |
424/535 ; 514/21;
514/23; 514/547 |
International
Class: |
A61K 038/17; A61K
031/70; A61K 031/225; A61K 035/20 |
Claims
We claim:
1. Use of a protein source including amino acids wherein threonine
comprises at least 5.5% by weight of the amino acids in the
preparation of a nutritional composition for the treatment of a
disease state characterized by alterations to the mucin levels in a
patient.
2. The use of claim 1 wherein threonine comprises at least 6% by
weight of the amino acids.
3. The use of claim 1 wherein the protein source comprises sweet
whey protein.
4. The use of claim 3 wherein the sweet whey protein is
hydrolyzed.
5. The use of claim 1 wherein the nutritional composition further
comprises a lipid source and a carbohydrate source.
6. The use of claim 5 wherein the lipid source comprises a mixture
of medium chain triglycerides and long chain triglycerides.
7. The use of claim 6 wherein the lipid source comprises about 30%
to about 80% by weight of medium chain triglycerides.
8. The use of a protein source including amino acids wherein
threonine comprises at least 5.5% by weight of the amino acids for
the preparation of a nutritional composition for maintaining the
synthesis of mucins in a patient.
9. The use of claim 8 wherein the protein source comprises sweet
whey protein.
10. The use of claim 9 wherein the sweet whey protein is
hydrolyzed.
11. The use of claim 8 wherein the nutritional composition further
comprises a lipid source and a carbohydrate source.
12. The use of claim 11 wherein the lipid source comprises a
mixture of medium chain triglycerides and long chain
triglycerides.
13. The use of claim 12 wherein the lipid source comprises about
30% to about 80% by weight of medium chain triglycerides.
14. A method for maintaining the synthesis of mucins in a patient,
the method comprising enterally administering to the patient a
nutritional composition which includes a protein source containing
a therapeutically effective amount of threonine, a carbohydrate
source and a lipid source including a mixture of medium chain
triglycerides and long chain triglycerides.
15. The method of claim 14 wherein the amount of threonine
comprises at least 5.5% by weight of amino acids of the protein
source.
16. The method of claim 14 wherein the protein source comprises
sweet whey protein.
17. The method of claim 14 wherein the sweet whey protein is
hydrolyzed.
18. The method of claim 14 wherein the lipid source comprises about
30% to about 80% by weight of medium chain triglycerides.
19. The method of claim 14 wherein the protein source provides
about 10% to about 20% of the energy of the nutritional
composition.
20. A nutritional composition that has a protein source including
amino acids wherein threonine comprises at least 7.4% by weight of
the amino acids for treating a disease state characterized by
alterations to the mucin levels in a patient.
21. The composition of claim 20 wherein threonine comprises at
least 14% by weight of the amino acids.
22. The composition of claim 20 wherein the protein source
comprises a sweet whey protein.
23. The composition of claim 20 wherein the protein source
comprises a caseino-glyco-macropeptide.
24. A method for maintaining the synthesis of mucins in a patient,
the method comprising enterally administering to the patient a
nutritional composition which has a protein source including amino
acids wherein threonine comprises at least 7.4% by weight of the
acids.
25. The method of claim 24 wherein threonine comprises at least 14%
by weight of the amino acids.
26. The method of claim 24 wherein the protein source comprises a
sweet whey protein.
27. The method of claim 24 wherein the protein source comprises
caseino-glyco-macropeptide.
28. A method for increasing the synthesis of mucins, the method
comprising supplementing a diet of a patient by adding a
therapeutically effective amount of threonine to the diet.
29. The method of claim 28 wherein the amount of threonine is at
least 0.2 mM.
30. The method of claim 28 wherein the amount of threonine is at
least 0.8 mM.
31. The method of claim 22 wherein the amount of threonine ranges
from about 0.2 mM to about 0.8 mM.
32. A method for increasing the synthesis of mucins in a patient,
the method comprising administering to the patient a nutritional
composition which has a protein source containing threonine at
least 30% of a daily recommended amount of threonine.
33. The method of claim 32 wherein the amount of threonine
comprises at least 60% of the daily recommended amount of
threonine.
34. The method of claim 32 wherein the amount of threonine
comprises at least 100% of the daily recommended amount of
threonine.
35. The use of a therapeutically effective amount of threonine to
prepare a composition for treating intestinal inflammation in a
patient.
36. The use of claim 35 wherein the threonine is provided as a
nutritional supplement.
