Nutritional intervention for improving muscular function and strength

Abstract

The present invention provides a composition comprising HMB and Vitamin D. Methods of administering HMB and Vitamin D to an animal are also described. Vitamin D and HMB are administered to increase muscle mass, strength, and functionality. The combination of Vitamin D and HMB together has a synergistic effect, which results in a surprising and unexpected level of improvement in muscle mass, strength and functionality.

Claims

1. A composition comprising a synergistic combination of from about 0.01 grams to 0.2 grams of β-hydroxy-β-methylbutyric acid (HMB) per kilogram body weight and Vitamin D in an amount sufficient to raise blood levels of Vitamin D to at least about 25 ng/ml, wherein the composition is administered to a non-human animal.

2. The composition of claim 1, wherein said HMB is selected from the group consisting of its free acid form, its salt, its ester and its lactone.

3. The composition of claim 2, wherein said HMB is in free acid form.

4. The composition of claim 2, wherein said salt is a calcium salt.

5. The composition of claim 1, wherein said composition is administration form is selected from the group consisting of a foodstuff, a treat, and a chew.

6. The composition of claim 5, wherein the foodstuff is commercial animal food.

7. The composition of claim 1, wherein the non-human animal is selected from the group consisting of a canine, feline and a horse.

8. A composition comprising a synergistic combination of from about 0.01 grams to 0.2 grams of β-hydroxy-β-methylbutyric acid (HMB) per kilogram body weight and Vitamin D in at least the recommended intake, wherein the composition is administered to a non-human animal.

9. The composition of claim 8, wherein said HMB is selected from the group consisting of its free acid form, its salt, its ester and its lactone.

10. The composition of claim 9, wherein said HMB is in free acid form.

11. The composition of claim 9, wherein said salt is a calcium salt.

12. The composition of claim 8, wherein said composition is administration form is selected from the group consisting of a foodstuff, a treat, and a chew.

13. The composition of claim 12, wherein the foodstuff is commercial animal food.

14. The composition of claim 8, wherein the non-human animal is selected from the group consisting of a canine, a feline and a horse.

15. The composition of claim 14, wherein HMB is in an amount from about 0.01 grams to 0.2 grams of β-hydroxy-β-methylbutyric acid (HMB) per kilogram body weight and Vitamin D is in an amount sufficient to raise blood levels of Vitamin D to at least about 25 ng/ml.

16. A method for increasing the muscle mass of a non-human animal in need thereof comprising the steps of administering to said animal a synergistic combination of from about 0.01 grams to 0.2 grams of β-hydroxy-β-methylbutyric acid (HMB) per kilogram body weight and Vitamin D in an amount sufficient to raise blood levels of Vitamin D to at least about 25 ng/ml, wherein upon said administration of said synergistic combination of HMB and Vitamin D to the animal, said muscle mass is increased.

17. The method of claim 16, wherein said HMB is selected from the group consisting of its free acid form, its salt, its ester and its lactone.

18. The method of claim 17, wherein said HMB is in free acid form.

19. The method of claim 17, wherein said salt is a calcium salt.

20. The method of claim 16, wherein said composition is administration form is selected from the group consisting of a foodstuff, a treat, and a chew.

21. The method of claim 20, wherein the foodstuff is commercial animal food.

22. The method of claim 16, wherein the non-human animal is selected from the group consisting of a canine, a feline and a horse.

23. A method for improving muscle function of a non-human animal in need thereof comprising the steps of administering to said animal a synergistic combination of from about 0.01 grams to 0.2 grams of β-hydroxy-β-methylbutyric acid (HMB) per kilogram body weight and Vitamin D in an amount sufficient to raise blood levels of Vitamin D to at least about 25 ng/ml, wherein upon said administration of said synergistic combination of HMB and Vitamin D to the animal, said muscle function is improved.

24. The method of claim 23, wherein said HMB is selected from the group consisting of its free acid form, its salt, its ester and its lactone.

25. The method of claim 24, wherein said HMB is in free acid form.

26. The method of claim 24, wherein said salt is a calcium salt.

27. The method of claim 23, wherein said composition is administration form is selected from the group consisting of a foodstuff, a treat, and a chew.

28. The method of claim 27, wherein the foodstuff is commercial animal food.

29. The method of claim 23, wherein the non-human animal is selected from the group consisting of a canine, a feline and a horse.

30. A method for increasing strength of a non-human animal in need thereof comprising the steps of administering to said animal a synergistic combination of from about 0.01 grams to 0.2 grams of β-hydroxy-β-methylbutyric acid (HMB) per kilogram body weight and Vitamin D in an amount sufficient to raise blood levels of Vitamin D to at least about 25 ng/ml, wherein upon said administration of said synergistic combination of HMB and Vitamin D to the animal, said strength is increased.

31. The method of claim 30, wherein said HMB is selected from the group consisting of its free acid form, its salt, its ester and its lactone.

32. The method of claim 31, wherein said HMB is in free acid form.

33. The method of claim 31, wherein said salt is a calcium salt.

34. The method of claim 30, wherein said composition is administration form is selected from the group consisting of a foodstuff, a treat, and a chew.

35. The method of claim 34, wherein the foodstuff is commercial animal food.

36. The method of claim 30, wherein the non-human animal is selected from the group consisting of a canine, a feline and a horse.

37. A method for supplementing the nutrition of a non-human animal, comprising feeding the animal a composition comprising a synergistic combination of from about 0.01 grams to 0.2 grams of β-hydroxy-β-methylbutyric acid (HMB) per kilogram body weight and Vitamin D in an amount sufficient to raise blood levels of Vitamin D to at least about 25 ng/ml.

38. A method of improving the efficacy of β-hydroxy-β-methylbutyric acid (HMB) in a nutritional composition for non-human animals, comprising combining Vitamin D in an amount sufficient to raise blood levels of Vitamin D to at least about 25 ng/ml with from about 0.01 grams to 0.2 grams of HMB per kilogram body weight, wherein the combination of HMB and vitamin D has a synergistic effect.

