MXPA99009242A - PROCESS FOR ENHANCING IMMUNE RESPONSE IN ANIMALS USING&bgr;-CAROTENE AS A DIETARY SUPPLEMENT - Google Patents

Abstract

A process for feeding a companion animal such as a dog or cat a diet containing an effective amount of&bgr;-carotene to enhance immune response and improve the overall health of the animal is provided. Preferably, the diet includes from about 1 to about 50 mg/day of&bgr;-carotene (from about 6 to about 315 mg&bgr;-carotene/kg diet). Such a diet provides sufficient&bgr;-carotene to be absorbed by the animal and supplied to the blood and blood leukocytes and neutrophils in the animal.

Description

PROCESS TO IMPROVE IMMUNE RESPONSE IN ANIMALS USING BETA-CAROTENE AS A DIETARY SUPPLEMENT BACKGROUND OF THE INVENTION This invention relates to a pet food supplement and a process for enhancing the immune response and improving the overall health of pets such as cats and dogs, and more particularly with a supplement for pet food and the process which includes beneficial amounts of beta-carotene in the diet of the animal. Carotenoids are naturally occurring plant pigments which are absorbed in varying degrees by different species. Common carotenoids include beta-carotene, lycopene, lutein, zeaxanthin, and astaxanthin. It is known that these carotenoids (the most studied is beta-carotene) play an important role in the modulation of the immune system and enhance the health of these species. It is known that beta-carotene is a precursor of vitamin A and is converted to vitamin A by enzymes in the bodies of certain animals that include humans and dogs. However, cats do not possess this enzyme and can not convert beta-carotene to vitamin A. It is also known that beta-carotene has powerful anti-oxidant activity and serves to protect cellular membranes and organelles from oxidative damage in certain species .

However, in order to be effective, beta-carotene must be present at critical sites in the cell such as the mitochondria, nucleus, and plasma membrane. Disease prevention is important in both humans and pets. A healthy immune system plays an important role both in preventing and fighting the disease. Some studies have reported only low to non-detectable amounts of beta-carotene in circulating blood and in the organs of dogs. In addition, due to the known inability of cats to convert beta-carotene to vitamin A, their diets have not included beta-carotene supplements. In accordance with the above, there remains a need in the art to promote a healthy immune system in domestic animals such as dogs and cats.