37. The use of claim 36 wherein the nutritional supplement contains
threonine in an amount of at least 0.2 mM.
38. The use of claim 36 wherein the nutritional supplement contains
a protein source including amino acids and wherein the threonine is
at least 5.5% by weight of amino acids.
39. The use of claim 36 wherein the nutritional supplement contains
a sweet whey protein.
40. The use of a therapeutically effective amount of threonine to
prepare a nutritional composition for treating intestinal bacterial
infection in a patient.
41. The use of claim 40 wherein the threonine is provided as a
nutritional supplement.
42. The use of claim 41 wherein the nutritional supplement contains
threonine in an amount of at least 0.2 mM.
43. The use of claim 41 wherein the nutritional supplement contains
a protein source including amino acids and wherein the threonine is
at least 5.5% by weight of amino acids.
44. The use of claim 41 wherein the nutritional supplement contains
a sweet whey protein.
45. The use of a therapeutically effective amount of threonine to
prepare a composition for reducing oxidative stress due to acute
intestinal inflammation.
46. The use of claim 45 wherein the threonine is part of a
nutritional composition.
47. The use of claim 46 wherein the nutritional composition
contains threonine in an amount of at least 0.2 mM.
48. The use of claim 46 wherein the nutritional composition
contains a protein source including amino acids and wherein the
threonine is at least 5.5% by weight of amino acids.
49. The use of claim 48 wherein the nutritional composition
contains a sweet whey protein.
Description
[0001] The present application is a continuation-in-part
application of U.S. patent application Ser. No. 09/498,905 filed on
Feb. 4, 2000.
BACKGROUND OF THE INVENTION
[0002] This invention generally relates to methods for maintaining,
improving or increasing the synthesis of mucins, especially in the
gastrointestinal tract and lungs. The invention further relates to
maintaining, improving or increasing the synthesis of mucins in a
patient by administering a nutritional composition to the patient
wherein the nutritional composition contains a therapeutically
effective amount of threonine. By increasing the synthesis of
mucins, a variety of disease states characterized by alterations to
the mucin levels, such as, intestinal inflammatory and bacterial
infections or other like disease states, can be effectively
treated.
[0003] Mucins are glycoproteins which are a primary component of
the viscoelastic gel, or mucus, which covers most of the mucosal
surfaces of the gastrointestinal tract and lungs. They are
continuously secreted from the surfaces of the lung and
gastrointestinal tract; for example from the goblet cells. They are
in the form of large marcomolecules composed of a peptide core and
oligosaccharide side chains. The oligosaccharide side chains are
linked via O-glycosidic bonds to serine and threonine residues in
the peptide core. The oligosaccharide side chains constitute
approximately 90% of the mass of the mucins. Threonine constitutes
about 22% by weight of the peptide core.
[0004] The mucus acts to protect the epithelial cells of the
gastrointestinal tract and lungs from toxins such as acids, bile,
digestive enzymes and from enteric bacteria and their toxins
(Neutra, M. R. and Forstner, J. F.; 1987; Physiology of the
Gastrointestinal Tract, second edition, Raven Press, NY, pages 975
to 1009). Hence the mucus functions as a major, local defense
barrier which acts to prevent the invasion and systemic spread of
bacteria and endotoxins normally present in the gastrointestinal
tract or lungs. Hence the healthy status of the mucus is important
to health.
[0005] However, many disease states are characterized by
alterations in the mucus composition. For example, histochemical
studies have demonstrated well characterized abnormalities in
mucins during malignancy, cystic fibrosis, chronic inflammatory
bowel diseases, ulcerative colitis and Crohn's disease and during
infection with intestinal nematode parasites (Tse, S-L, and Chadee,
K; 1992; Infection and Immunity, Vol. 60, No 4, pages 1603-1612).
Also, non-steroidal anti-inflammatory drugs are known to increase
the risk of damage to mucosa by acid and pepsin. Also, patients
undergoing inflammatory response may have impaired mucin
production. The results of these changes or impairments are a
variety of adverse effects; including bacterial translocation,
gastritis, gastric erosions, peptic ulceration, and invasions of
pathogenic bacteria.
[0006] There is therefore a need for a method of maintaining or
improving the synthesis of mucins.