39. A method of improving the efficacy of β-hydroxy-β-methylbutyric acid (HMB) comprising the steps of administering to a non-human animal in need thereof from about 0.01 grams to 0.2 grams of HMB per kilogram body weight of HMB and vitamin D in an amount sufficient to raise blood levels of Vitamin D to at least about 25 ng/ml, wherein the combination of HMB and vitamin D has a synergistic effect.

40. A composition comprising a synergistic combination of from about 0.01 grams to 0.2 grams of β-hydroxy-β-methylbutyric acid (HMB) per kilogram body weight and Vitamin D in an amount sufficient to raise blood levels of Vitamin D to at least about 30 ng/ml, wherein the composition is administered to a non-human animal.

41. A composition comprising a synergistic combination of from about 0.01 grams to 0.2 grams of β-hydroxy-β-methylbutyric acid (HMB) per kilogram body weight and from about 4 IU of Vitamin D per kilogram of body weight, wherein the composition is administered to a non-human animal.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1a is a graph showing the changes in muscle mass in subjects depending on Vitamin D status.

(2) FIG. 1b is a graph showing overall knee strength based on Vitamin D status with administration of HMB.

(3) FIG. 2a is a graph showing serum Vitamin D levels in subjects.

(4) FIG. 2b is a graph showing knee extension strength in subjects.

(5) FIG. 2c is a graph showing an eight week performance index in subjects.

(6) FIG. 3 shows the Protein:DNA ratio and protein degradation in C.sub.2C.sub.12 cells.

(7) FIG. 4a is a graph showing serum Vitamin D levels in subjects.

(8) FIG. 4b is a graph showing urine HMB excretion in subjects.

DETAILED DESCRIPTION OF THE INVENTION

(9) The present invention comprises a combination of HMB and Vitamin D that has a synergistic effect and improves overall muscle strength and function. The combination of HMB and Vitamin D results in significant enhancements in overall muscle mass, function and strength. This combination can be used on all age groups seeking enhancement in overall muscle mass, function and strength. The following methods describe and show increased overall muscle mass, function and strength even in non-exercising animals.

(10) One specific use HMB and Vitamin D is in the older or elderly population. Current estimates place a large portion of the older population at risk for falls with potential significant associated morbidities. The combination of HMB and Vitamin D specifically targets muscle mass, strength and function and consequently may produce significant improvement in health, quality of life, and in particular, decreased falls and injury in this group.

(11) The younger population also benefits from the administration of HMB and Vitamin D, in part due to the widespread occurrence of Vitamin D deficiency. Women also benefit from the administration of HMB and Vitamin D as women are prone to Vitamin D deficiency.

(12) An additional specific use of HMB and vitamin D is in non-human animals, including but not limited to pets such as dogs, cats, and horses.

(13) The present invention provides a composition comprising HMB and Vitamin D. The composition is administered to an animal in need of improvement in overall muscle mass, strength or function.

(14) The composition of HMB and Vitamin D is administered to an animal in any suitable manner. Acceptable forms include, but are not limited to, solids, such as tablets or capsules, and liquids, such as enteral or intravenous solutions. Also, the composition can be administered utilizing any pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and examples of such carriers include various starches and saline solutions. In the preferred embodiment, the composition is administered in an edible form.

(15) B-hydroxy-β-methylbutyric acid, or β-hydroxy-isovaleric acid, can be represented in its free acid form as (CH.sub.3).sub.2(OH)CCH.sub.2COOH. The term “HMB” refers to the compound having the foregoing chemical formula, in both its free acid and salt forms, and derivatives thereof. While any form of HMB can be used within the context of the present invention, preferably HMB is selected from the group comprising a free acid, a salt, an ester, and a lactone. HMB esters include methyl and ethyl esters. HMB lactones include isovalaryl lactone. HMB salts include sodium salt, potassium salt, chromium salt, calcium salt, magnesium salt, alkali metal salts, and earth metal salts.

(16) Methods for producing HMB and its derivatives are well-known in the art. For example, HMB can be synthesized by oxidation of diacetone alcohol. One suitable procedure is described by Coffman et al., J. Am. Chem. Soc. 80: 2882-2887 (1958). As described therein, HMB is synthesized by an alkaline sodium hypochlorite oxidation of diacetone alcohol. The product is recovered in free acid form, which can be converted to a salt. For example, HMB can be prepared as its calcium salt by a procedure similar to that of Coffman et al. in which the free acid of HMB is neutralized with calcium hydroxide and recovered by crystallization from an aqueous ethanol solution. The calcium salt of HMB is commercially available from Metabolic Technologies, Ames, Iowa.

(17) CaHMB has historically been the preferred delivery form of HMB. Previously, numerous obstacles existed to both extensive testing and commercial utilization of the free acid form of HMB, and since it was thought there was no difference between the two forms from a pharmacokinetic perspective, the calcium salt was adopted as a commercial source of HMB. Until recently packaging and, in particular, distribution of dietary supplements has been better suited to handle nutrients in a powdered form and therefore the calcium salt of HMB was widely accepted. HMB-acid is a liquid and much more difficult to deliver or incorporate into products.

(18) Currently, the manufacturing process for HMB has allowed for HMB free acid to be produced in a purity that allows for oral ingestion of the HMB free acid. Besides having a commercial source that is pure enough for oral ingestion, the HMB-acid needs to be buffered for oral ingestion, a process which only recently was determined due to the factors listed above which precluded previous use of HMB-acid.

(19) It was assumed that ingestion of CaHMB would result in a rather quick dissociation of HMB from the calcium salt form. However, a recent study and corresponding patent application (U.S. App. Publication No. 20120053240) has shown that HMB in the free acid form has rather unique pharmacokinetic effects when compared to CaHMB ingestion. Use of HMB free acid (also called HMB-acid) improves HMB availability to tissues and thus provides a more rapid and efficient method to get HMB to the tissues than administration of CaHMB.