SUMMARY OF THE INVENTION
[0007] This invention provides methods for maintaining, improving
or increasing the synthesis of mucins in a patient by administering
a nutritional composition or supplement that contains a
therapeutically effective amount of threonine. It has been
surprisingly found that administering to a patient a
therapeutically effective amount of threonine has a beneficial
effect on the treatment of a variety of disease states
characterized by alterations to the mucin levels, the condition of
the mucus in general and other like beneficial effects.
[0008] In an embodiment, the present invention includes a method of
treating a disease state characterized by alterations to the mucin
levels in a patient wherein the method comprises enterally
administering to the patient a nutritional composition which has a
protein source including amino acids wherein threonine comprises at
least 5.5% by weight of the amino acids.
[0009] In an embodiment, the present invention includes a method
for maintaining the synthesis of mucins in a patient wherein the
method comprises enterally administering to the patient a
nutritional composition which has a protein source including amino
acids wherein threonine comprises at least 5.5% by weight of the
amino acids.
[0010] In an embodiment, the present invention includes a method
for maintaining the synthesis of mucins in a patient wherein the
method comprises enterally administering to the patient a
nutritional composition which includes a protein source containing
a therapeutically effective amount of threonine, a carbohydrate
source and a lipid source including a mixture of medium chain
triglycerides and long chain triglycerides.
[0011] In an embodiment, the present invention includes a method of
treating a disease state characterized by alterations to the mucin
levels in a patient. The method comprises enterally administering
to the patient a nutritional composition which has a protein source
including amino acids wherein threonine comprises at least 7.4% by
weight of the amino acids.
[0012] In an embodiment, the present invention includes a method
for maintaining the synthesis of mucins in a patient. The method
comprises enterally administering to the patient a nutritional
composition which has a protein source including amino acids
wherein threonine comprises at least 7.4% by weight of the amino
acids.
[0013] In an embodiment, the present invention includes a method
for increasing the synthesis of mucins wherein the method comprises
supplementing a diet of a patient by adding a therapeutically
effective amount of threonine to the diet.
[0014] In an embodiment, the present invention includes a method
for increasing the synthesis of mucins in a patient wherein the
method comprises administering to the patient a nutritional
composition that contains threonine in an amount of at least 30% a
daily recommended amount of threonine.
[0015] In an embodiment, the present invention provides a method
for treating intestinal inflammation in a patient. The method
includes administering a therapeutically effective amount of
threonine.
[0016] In an embodiment, the present invention provides a method
for treating intestinal bacterial infection in a patient. The
method includes administering a therapeutically effective amount of
threonine.
[0017] In an embodiment, the present invention provides a method
for reducing oxidative stress due to acute intestinal inflammation.
The method includes administering a therapeutically effective
amount of threonine.
[0018] An advantage of the present invention is hat it provides
improved methods for maintaining, improving or increasing the
synthesis of mucins in a patient.
[0019] A further advantage of the present invention is that it
provides methods for maintaining, improving or increasing the
synthesis of mucins in a patient by administering a nutritional
composition to the patient wherein the nutritional composition
contains a therapeutically effective amount of threonine.
[0020] Yet another advantage of the present invention is that it
provides methods for maintaining, improving or increasing the
synthesis of mucins in a patient by increasing the food efficiency
in a diet administered to the patient wherein the diet contain a
therapeutically effective amount of threonine.
[0021] A still further advantage of the present invention is that
it provides methods for treating a variety of disease states.
[0022] Another advantage of the present invention is that it
provides methods for treating intestinal inflammation.
[0023] Still another advantage of the present invention is that it
provides methods for treating intestinal bacterial infection.
[0024] An additional advantage of the present invention is that it
provides methods for reducing oxidative stress due to acute
intestinal inflammation by increasing the synthesis of mucins in a
patient.
[0025] Additional features and advantages of the present invention
are described in, and will be apparent from, the detailed
description of the presently preferred embodiments.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 illustrates graphically the effect of threonine
concentration on the fractional synthesis rate of mucoproteins.
[0027] FIG. 2 illustrates graphically the effect of a threonine
requirement on the food efficiency of a diet.
[0028] FIG. 3 illustrates graphically the effect of a threonine
requirement on the fractional synthesis rates of mucoproteins.
DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
[0029] The present invention provides methods for maintaining,
improving or increasing the synthesis of mucins by administering to
a patient a nutritional composition that contains a therapeutically
effective amount of threonine. The invention provides methods of
treating a variety of disease states characterized by alterations
to the mucin levels, such as, intestinal inflammatory and bacterial
infections or other like disease states.
[0030] The invention is based on the finding that enterally
administering nutritional composition which contains a protein
source enriched in threonine has a beneficial effect on the
synthesis of mucins. The protein source may be any suitable source
of is amino acids that is enriched in threonine. For example, the
protein source may be milk protein, egg white,
aseino-glyco-macropeptide, whey protein, casein protein, soy
protein, rice protein, pea protein or oat protein, or mixtures of
these proteins. Also the protein source may be in the form of
intact protein, hydrolyzed protein, or mixtures thereof. Further,
if desired, the protein source may be in the form of free amino
acids. In an embodiment, the protein may be in the form of mixtures
of intact protein or hydrolyzed protein, with free amino acids.
[0031] In an embodiment, threonine provides at least 5.5% by weight
of the amino acids of the protein source. The amount of threonine
can also include at least 6% by weight of the amino acids and
preferably at least 7.4% by weight of the amino acids. This may be
obtained by using a protein source which is naturally enriched in
threonine or by supplementing other protein sources with threonine.
A protein source which is naturally enriched in threonine is
particularly preferred; for example sweet whey protein or
caseino-glyco-macropeptide. Sweet whey protein has a threonine
content of about 7.4% by weight of amino acids and
caseino-glyco-macropeptide has a threonine content of about 14% by
weight of amino acids.
[0032] For patients suffering from malabsorption or intolerance to
intact proteins, the protein source is preferably based upon
hydrolyzed protein. Especially preferred in this regard is
hydrolyzed sweet whey protein. Using hydrolyzed sweet whey provides
the advantage of a naturally enriched threonine content while
providing nutrition to malabsorbing patients or patients with
intolerance to intact proteins.
[0033] The protein source preferably provides about 10% to about
20% of the energy of the nutritional composition. For example, the
protein source may provide about 15% to about 18% of the energy of
the nutritional composition.
[0034] The nutritional composition may also include a carbohydrate
source. The carbohydrate source preferably provides about 35% to
about 65% of the energy of the nutritional composition, preferably
40% to 60% of the energy of the nutritional composition. For
example, the carbohydrate source may provide about 51% of the
energy of the composition. Several carbohydrates may be used
including maltodexrin, corn starch, modified starch, or sucrose, or
mixtures thereof. Preferably the composition is free from
lactose.
[0035] The nutritional composition may further include a lipid
source. Preferably the lipid source provides about 20% to about 50%
of the energy of the nutritional composition; especially 25% to
about 40% of the energy of the nutritional composition. For
example, the lipid source may provide about 33% of the energy of
the nutritional composition
[0036] The lipid source may comprise a mixture of medium chain
triglycerides (MCT) and long chain triglycerides (LCT). The lipid
source preferably includes at least 30% to about 80% by weight of
medium chain triglycerides. For example, medium chain triglycerides
may make up about 70% by weight of the lipid source. Suitable
sources of long chain triglycerides are sunflower oil, rapeseed
oil, soy oil, milk fat, corn oil and soy lecithin. Fractionated
coconut oils are a suitable source of medium chain
triglycerides.
[0037] The lipid profile of the enteral composition is preferably.
designed to have a polyunsaturated fatty acid omega-6 (n-6) to
omega-3 (n-3) ratio of about 1:1 to about 10:1. For example, the
n-6 to n-3 fatty acid ratio may be about 6:1 to about 9:1.
[0038] The nutritional composition preferably includes a complete
vitamin and mineral profile. For example, sufficient vitamins and
minerals may be provided to supply about 75% to about 250% of the
recommended daily allowance of the vitamins and minerals per 1000
calories of the nutritional composition.
[0039] The nutritional composition preferably has an energy content
of about 800 kcal/l to about 1200 kcal/l; for example an energy
content of about 1000 kcal/l.
[0040] The nutritional composition may be in any suitable form. For
example, the nutritional composition may be in the form of a
soluble powder, a liquid concentrate, or a ready-to-drink
formulation. Alternatively, the nutritional composition may be in
solid form; for example in the form of a ready-to-eat bar or
breakfast cereal. Ready to drink formulations are particularly
preferred. The composition may be fed to a patient via a
nasogastric tube, jejunum tube, or by having the patient drink or
eat it. Various flavors, fibers, sweeteners, and other additives
may also be present.