(20) Vitamin D is present in the composition in any form. In the preferred embodiment, Vitamin D.sub.3 (cholecalciferol) is administered, but the invention is not limited to that form of Vitamin D. While Vitamin D.sub.3 is the synthesized and preferred form of Vitamin D for mammals, mammals can also use supplemental Vitamin D.sub.2. Vitamin D.sub.2 may be less potent than Vitamin D.sub.3, hence additional D.sub.2 may be required in order to raise blood levels of 25-OH VitD.sub.2.

(21) When the composition is administered orally in an edible form, the composition is preferably in the form of a foodstuff or pharmaceutical medium, more preferably in the form of a foodstuff. Any suitable foodstuff comprising the composition can be utilized within the context of the present invention. In order to prepare the composition as a foodstuff, the composition will normally be blended with the appropriate foodstuff in such a way that the composition is substantially uniformly distributed in the foodstuff. Alternatively, the composition can be dissolved in a liquid, such as water. Although any suitable pharmaceutical medium comprising the composition can be utilized within the context of the present invention, preferably, the composition is blended with a suitable pharmaceutical carrier, such as dextrose or sucrose, and is subsequently tabulated or encapsulated as described above.

(22) Furthermore, the composition can be intravenously administered in any suitable manner. For administration via intravenous infusion, the composition is preferably in a water-soluble non-toxic form. Intravenous administration is particularly suitable for hospitalized patients that are undergoing intravenous (IV) therapy. For example, the composition can be dissolved in an IV solution (e.g., a saline or glucose solution) being administered to the patient. Also, the composition can be added to nutritional IV solutions, which may include amino acids and/or lipids. The amounts of the composition to be administered intravenously can be similar to levels used in oral administration. Intravenous infusion may be more controlled and accurate than oral administration.

(23) The HMB itself can be present in any form; for example, CaHMB is typically a powder that can be incorporated into any delivery form, while HMB-acid is typically a liquid or gel that can be incorporated into any delivery form. Non-limiting examples of delivery forms include pills, tablets, capsules, gelcaps, liquids, beverages, solids and gels. For animals such as companion animals, HMB and Vitamin D can be incorporated into either feed, including commercial pet food, chews and treats, or along with the previously listed delivery forms.

(24) Methods of calculating the frequency by which the composition is administered are well-known in the art and any suitable frequency of administration can be used within the context of the present invention (e.g., one 6 g dose per day or two 3 g doses per day) and over any suitable time period (e.g., a single dose can be administered over a five minute time period or over a one hour time period, or, alternatively, multiple doses can be administered over an eight week time period). The combination of HMB and Vitamin D can be administered over an extended period of time, such as months or years.

(25) It will be understood by one of ordinary skill in the art that HMB and Vitamin D do not have to be administered in the same composition to perform the claimed methods. Stated another way, separate capsules, pills, mixtures, etc. of Vitamin D and of HMB may be administered to a subject to carry out the claimed methods. In the instance of non-human animals such as dogs and cats, where commercial food sources have the animal's total daily requirement of vitamin D, supplementation with a separate source of HMB, such as HMB in the form of a treat, chew, pill, capsule, tablet, etc. without vitamin D, is within the scope of the present invention.

(26) Any suitable dose of HMB can be used within the context of the present invention. Methods of calculating proper doses are well known in the art. The dosage amount of HMB can be expressed in terms of corresponding mole amount of Ca-HMB. The dosage range within which HMB may be administered orally or intravenously is within the range from 0.01 to 0.2 grams HMB (Ca-HMB) per kilogram of body weight per 24 hours. For adults, assuming body weights of from about 100 to 200 lbs., the dosage amount orally or intravenously of HMB (Ca-HMB basis) can range from 0.5 to 30 grams per subject per 24 hours.

(27) The amount of Vitamin D in the composition can be selecting an amount of Vitamin D within the range of greater than 500 IU, as the below examples indicate that 500 IU is the lower threshold for an effective amount in individuals with inadequate levels of Vitamin D in the bloodstream, yet not too much Vitamin D as to be toxic. While the examples indicate a threshold of 500 IU, lower amounts such as 400 IU, may be appropriate, in some individuals, to raise blood Vitamin D levels to an appropriate amount. In another embodiment, the amount of Vitamin D in the composition can be selecting an amount of Vitamin D within the range of greater than 400 IU, yet not too much Vitamin D as to be toxic. The toxic level of vitamin D is a person-specific amount and depends on a person's blood level of vitamin D. For example, administration of 100,000 IU of vitamin D may be toxic for healthy individuals, but not toxic for a person suffering from rickets. One of skill in the art will recognize toxicity levels for an individual. Further, the composition may include Vitamin D in amounts sufficient to raise blood levels of Vitamin D to at least around 25 ng/ml.

(28) In the preferred embodiment, the composition comprises HMB in the form of its calcium salt, and Vitamin D in the form of 25-OH Vit D.sub.3. Preferably, a composition in accordance with the present invention comprises HMB in an amount from about 0.5 g to about 30 g and Vitamin D in an amount greater than 500 IU, but not in an amount high enough to be toxic. One range of Vitamin D in accordance with this invention is around 1000 IU to around 4000 IU. For examples, 1001 IU, 1002 IU, 1003 IU . . . 1995 IU, 1996 IU, 1997 IU, 1998 IU, 1999 IU, 2000 IU, 2001 IU, 2002 IU, 2003 IU, 2004 IU, 2005 IU . . . 3997 IU, 3998 IU, 3999 IU, and all numbers between around 1000 IU and 4000 IU and not otherwise stated.

(29) In another example, a range of Vitamin D in accordance with this invention is around 400 IU to around 100,000 IU. The specific amount of vitamin D that is appropriate to administer to a particular individual routinely varies. A healthy individual likely requires supplementation with vitamin D in an amount lower than an individual with certain disease conditions. For example, it would be appropriate in some circumstances to administer vitamin D to an individual with rickets in an amount of 100,000 IU daily. One of skill in the art is able to readily determine the amount of vitamin D that should be given to a particular individual without causing toxicity.