[0041] The nutritional composition may be used as a nutritional
support for patients suffering from, or at risk of, impaired or
reduced mucin production. For example, the nutritional composition
may be used as a nutritional support for patients undergoing an
inflammatory response, suffering from malnutrition, suffering from
cystic fibrosis, malignancy, chronic inflammatory bowel diseases,
ulcerative colitis and Crohn's disease, undergoing treatment which
includes the administration of non-steroidal anti-inflammatory
drugs, and other like disease states. The nutritional composition
may also be used to restore mucin production after total parenteral
nutrition.
[0042] The amount of the nutritional composition required to be fed
to a patient will vary depending upon factors such as the patient's
condition, the patient's body weight, the age of the patient, and
whether the nutritional composition is the sole source of
nutrition. However the required amount may be readily set by a
medical practitioner. In general, a sufficient amount of the
nutritional composition is administered to provide the patient with
about 1 g protein to about 4.0 g protein per kg of body weight per
day and about 0.05 g to about 0.4 g of threonine per day. For
example, an adult patient may be administered about 1.5 g protein
to about 2.0 g protein per kg of body weight per day and about 0.07
g to about 0.2 g of threonine per day. If the nutritional
composition is used as a supplement to other foods, the amount of
the nutritional composition that is administered daily may be
decreased accordingly.
[0043] The nutritional composition may be taken in multiple doses,
for example 2 to 5 times, to make up the required daily amount or
may be taken in a single dose.
[0044] In an embodiment, the present invention includes a method of
treating a disease state characterized by alterations to the mucin
levels in a patient. The method includes enterally administering to
the patient a nutritional composition which has a protein source
including amino acids wherein threonine includes at least 5.5% by
weight the amino acids. The amount of threonine can also include at
least 6.0% and preferably at least 7.4% by weight of the amino
acids.
[0045] In an embodiment, the present invention includes increasing
the synthesis of mucins in a patient by supplementing a diet of a
patient with a therapeutically effective amount of threonine. The
amount of threonine added to the diet can include at least 0.2 mM.
Preferably, the amount of threonine also ranges from about 0.2 mM
to about 0.8 mM.
[0046] The amount of threonine to be added can be based on the
recommended daily dosages or requirements for threonine. In an
embodiment, the amount of threonine added to the diet includes at
least 30% of the recommended daily amount, preferably at least 60%,
and most preferably at least 100% of this amount.
[0047] In an embodiment, the present invention provides a method
for increasing the synthesis of mucins by increasing the food
efficiency in a diet of a patient. The food efficiency represents
the ratio between the body weight gain and the quantity of ingested
food during a diet period as detailed below in Example 4. The food
efficiency and thus the synthesis of mucins can be increased by
administering to the patient a nutritional composition that
contains a therapeutically effective amount of threonine. For
example, the amount of threonine can be based on the daily
recommended amount of threonine as previously discussed.
[0048] An embodiment of the invention provides methods of treating
disease states characterized by alterations to mucin levels in a
patient. The disease states can include a variety of disease
states, such as intestinal inflammation and bacterial infection or
other like disease states, as previously discussed. In an
embodiment, the invention provides methods for treating intestinal
inflammation, for treating intestinal bacterial infections, for
reducing oxidative stress due to acute intestinal inflammation and
for treating other like disease states. The methods for treating
disease states provide administering to a patient a therapeutically
effective amount of threonine as previously discussed. The
threonine can be administered as part of a nutritional composition,
supplement or other like nutritional substance.
[0049] By way of example and not limitation, examples of the
present invention are as follows:
EXAMPLE 1
[0050] Twenty four male Wistar rats, each weighing about 200 g, are
used. The rats have free access to water. The rats are divided into
3 groups of 8 rats with each rat in a separate cage.
[0051] Each group of rats has free access, for 3 days, to a control
diet based upon soy protein supplemented with Met. Thereafter, each
group is fed, once a day, an amount of the control diet sufficient
to meet 80% of the physiological requirements for growing of each
rat. Thereafter, one group (the control group) is fed in the same
fashion for the remainder of the trial. The remaining groups are
then starved for a period of three days. The rats have free access
to water during this time.
[0052] The remaining two groups of rats are then each fed a
separate diet, once a day, in an amount of the control diet
sufficient to meet 80% of the physiological requirements for the
growing of each rat. One group (group A) is fed an enteral formula
based upon acid whey supplemented with 5% cellulose. The second
group (group 1) is fed a freeze dried enteral formula based upon
sweet whey (available from Nestle Clinical Nutrition under the
trademark PEPTAMEN.RTM.) supplemented with 5% cellulose.
[0053] The protein content of the diets are as follows: the control
diet contains 19.02 g of protein per gram of diet, enteral formula
based upon acid whey contains 15.67 g of protein per gram of diet,
and the enteral formula based upon sweet whey contains 15.63 g of
protein per gram of diet. Threonine provides about 7.4% by weight
of protein in the enteral formula based upon sweet whey. In the
enteral formula based upon acid whey, threonine provides less than
5% by weight of protein.
[0054] Results 1
[0055] After three days, gastro-intestinal mucosa is collected from
each rat. The threonine content of the mucosa is then determined as
follows:
1 Protein Intake Thr Intake Thr content in Mucosa Protein Group
(g/3 days) (mg/3 days) (mg/g mucosa protein) Control 8.2 270 4.69 A
6.2 258 3.81 1 6.3 410 4.30
[0056] The results of Example 1 indicate that the starved rats of
group have a higher content of threonine in the mucosa protein than
those of group A and a threonine content slightly lower than the
normal content of those of the control group. This indicates a rise
in the production of mucins in the rats of group 1.
EXAMPLE 2
[0057] An isotonic liquid diet is used. The diet is available from
Nestle Clinical Nutrition under the trademark PEPTAMEN.RTM.. The
diet has the following components:
2 Nutrient Amount per 1000 ml Protein (hydrolyzed sweet whey) 40 g
Carbohydrate (maltodextrin, corn starch) 127 g Lipid (medium chain
triglycerides, 39 g sunflower oil, soy lecithin) Vitamin A 4000 IU
Vitamin D 280 IU Vitamin E 28 IU Vitamin K 80 micrograms Vitamin C
140 mg Thiamin 2 mg Riboflavin 2.4 mg Niacin 28 mg Vitamin B6 4 mg
Folic acid 540 micrograms Pantothenic acid 14 mg Vitamin B 12 8
micrograms Biotin 400 micrograms Choline 450 mg Taurine 80 mg
L-carnitine 80 mg Minerals Calcium, Phosphorus, Magnesium, Zinc,
Iron, Copper, Manganese, Iodine, Sodium, Potassium Chloride,
Chromium, Molybdenum, Selenium
[0058] The diet has an energy density of 1000 kcal/l and the
protein provides 16% of energy, the carbohydrate provides 51% of
energy, and the lipid provides 33% of energy. Threonine provides
about 7.4% by weight of the protein source. Ten patients, of both
sexes and between the ages of 20 to 60 years, are recruited for the
study. All patients have been diagnosed as suffering from Crohn's
disease, and the diagnosis has been confirmed by histology,
radiology, or both. All patients are given barium meal, and
radiography is used to assess the disease site in the small bowel.
The patients are also are subjected to endoscopic assessment. A
score of 0 to 3 (0=normal, 1=mild inflammation, 2=moderate
inflammation, and 3=severe inflammation) is assigned to each area
of the bowel which is inspected. The bowel areas include) for
example, the terminal ileum, caecum, descending colon, transverse
colon, ascending colon, sigmoid colon and rectum. Also, an
assessment is made upon the condition of the mucus in area of the
bowel which is inspected. A score of 0 to 3 (0=normal, 3=severe
reduction) is assigned to each area.
[0059] Each patient in the study is fed the PEPTAMEN.RTM. product
as the sole source of nutrition for a period of 8 weeks.
Administration of the nutritional composition is under the
supervision of a dietitian and is effected orally or by nasogastric
tube, as desired by the patient. The intake of the nutritional
composition is adjusted on the basis of tolerance, palatability and
weight gain. After the 8 weeks, each patient again undergoes
endoscopic assessment and inflammation and mucus values are
assigned
[0060] Results 2
[0061] In general, the endoscopic assessment and mucus conditions
in all of patients have improved after administering the
nutritional composition to the patients as discussed in Example 2.
Therefore, the treatment effectively resulted in the remission of
Crohn's disease in most cases.
EXAMPLE 3 (IN VITRO)
[0062] The effect of threonine supplementation on the mucin
synthesis rate was analyzed in vitro in HT29-MTX cells (human
colonic carcinoma cells). HT29-MIX cells were grown in normal
Dulbecco's modified minimum essential medium (DMEM) supplemented
with 10% (v/v) heat-inactivated fetal bovine serum (Life
Technologies) and containing 25 mM glucose, 1% non-essential amino
acids (Life Technologies), 2 mM glutamine (AJINOMOTO CO), 85 mg/L
penicillin-streptomycin, 50 mg/L gentamicin, and 1.5 mg/L
Amphotericin B (Life Technologies). Cells were cultured at
37.degree. C. in an atmosphere containing 10% CO.sub.2:90% air. The
culture medium was changed daily. Experiments were carried out in
T75 cm.sup.2 culture flasks between passages 10 to 20. Studies were
performed in the late post-confluent period (21 days after
seeding), when all cells display a mucin-secreting phenotype.