(30) The amount of vitamin D used in the present invention depends on the individual's vitamin D status. In some individuals, around 400-500 IU of vitamin D is all that would be required to achieve a serum blood level of around 25 ng/ml. In others, 2,000, 4,000 or even 100,000 IU of vitamin D may be required. For example, 400 IU, 40 IU, 405 IU, 450 IU, 500 IU, 550 IU, 1000 IU, 1001 IU, 2000 IU, 5000 IU, 10,000 IU, 20,000 IU, 50,000 IU, 75,000 IU and 100,000 IU and all numbers around and between 400 IU and 100,000 IU that have not been otherwise stated are included in this invention.

(31) The amount of vitamin D needed to reach appropriate blood serum levels of vitamin D in accordance with the present invention may routinely vary from person to person, and determination of the optimum amount in each instance can be readily obtained by routine procedures. “Person” is defined to include non-human animals, as well has humans.

(32) The National Research Council has established recommended vitamin intake amounts for cats and dogs. Requirements expressed relative to dry matter change with the energy density of the diet. In addition, nutritional requirements can change with body weight. The following table illustrates the vitamin D requirement of a mature dog of normal activity.

(33) TABLE-US-00001 TABLE 2 Recommended Vitamin D Intake, IU/d Daily Vit D per Body Mature Dog Energy Weight (0.45 Weight ME, ug * BW{circumflex over ( )}0.75) lbs. kg kcal/d Vit D 3.4 ug/1,000 kcal converted to IU 5 2.3 240 33 33 10 4.5 404 55 56 20 9.1 679 92 94 30 13.6 921 125 128 40 18.1 1143 155 158 60 27.2 1549 211 214 80 36.3 1922 261 266 100 45.4 2272 309 315

(34) Similarly, the National Research Council has also published vitamin D recommendations for cats, as shown in Table 3.

(35) TABLE-US-00002 TABLE 3 Adult Lean Domestic Cat (ME per Day 100 kcal * BW{circumflex over ( )}0.67) Recommended Vitamin D Intake, IU Vit D per Body Mature Cat Weight (0.17 Weight Daily Energy ug * BW{circumflex over ( )}0.67) lbs. kg ME, kcal/d Vit D 1.75 ug/1,000 kcal converted to IU 3 1.4 123 9 8 6 2.7 196 14 13 9 4.1 257 18 17 12 5.4 311 22 21 15 6.8 361 25 25

(36) Further, vitamin D adequacy in canines is similar to the level considered adequate for humans. For example, the prevalence of cancer in dogs has been found to be significantly increased if the serum blood levels of Vitamin D are below 40 ng/ml, which correlates with research in humans indicating an increased risk for many cancers in individuals with Vitamin D levels below 40 ng/ml. With respect to adequacy as it relates to the present invention and as described for humans, adequacy for companion animals, including dogs, as included in this invention, is a circulating blood level of Vitamin D of at least about 25 ng/ml.

(37) Additionally, adequate amounts of Vitamin D in canines and felines can be determined based on the Recommended Daily Allowance for Vitamin D in humans, which is 600 IU per day. In the United States the average human is approximately 81 kg, therefore the recommended human dosage would be approximately 7.41 IU of vitamin D per kg body weight. The National Research Council (NRC) daily recommended intake for vitamin D in dogs is based upon the formula 0.45 μg*Body weight.sup.0.75. Using this formula smaller dogs require more vitamin D per body weight unit than larger canines. When converted to IU/day, a small dog would require up 15 to 15 IU/kg body weight, whereas, a large dog would require amounts similar to humans of 7-8 IU/kg body weight. Similarly for cats the NRC formula is 0.17 μg*Body weight.sup.0.67. Therefore the daily requirement for smaller cats would be about 6 IU/kg body weight, whereas, most typical adult cats would require 4 IU/kg body weight.

(38) In an additional embodiment, the composition in accordance with the present invention comprises HMB in an amount from about 0.5 g to about 30 g and Vitamin D in an amount sufficient to increase circulating blood levels of 25OH-VitD.sub.3 or 25-OH VitD.sub.2, depending on the form supplemented, to at least about 25 ng/ml.

(39) In general, an amount of HMB and vitamin D in the levels sufficient to improve overall muscle strength, function, and overall mass is administered for an effective period of time.

(40) The invention provides a method of administering a composition of HMB and Vitamin D to an animal such that the animal's muscle mass increases. The animal may or may not engage in exercise. Exercising in conjunction with the administration of HMB and Vitamin D results in an even greater improvement in strength and muscle function, but exercise is not necessary to improve strength and muscle function. The amount of HMB and Vitamin D in the composition administered that are effective for increasing the animal's muscle mass can be determined in accordance with methods well-known in the art. In one embodiment, the effective amount of HMB in the composition may be from about 0.5 g to about 30 g and the effective amount of Vitamin D in the composition may be from greater than about 500 IU per 24 hour period. In another embodiment, the effective amount of HMB is the same, and the effective amount of Vitamin D is that which is sufficient to increase blood levels of Vitamin D to at least about 25 ng/ml.

(41) The invention provides a method of administering a composition of HMB and Vitamin D to an animal such that the animal's strength increases. The animal may or may not engage in exercise. The amount of HMB and Vitamin D in the composition administered that are effective for increasing the animal's muscle mass can be determined in accordance with methods well-known in the art. In one embodiment, the effective amount of HMB in the composition may be from about 0.5 g to about 30 g and the effective amount of Vitamin D in the composition may be from greater than about 500 IU per 24 hour period. In another embodiment, the effective amount of HMB is the same, and the effective amount of Vitamin D is that which is sufficient to increase blood levels of Vitamin D to at least about 25 ng/ml.

(42) The invention further comprises a method of administering a composition of HMB and Vitamin D in an effective amount for improving muscle function. The amount of HMB and Vitamin D in the composition administered that are effective for increasing the animal's muscle mass can be determined in accordance with methods well-known in the art. In one embodiment, the effective amount of HMB in the composition may be from about 0.5 g to about 30 g and the effective amount of Vitamin D in the composition may be from greater than about 500 IU. In another embodiment, the effective amount of HMB is the same, and the effective amount of Vitamin D is that which is sufficient to increase blood levels of Vitamin D to at least about 25 ng/ml.

EXAMPLES

(43) The following examples further illustrate the invention but should not be construed as in any way limiting its scope. For example, the amounts of HMB and Vitamin D administered and the duration of the supplementation are not limited to what is described in the examples.

(44) These examples demonstrate the surprising result that the combination of vitamin D and HMB improves strength and muscle function. It was previously known that HMB supplementation increases muscle mass, but no corresponding improvement in strength and muscle function was seen with HMB alone. The examples demonstrate that when serum levels of Vitamin D reach appropriate levels, most typically through supplementation, muscle strength and function improve. The increases in strength, muscle mass, and improved muscle function described and observed in the examples below demonstrate that HMB and Vitamin D are synergistic; when vitamin D levels reach an adequate amount, administration of HMB works better, more effectively or more efficiently than HMB when administered without adequate vitamin D levels. A composition containing HMB and Vitamin D in sufficient amounts will be more efficient and more effective than a composition containing HMB that does not also include adequate amounts of Vitamin D. The studies below examine the effects of vitamin D levels on the efficacy of HMB as related to muscle function, strength and muscle mass, but the improved efficacy of HMB as described in this invention includes all known uses of HMB, including but not limited to the use of HMB for disease associated wasting, aging, cachexia, and nitrogen retention. Further, the efficacy of HMB as related to immune function and lowering cholesterol are also within the scope of this agreement.

Example 1

(45) It is known that administration of HMB and amino acids, specifically arginine and lysine, administered to the non-exercising population results in significant increases in lean mass and improvement in protein turnover. Due to the increases in lean mass and improvement in protein turnover, it would be expected that administration of HMB and amino acids would also improve strength and function. Administration of HMB and amino acids alone, however, does not result in improvement in strength, function, or both. Instead, a gradual loss of handgrip and leg strength is observed. The following example demonstrates that the amount of Vitamin D in the bloodstream affects whether administration of HMB shows improvement in muscle strength and/or function. The data demonstrate that administration of HMB, arginine, and lysine (HMB/ARG/LYS) and Vitamin D is superior to administration of either HMB/ARG/LYS alone or Vitamin D alone. These results show a synergistic effect between HMB and Vitamin D for improving muscle strength and function.

(46) Methods

(47) The effects of HMB, arginine, and lysine (HMB/ARG/LYS) on strength and muscle function in the elderly was studied in both elderly men (n=38) and women (n=39) with an average age of 76.0±1.6 years (39). Subjects were randomly assigned to either treatment with HMB/ARG/LYS (n=40) or to an isonitrogenous control group (n=37) for a one year study. Supplements were taken once per day in the morning with breakfast and all supplements supplied 0.1 g ascorbic acid per day. The HMB/ARG/LYS contained 2 g CaHMB, 5 g arginine, and 1.5 g lysine. The control contained 5.6 g alanine, 0.9 g glutamic acid, 3.1 g glycine, and 2.2 g serine. The supplements were supplied as a ready to mix orange-flavored powder. Subjects weighing more than 68 kg were given supplements containing 1.5 times the dosage above to maintain close to 38 mg/kg body weight per day CaHMB intake as this has been shown to be an effective intake of HMB per day. Lean tissue mass was measured using two independent methods, bioelectrical-impedance analysis (BIA) and dual energy x-ray absorptiometry (DXA). Strength was measured in the upper and lower extremities.

(48) The retrospective analysis of serum 25OH-VitD.sub.3 in the same cohort was performed and the data were stratified based upon the Vitamin D status of the subjects within each original treatment group. An “adequate Vitamin D status” was defined as serum 25OH-VitD.sub.3 level of ≧30 ng/mL (41; 54; 90). Consequently four cohorts were identified: (1) control-subjects with a 25OH-VitD.sub.3 level<30 ng/mL (n=25); (2) HMB/ARG/LYS subjects with a 25OH-VitD.sub.3 level<30 ng/mL (n=29); (3) control subjects with a 25OH-VitD.sub.3 level>30 ng/mL (n=12); and (4) HMB/ARG/LYS subjects with a 25OH-VitD.sub.3 level>30 ng/mL (n=11).

(49) Statistics

(50) The data, means and changes in means over the 12-month period, were analyzed using a mixed models procedure of the Statistical Analysis System for Windows (Release 8.02, SAS Institute, Cary, N.C.). A repeated measures analysis of variance (ANOVA) that included the initial time 0 values for the variable measured as a covariate and main effects of site, gender, and treatment. Lean body mass was analyzed using linear and quadratic time by treatment contrasts.

(51) Results and Analysis

(52) Supplementation with HMB/ARG/LYS resulted in statistically significant increases in muscle mass irrespective of the Vitamin D status (FIG. 1a). The 12-month increase in lean mass within the cohorts with 25OH-VitD.sub.3>30 ng/mL was 0.9 kg, and 0.7 kg in the cohorts with <30 ng/mL (FIG. 1a), which were not significantly different. However, regardless of Vitamin D status, there was a significant increase in lean mass over the year-long supplementation (FIG. 1a inset, p=0.02). On the other hand there was significant divergence in muscle strength based upon Vitamin D status (FIG. 1b). Measurements of “Overall Knee Strength” showed that the HMB/ARG/LYS-supplemented group with 25OH-VitD.sub.3 level>30 ng/mL had significant improvements in muscle strength. When this group was compared with the other three cohorts, there was a 21% linear increase in strength (p<0.003).

(53) Taken together, these results suggest a synergistic effect between the HMB-supplemented cocktail and Vitamin D. Although HMB/ARG/LYS supplementation increased muscle mass (fat-free-mass) regardless of Vitamin D status, strength was only increased with HMB/ARG/LYS supplementation when subjects had adequate Vitamin D status.