[0063] For the experiment, cells were grown in the normal DMEM
until confluency (7 days after seeding). Then from day 7 to day 21
after seeding, several concentrations of threonine in the DMEM were
tested, namely, 0.2 mM, 0.4 mM, 0.8 mM and 1.6 mM. At day 20,
L-(3-.sup.3H) threonine (30 microCi/Flask) was added to a fresh 10
mL culture medium. The incorporation of the radioactive tracer to
newly synthesized mucoproteins was determined for the following 24
hour period.
[0064] After 24 hours of metabolic labeling, the culture medium of
cells was removed. Cells were washed twice with 10 ml of
1.times.PBS and recovered using a rubber scraper. Cells were
homogenized in 0.05M Tris/HCl buffer pH 7.50 using a polytron at a
low setting (6.000 rpm, 30 sec, 4.degree. C.). An aliquot of each
homogenate was removed for the measurement of the specific
radioactivity of the intracellular free threonine considered as the
precursor pool. Mucoproteins were purified from the cell
homogenates by a size exclusion chromatography after a partial
enzymatic digestion of non-highly glycosylated and thus protected
proteins. Purified mucins were hydrolyzed with 6M HCl (24 h at
100.degree. C.), and their amino acid composition was determined by
HPLC. The outlet of the UV detector was connected to a radioactive
detector (Radiomatic 500TR, Packard) to measure the
.sup.3H-threonine incorporated in mucoproteins. The fractional
synthesis rate (FSR) of mucoproteins was calculated and expressed
in percent/day (%/d): (FSR=(Specific radioactivity of mucoprotein
bound threonine/Specific radioactivity of intracellular free
threonine)*100).
[0065] Results 3
[0066] The results of Example 3 are. shown in FIG. 1 which
demonstrates the effect of the threonine concentration on the
fractional synthesis rate of mucoproteins ("mucoprotein FSR"). The
mucoprotein FSR was determined for each of the varying
concentrations of threonine (mM) in the culture medium of HT29-MTX
cells as further shown in FIG. 1. The data values are mean
values.+-.SEM (n=6) wherein the data values that do not share a
common letter are significantly different (p<0.05).
[0067] The mucoprotein FSR increased significantly from 0.2 mM
(26.4.+-.1.4%) to 0.8 mM (37.3.+-.1.4%) of threonine and reached a
plateau at about 0.8 mM of threonine. The mucoprotein FSR generally
increased with the level of threonine in the culture medium under
conditions which are not limiting in threonine for the cell culture
as the cell number and viability are not disturbed.
[0068] These results clearly indicate that the threonine
bioavailability in the culture medium can significantly influence
the mucoprotein synthesis rate in vitro. Thus, whatever the disease
state considered (Inflammatory Bowel Disease, infection, Crohn's
disease, or other like disease states), the threonine requirement
is increased for the synthesis of threonine rich inflammatory
proteins at the expense of the mucoprotein synthesis. Thus,
threonine supplementation can be an efficient and effective
nutritional intervention to stimulate or to restore the mucoprotein
synthesis rate, and thus to facilitate improved epithelial cell
protection.
EXAMPLE 4 (IN VIVO)
[0069] The effect of the threonine content in the diet on the mucin
synthesis rate was analyzed in vivo in growing rats. Thirty two
male Sprague-Dawley rats (90 g) were used. They were randomly
divided into 4 groups of 8 rats and separated in individual cages.
Rats had free access to water during all the experiments.
[0070] After an adaptation period of 6 days on a chow diet, rats
were adapted (free access) to their experimental diets for 3 days.
Experimental diets were based on free amino acids (semi-synthetic
diet, 12.5% proteins) and varying amounts of threonine: A) a
threonine requirement for growing rats according to the
recommendations of the National Research Council (revised edition
of 1995); B) 60% of the threonine requirement; C) 30% of the
threonine requirement; and D) 150% of the threonine requirement.
Rats of each group were fed with the experimental diets for 14
days. Groups A, B and D were pair-fed with group C.