(54) The combination of HMB/ARG/LYS and adequate Vitamin D status is necessary for and superior to either HMB/ARG/LYS alone or adequate Vitamin D alone based upon the controls with adequate Vitamin D in improving the strength and functionality of muscle. These results show a synergistic effect between HMB and Vitamin D for improving muscle strength and function.

(55) Prior studies examining the effect of HMB on increasing muscle mass were not significantly improved in all study designs (published and unpublished). It is believed that Vitamin D status may not have been adequate to maximize muscle mass gains in these earlier studies in the HMB-supplemented subjects. This may be especially true in populations where low vitamin D status due to age, geographical location, dietary intake, or disease was in question. Consequently, supplementation with a combination of HMB and Vitamin D may not only increase muscle strength and functionality but also restore or increase muscle mass compared to supplementation of HMB alone.

Example 2

(56) In this example, subjects were administered a combination of HMB and vitamin supplements.

(57) Materials and Methods:

(58) Elderly women (n=30) and men (n=16) were recruited into a double-blinded controlled study. The older adults were recruited from two locations in South Dakota: Brookings and Sioux Falls. The subjects underwent an initial screening and were randomized to treatments. Testing consisted of an initial (0 weeks) and follow-up testing at 4, 8 and 12 weeks over the course of the 12-week study.

(59) Prior to the start of the experimental period, we randomly assigned nutritional supplements to each subject using computer-generated random numbers in a double-blind fashion. Treatments were arranged in a 2×2 factorial design with two levels of HMB (0 & 3.0 g/d) and two levels of Vitamin D (0 & 2,000 IU/d). We assigned subjects to one of the following four treatments:

(60) (1) Control

(61) (2) HMB (calcium salt), 3.0 g/day

(62) (3) 2,000 IU Vitamin D/day

(63) (4) HMB, 3.0 g/day+2,000 IU Vitamin D.

(64) The treatments were supplied in capsules of equal size and color and contained equal amounts of calcium and phosphorus. The subjects were instructed to take three capsules two times a day. Each subject was supplied with a one-week supply of the supplement, allocated by subject number and returned each week for an additional 1-week supply.

(65) The exercise testing and training session was supervised by trained research associates. Resting heart rate and blood pressure measurements were obtained prior to strength measurements. Enrolled individuals participated in an exercise training program consisting of strength training exercises with Theraband® stretch cords (resistance training) and jumping. The equipment consisted of items that can easily be used at home. Exercise sessions were 3 times a week for 12 weeks. Each exercise session was about 45-60 minutes. Testing sessions were performed at 0, 4, 8, and 12 weeks. Each testing session lasted ˜60 minutes.

(66) The strength program incorporated the following exercises: bicep curls, triceps extensions, chair squats, calf raises, ankle dorsiflexion, shoulder front raises and lateral raises, latissimus dorsi pull-down, chest press, seated row, knee flexion and extension, and hip flexion. For each of the 12 exercises, the participants completed 3 sets of isotonic movement, 2 sets of 20 repetitions, and a final set to failure. When the 3.sup.rd set could be performed for 20 repetitions in good form, the resistance was increased by moving to the next color of the resistance band. Between each set, participants performed a set of hops or small jumps. Initially, 5 hops/jumps were performed following each set. The number of hops/jumps was increased by 5 every 3 weeks until 25 hops/jumps were achieved. Subjects remained at 25 hops/jumps between sets for the remainder of the study. The number of hops/jumps was reduced or omitted if there are any complaints regarding joint pain. The resistance bands had been shown to safely increase strength and functionality when used in an older adult population (91-94).

(67) Body composition measurements were obtained at 0, 4, 8, and 12 weeks. Measurements of strengths of quadriceps (extension/flexion) and elbows (extension/flexion) were obtained using the BIODEX Isokinetic Dynamometer. Handgrip strength was measured using a handgrip dynamometer. Peak torque for knee extension and flexion was measured at 60, 90 and 120°/sec. Peak torque for elbow extension and flexion was measured at 60 and 120°/sec.

(68) Functionality tests included: “Get-up-and-Go” performance (speed and gate), and “Get-up” performance. The “Get-up-and-Go” test consisted of timed measurements of the subject's starting from a seated position, standing, walking forward 3 meters, turning around, walking back to the chair, and sitting down. The “Get-up” test consisted of the subject standing upright from a seated position as many times as possible within 30 seconds. The complete descriptions and standardization of the tests are outlined in the Physical Dimensions of Aging by Waneen W. Spirduso (Human Kinetics, 1995).

(69) A muscular performance index was calculated by summing the percentage change from baseline for Get-up-and-Go, Get-up, handgrip, peak torque for knee extension and flexion (60, 90 and 120°/sec) and peak torque for elbow extension and flexion (60°/sec).

(70) At 0, 4, 8, and 12 weeks blood samples were taken from a superficial arm vein into Vacutainers™ (Becton Dickinson, Vacutainer Systems, Rutherford, N.J.) after an overnight fast. Serum was collected and used to measure 25OH-VitD3 using a fully automated antibody-based chemiluminescence assay (DiaSorin Inc., Stillwater, Minn.).

(71) Least Squares Mean±SEM were calculated strength and functionality changes in each variable over the 12-week period. The mixed models procedure of the Statistical Analysis System for Windows (Release 9.1, SAS Institute, Cary, N.C.) was used to analyze the data. Analysis of Covariance was used with main effects of HMB, Vitamin D and the HMB*Vitamin D interaction. Data were adjusted using the initial serum 25OH-VitD.sub.3 concentration as the covariate. The hypothesize was that the combination of HMB and Vitamin D supplementation will result in a greater improvements in strength and functionality as compared to control, HMB, or Vitamin D-treated subjects. This synergistic effect was tested with pre-planned one-tail t-test in a post-hoc analysis. Statistical significance was determined for p<0.05 and a trend was determined for 0.05<p<0.10.

(72) Results and Discussion:

(73) A total of 43 subjects completed the 12-week and one subject completed the 8-weeks of the study and was included in the analysis. Two subjects dropped from the study prior to the 4-week follow-up visit and were not used in the analysis. The subjects that dropped from the study did so because of the demanding commitment of a 12-week training study and not due to any adverse events. The subject characteristics of the 44 subjects used in the analysis are presented in Table 1.

(74) TABLE-US-00003 TABLE 1 Subject Characteristics HMB + Item Control HMB VitD.sub.2000 VitD.sub.2000 n 11 11 11 11 Age 73.4 ± 2.6 73.5 ± 3.0 72.1 ± 2.8  68.5 ± 2.5   (62-87)  (60-89) (61-96) (60-84) M/F 3/8 4/7 4/7 4/7 *25OH- 29.2 ± 3.0 25.4 ± 3.0 24.0 ± 2.0  27.4 ± 3.0  VitD.sub.3  (12.4-46.5)  (12.2-39.7) (11.7-34.3) (15.1-42.9) #Leg 73.9 ± 9.6 64.1 ± 7.4 74.8 ± 10.0 83.1 ± 10.6 Strength,  (33-148)   (23-98.3) (24.7-133)   (40.5-146.1) Extension Flexion 51.4-6.2 43.6-5.3 55.9-6.1  51.8-7.7   (23-95)  (14-61) (13.8-83)   (18-99) Data are expressed as Mean ± SE (min-max) *Baseline value #Baseline Peak Torque @ 60°/sec
The average 25OH-VitD.sub.3 concentration at wk 0 was 26.5 ng/ml. Subjects treated with 2000 IU of Vitamin D per day increased their serum 25OH-VitD.sub.3 level by 10.7 ng/mL (FIG. 2a). However, subjects either supplemented with a control or HMB alone decreased their serum 25OH-VitD.sub.3 level by 2.0 ng/mL. A clean catch urine sample was used to test for supplement compliance. Subjects supplemented with HMB had a 50 fold increase in urinary HMB concentration whereas the subjects not consuming a HMB supplement had no increase urinary HMB concentration.

(75) The effects of the combination of HMB and Vitamin D on knee extension strength (peak torque, 600/sec is presented in FIG. 2b. Supplementation with HMB+Vitamin D (7.28±5.03 nm) in older adults results in a statistically greater eight week increase in knee extension strength as compared to control-treated subjects, P<0.05 (−6.28±5.02 nm). Neither the HMB alone nor the Vitamin D-treated subjects increased knee extension strength when compared to the control-treated subjects. These data support our hypothesis that the combination of HMB+Vitamin D would be synergistic on muscular strength.

(76) Although not significant the performance index tends supports these observations (see FIG. 2c). The performance index not only includes leg strength measurements but also functionality, hand grip strength, and elbow strength. These strength and function measurements are summed into one overall performance index. The combination of HMB+Vitamin D resulted in a 146% increase in the performance index over the control. Whereas HMB and Vitamin D supplementation resulted in only a 35 and 58% increase, respectively, when compared to older adults supplemented with the control treatment.

(77) This experimental example demonstrates the synergistic effect of HMB and vitamin D on muscle when vitamin D was supplemented at approximately 24 IU/kg of body weight (2,000 IU daily in an average 82.3 kg human). A similar effect of supplemental vitamin D on the muscles of dogs and cats occurs at similar supplemental rates over the minimal required vitamin D dosage.

(78) Implications:

(79) These data from a prospective study in older adults support the hypothesis that the combination of HMB and Vitamin D supplementation is synergistic, and resulted in significant improvements in knee muscle strength and overall performance index. These data support the observation from our retrospective study in an elderly population supplemented with HMB, Arginine, and Lysine. Muscular strength increases only occur with HMB, Arginine, and Lysine in older adults with adequate Vitamin D. In conclusion, the combination HMB and Vitamin D is superior to either HMB or Vitamin D alone and is therefore synergistic. These findings are important as the Vitamin D status in approximately 66% of the elderly population is low and leads to a decrease in muscular strength and function. The loss of strength and function leads to an increase in falls and fractures, poor quality of life and will ultimately impact health care costs.

Example 3

(80) In this example, cell culture studies were performed to analyze the effects of HMB and vitamin D on protein, DNA and protein turnover in muscle cell cultures.

(81) Recent in vitro studies have shown that HMB decreases protein breakdown. In one study HMB attenuated proteolysis-inducing factor (PIF) activation and increased gene expression of the ubiquitin-proteasome pathway in murine myotubes, thereby reducing protein degradation (31). Other studies also suggest HMB may influence protein synthesis through the mTOR pathway (29). Early studies have also suggested that vitamin D affects protein metabolism (59). Vitamin D also binds a VDR found in the nucleus of muscle cells and regulates gene expression (95). We hypothesize that HMB and vitamin D both have effects on muscle cells and that if they act through independent mechanisms there could be a synergistic effect on either protein, DNA or protein turnover in the cells.

(82) Methods

(83) Mouse C2C12 skeletal muscle cell myoblasts were grown in culture under standard conditions and fused into myotubes following the methods of Menconi et al. (96). Four separate studies were conducted during different weeks. The first 3 studies consisted of 4 replicate dishes per treatment and the fourth study consisted of 6 replicate dishes per treatment. The treatments were applied to the cells during the 24 h measurement period. Treatments applied were control, 25OH-VitD.sub.3 (200 ng/mL), HMB (200 μM HMB as Ca(HMB).sub.2, and the combination of HMB and 25OH-VitD.sub.3. Dexamethasone (100 nM) was added to the culture medium during the treatment/measurement period to stimulate protein degradation. Calcium in the form of calcium chloride was added to the control and 25OH-VitD.sub.3 treatments so that all the treatments were balanced for calcium. For measurement of protein degradation during the treatment period, leucine free medium was obtained to which [5,5,5].sup.2H.sub.3-leucine was added so that the isotopes of leucine (natural leucine and [1-.sup.13C]-leucine) could be measured as they were released into the medium through the protein degradation process. The measurement medium was sampled at 2 and 24 h and samples stored at −80° C. until analyzed. The samples were analyzed for leucine using a gas chromatography (model 6890, Hewlett Packard, Palo, Calif.) mass spectrometry (model 5973, Hewlett Packard, Palo, Calif.) and the gas chromatography column used for the analysis was a Zebron ZB-5. (Phenomenex, Torrence, Calif.). Natural leucine, [1-.sup.13C]-leucine, and [5,5,5.sup.2H.sub.3]-leucine were monitored at 302, 303, and 305 AMU, respectively. Corrections for sampling at 2 h and for leucine utilization from the measurement media were made. The amount of the leucine isotopes released was also corrected for utilization (transamination and/or protein synthesis) (97). Concentrations of proteins were analyzed following the microplate assay instructions using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, Ill.). DNA was measured flourometrically using the Quant-iT™ dsDNA assay kit from Invitrogen (Carlsbad, Calif.).