[0071] Before the sacrifice of rats, a flooding dose of
1-.sup.13C-Valine was performed in the lateral tail vein of
animals. Each animal received a bolus of 150 micromole
L-.sup.13C-Valine (99% .sup.13C)/100 g body weight Animals were
sacrificed after anesthesia by exsanguination from the abdominal
aorta between 35 to 53 minutes after the 1-.sup.13C-Valine
injection. Intestinal mucosal samples were obtained by scrapping
with a glass slide and thereafter immediately freezing them in
liquid nitrogen.
[0072] Rat mucosal samples were gently homogenized in 0.05M
Tris/HCl buffer pH 7.50 using a polytron at a low setting (6.000
rpm, 30 sec, 4.degree. C.) An aliquot of each sample homogenate was
used to measure the 1-.sup.13C-Valine enrichment in the
intracellular pool that was considered as .sup.13C-enrichment of
the precursor pool. Thereafter, mucoproteins were purified as
described previously for the in vitro experiment. 1-.sup.13C-Valine
enrichments in mucoproteins were measured by mass spectrometry.
[0073] Results 4
[0074] As shown in FIG. 2, the effect of the threonine requirement
and percentages thereof on the food efficiency of a diet was
evaluated. As discussed in Example 4, the diets contained 30%, 60%,
100%, and 150% of the threonine requirements of rats. The food
efficiency was calculated as the ratio between the body weight gain
(grams) and the quantity of ingested food (grams) during the 14
days, and expressed in percentage. The data values are mean
values.+-.SEM (n=8). The data values that do not share a common
letter are significantly different (p<0.05).
[0075] As further illustrated in FIG. 2, the food efficiency of the
four diets varied with respect to the percentage of threonine
requirement in the diet. It was significantly lower for the diet
containing 30% of the threonine requirements of rats (11.8.+-.1.2%)
than for diets containing 60% (17.5.+-.0.5%), 100% (18.8.+-.0.8%)
and 150% (20.6.+-.1.3%) of the threonine requirements.
[0076] As shown in Table 1 (below) and FIG. 3, the effect of the
percentage of the threonine requirement on the fractional synthesis
rates of mucoproteins was evaluated.
3 TABLE 1 % Threonine Diet requirements FSR (%/d) Average .+-. SEM
C 30% 123.5 101.8 .+-. 9.4 109.8 80.4 93.6 B 60% 178.3 139.8 .+-.
19.3 123.9 117.4 A 100% 157.3 143.5 .+-. 6.3 146.2 135.3 D 150%
117.6 138.5 .+-. 12.0 159.1 138.9
[0077] Table 1 compares the fractional synthesis rate of
mucoproteins in jejunum of growing rats with respect to Diet A
(100% of threonine requirements), Diet B (60% of threonine
requirements), Diet C (30% of threonine requirements), and Diet D
(150% of threonine requirements). FIG. 3 is a graphical
illustration of Table 1. The data values are mean values.+-.SEM as
further illustrated in FIG. 3.
[0078] As shown in Table 1 and FIG. 3, the synthesis rate of
mucoproteins was the lowest in the jejunum of rats fed with the
diet that is the most deficient in threonine (30% of threonine
requirements). As shown, the mucoprotein fractional synthesis rate
in jejunum was 143%/day with the control diet (100% of threonine
requirements), and it decreased to 101%/day with the diet
containing 30% of threonine requirements. However, the mucoprotein
synthesis rate did not increase with the diet containing 150% of
threonine requirements than what was observed in the control
diet.
[0079] These results indicate that if threonine requirements for
growth are not covered by the diet, the mucoprotein synthesis rate
is decreased Thus, the threonine content of the diet has an effect
on both growth and mucoprotein synthesis rate in vivo in rats. This
demonstrates that not only growth but also mucoproteins synthesis
is effected when the diet is deficient in threonine. Accordingly,
if threonine requirements are not adequately provided by the diet,
threonine supplementation can be an effective and efficient
nutritional strategy to increase or to restore the mucoprotein
synthesis rate, and thus to assure a better epithelial cell
protection.
[0080] It should be understood that various changes and
modifications to the presently preferred embodiments described
herein will be apparent to those skilled in the art. Such changes
and modifications can be made without departing from the spirit and
scope of the present invention and without diminishing its
attendant advantages. It is therefore intended that all such
changes and modifications be covered by the appended claims.
* * * * *