(84) Statistics

(85) Each cell culture dish was considered an experimental unit for analysis. Data presented are least square means and data were analyzed using General Linear Models in the Statistical Analysis System for Windows (Release 8.02, SAS Institute, Cary, N.C.). Main effects of experiment, HMB, vitamin D, and the interaction of HMB and vitamin D were included in the model. Post-hoc least square means analysis was performed for individual treatment means.

(86) Results and Analysis

(87) After the 24 h treatment period there were no treatment main effects on total protein content in the culture dishes. There were no significant treatment main effects for DNA content. Protein:DNA ratio with the combination of HMB and 25OH-VitD.sub.3 (FIG. 3) was significantly greater than either the control group or either treatment group alone and the interaction was significant (p<0.003). This significant interaction response for the main effects would indicate a synergistic response of HMB and 25OH-VitD.sub.3 on protein:DNA ratio. Measures of protein degradation and protein synthesis showed no significant main effects of treatment on protein degradation measured either by natural or [1-.sup.13C]-leucine release into the medium from the cells. There was, however, a strong trend for the interaction between HMB and 2=50H VitD.sub.3 on protein degradation measured by [1-.sup.13C]-leucine (p<0.08, FIG. 3). Post-hoc least square means analysis of the effects of HMB alone showed a significant decrease in protein degradation of 7.6 and 6.3% as measured by natural leucine and [1-.sup.13C]-leucine, respectively (p<0.06). HMB and 25OH-VitD.sub.3 combination treatment also decreased protein degradation, but the lack of neither a significant interaction nor a 25OH-VitD.sub.3 main treatment effect would indicate the effect was due primarily to HMB. Protein synthesis and leucine utilization as measured by disappearance of [5,5,5-.sup.2H.sub.3]-leucine from the media showed no differences among treatments. The current series of studies show that HMB and 25OH-VitD.sub.3 combination increases protein:DNA ratio and that HMB decreases protein degradation. These both are indications that the cell is producing more proteins, possibly of a functional nature. The data also show that the HMB decreases dexamethasone stimulated protein degradation in both slower and faster turnover protein pools natural and [1-.sup.13C]-leucine labels, respectively. Vitamin D tended to decrease dexamethasone stimulated protein degradation in only the faster turnover protein pool. These could indicate that vitamin D affects synthesis of functional proteins as vitamin D has been shown in other cell types to increase cell adhesion proteins (98; 99). In conclusion, the HMB+vitamin D combination was synergistic in increasing protein:DNA ratio in the cells and supports our hypothesis.

Example 4

(88) The amount of Vitamin D administered with HMB must be in an effective amount to raise the blood level of Vitamin D. In this example, it is demonstrated that 500 IU of Vitamin D does not sufficiently raise the blood level of Vitamin D; this finding however, is based on the subjects in this study. As stated hereinabove, the amount of Vitamin D necessary to raise blood serum levels of Vitamin D to an adequate amount depends on the individual's Vitamin D status; in some instances, as little as 400 IU of Vitamin D is an appropriate amount to raise blood levels to around at least 25 ng/ml.

(89) Subjects with adequate Vitamin D (plasma 25OH-VitD.sub.3 levels>30 ng/ml) manifested significant improvements in muscle function, while those with biochemical evidence of Vitamin D deficiency (25OH-VitD.sub.3 levels<30 ng/ml) failed to show improvements in muscle strength and functionality. While muscular weakness associated with Vitamin D may not be surprising at classical Vitamin D deficiency levels (blood 25OH-VitD.sub.3 of <15 ng/mL), Bischoff-Ferrari et al continued to see improvement in lower extremity function up to and beyond 40 ng 25OH-VitD.sub.3/mL which are levels well above what previously might have been thought necessary for maximal benefit (52). Therefore, supplementation Vitamin D should be adequate to raise 25OH-VitD.sub.3. FIG. 4a demonstrates the blood 25OH-VitD3 response when a control, or supplement containing either 500 or 2000 IU of Vitamin D were given daily for 12 weeks in older adult over 65 years of age. Subjects supplemented with either the control or 500 IU of Vitamin D for 12 weeks did not increase blood 25OH-VitD.sub.3. Whereas subjects supplemented with 2000 IU of Vitamin D increased blood levels by over 10 ng/mL in 12 weeks. This supplementation established a blood level of 25OH-VitD.sub.3 level>30 ng/ml. FIG. 4b shows the urine levels of HMB in these subjects. In conclusion, 2000 IU of Vitamin D is sufficient to raise blood levels 25OH-VitD.sub.3, where 500 IU is inadequate. When Vitamin D levels are adequately raised, the use of HMB results in an increase in strength and muscle function.

(90) The foregoing description and drawings comprise illustrative embodiments of the present inventions. The foregoing embodiments and the methods described herein may vary based on the ability, experience, and preference of those skilled in the art. Merely listing the steps of the method in a certain order does not constitute any limitation on the order of the steps of the method. The foregoing description and drawings merely explain and illustrate the invention, and the invention is not limited thereto, except insofar as the claims are so limited. Those skilled in the art who have the disclosure before them will be able to make modifications and variations therein without departing from the scope of the invention.

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