SUMMARY OF THE INVENTION The present invention satisfies the need by providing a process for feeding a pet such as a dog or cat with a diet containing an effective amount of beta-carotene to enhance the immune response and improve the overall health of the animal. . Preferably the animal is fed a diet that includes from about 1 to about 50 milligrams / day of beta-carotene (from about 6 to about 315 milligrams of beta-carotene / kilogram of diet). This diet provides enough beta-carotene to be absorbed by the animal and supplied to the blood and to the leukocytes and neutrophils of the blood in the animal. In accordance with the above, it is a feature of the present invention to provide a pet food supplement and a process to enhance the immune response and improve the overall health of domestic animals such as cats and dogs * by providing an effective amount of beta- carotene in the diet of the animal. This, and other features and advantages of the present invention, will become apparent from the following detailed description and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph of the concentration of beta-carotene in blood plasma in dogs (μg / l) against time, for dogs given a single oral dose of beta-carotene; Figure 2 is a graph of the concentration of beta-carotene in blood plasma in dogs (μg / l) against time, for dogs given repeated doses of beta-carotene, - Figure 3 is a graph of Q assimilation beta-carotene from the diet by blood lymphocytes (ng / 10 cells) against time, from dogs fed beta-carotene daily for 30 days; Figure 4 is a graph of dietary beta-carotene assimilation by the nucleus, mitochondria, microsomes, o and cytosol of blood lymphocytes (ng / 10 cells) of dogs fed beta-carotene daily for 30 days; Figure 5 is a graph of the relative assimilation (percent) of beta-carotene by the subcellular fractions of blood lymphocytes (mitochondria, microsomes, cytosol, and nucleus) of dogs fed beta-carotene daily for 30 days; Figure 6 is a graph of diet assimilation of beta-carotene by blood neutrophils of dogs (ng / 10 cells) fed beta-carotene daily for 30 days, - Figure 7 is a graph of beta-carotene assimilation in the diet by the nucleus (nuci), mitochondria (myth), microsomes (micro), and cytosol (cyto) of blood neutrophils from dogs fed by beta-carotene for 30 days; Figure 8 is a graph of the relative assimilation (percent) of beta-carotene by subcellular fractions of blood neutrophils (mitochondria, microsomes, cytosol, and nucleus) of dogs fed beta-carotene daily for 30 days; Figure 9 is a graph of changes in plasma beta-carotene concentrations in dogs fed 0, 2, 20, or 50 milligrams of beta-carotene daily for 8 weeks; Figure 10 is a graph of the delayed type hypersensitivity response (DTH) for PHA in dogs fed 0, 2, 20 or 50 milligrams of beta-carotene daily for 7 weeks, - Figure 11 is a graph of the DTH response to the vaccine in dogs fed 0, 2, 20, or 50 milligrams of beta-carotene daily for 7 weeks; Figure 12 is a graph of changes in the subset of CD4 lymphocytes in dogs fed 0, 2, 20, or 50 milligrams of beta-carotene daily for 8 weeks, - Figure 13 is a graph of changes in total immunoglobulin in plasma in dogs fed 0, 2, 20, or 50 milligrams of beta-carotene daily for 8 weeks; Figure 14 is a graph of beta-carotene concentration in blood plasma in cats (μg / l) versus time for cats given a single oral dose of beta-carotene; Figure 15 is a plot of beta-carotene concentration in blood plasma in cats (μg / l) versus time for cats given repeated doses of beta-carotene; Figure 16 is a graph of the assimilation of beta-carotene in the diet by blood lymphocytes (ng / 10 cells) against the time of cats fed beta-carotene daily for 14 days, - Figure 17 is a graph of the assimilation of beta-carotene in the diet by the nucleus (nucí), mitochondria (mito), microsomes (micro), and cytosol (cyto) of blood lymphocytes of cats fed beta-carotene daily for 7 days, - Figure 18 is a graph of the beta-carotene assimilation of the diet by the nucleus (nuci), mitochondria (micro), microsomes (micro), and cytosol (cyto) of the blood lymphocytes of cats fed beta-carotene daily for 14 days; Figure 19 is a graph of changes in the relative proportion (percent) of beta-carotene in subcellular fractions of lymphocytes in cats fed 5 milligrams of beta-carotene daily for 7 and 14 days, and Figure 20 is a graph of changes in the relative proportion (percentage) of beta-carotene in subcellular fractions of lymphocytes (mitochondria, microsomes, cytosol, and nucleus) in cats fed 10 milligrams of beta-carotene daily for 7 and 14 days.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The present invention uses a pet food composition containing a source of beta-carotene as a supplement in an amount between about 1 to about 50 milligrams / day of beta-carotene (from about 6 to about 315 milligrams of beta-carotene / kilogram of diet). This diet provides beta-carotene so that it is absorbed by the animal and is supplied to the blood and to the leukocytes and neutrophils of the blood in the animal. It has been found that both dogs and domestic cats are able to absorb dietary beta-carotene. further, circulating beta-carotene is significantly absorbed by both lymphocytes and peripheral blood neutrophils in these animals. Beta-carotene is also distributed in the various subcellular organelles. This beta-carotene in the various leukocyte organelles is believed to (1) protect these cells from attack by oxygen free radicals and / or (2) directly regulate nuclear events. In this way, feeding dogs and cats with effective amounts of beta-carotene provides beta-carotene at important cell cycles in the animal's body tissues, which can result in an up-regulation of immune function and improve the immune function. health in these animals. The pet food composition can be any convenient pet food formula that also provides adequate nutrition for the animal. For example, a typical canine diet for use in the present invention may contain about 30% crude protein, about 20% fat, and about 10% total dietary fiber. However, no specific proportions or percentages of these or other nutrients are required. Beta-carotene can be mixed with this pet food to provide the necessary beneficial amounts. With the final purpose. of the invention being understood more easily, reference is made to the following examples which are intended to illustrate the invention, but do not limit the scope thereof. Example 1 - Dogs - Assimilation in the blood after a single dose Beagle dogs (from 18 to 19 months of age from 7 to 9 kilograms of body weight) were used in examples 1 to 3 and fed a basal diet ( The Ia Co., Lewisburg, OH) which met or exceeded the requirements of all essential nutrients. The animals were housed indoors with rooms with light (14 hours of light, - 10 hours of darkness) and temperature controlled. A test was conducted to study the assimilation profile of beta-carotene after a single oral dose of beta-carotene. To study the oral beta-carotene assimilation in dogs given a single dose of beta-carotene orally, the dogs (n = 6 / treatment) were fed one time orally with 0, 50, 100 or 200 milligrams of beta-carotene (10 % dissolvable in cold water; GASF Corp., Ludwigshafen, Germany). The appropriate dose of beta-carotene was dissolved in 5 milliliters of water orally fed using a feeding syringe. In order to establish adequate sampling times, two dogs were used in a preliminary study. These dogs were fed once with 50 milligrams of beta-carotene and the blood was sampled at 0 (immediately before feeding with beta-carotene), 3, 6, 9, 12, 15, 18, 21 and 24 hours. The blood plasma was separated by centrifugation and the beta-carotene concentrations were analyzed using high performance liquid chromatography (HPLC) as follows. All procedures were carried out under dim light. Duplicate plasma aliquots, each leukocyte homogenate, and each subcellular leukocyte fraction were extracted with a 1: 1 mixture of diethyl ether and petroleum ether in the presence of BHT. The ether phase was removed and dried under a stream of nitrogen. The residue was reconstituted in mobile phase for the determination by high performance liquid chromatography of beta-carotene. Samples (50 μl) were injected on a 5 μl spherical C-18 reverse phase column (3.9 x 150 millimeters; Resolve) and eluted with a 47: 47: 6 (volume / volume / volume) mixture of acetonitrile, methanol , and chloroform at an expense of 1.0 milliliter / minute. The results of this example are illustrated in Figure 1 and show that the peak concentrations of beta-carotene occur between 3 and 6 hours after dosing and were not detectable at 24 hours. Subsequently, the blood of the remaining dogs was sampled in the same time periods. The plasma was separated in a similar manner and analyzed by high performance liquid chromatography. The concentrations of beta-carotene in plasma were undetectable in dogs without supplement in all the periods of time studied. In contrast, there was a dose-dependent increase (P <0.01) in plasma beta-carotene in dogs given an oral dose of beta-carotene (Figure 1) . Peak concentrations were observed at six hours after dosing and were consistent in all treatment groups. After that, there was a rapid decrease (P <0.01) in beta-carotene concentrations in all dogs supplemented with beta-carotene. The concentrations were undetectable at 24 hours after dosing. The half-life of beta-carotene in the plasma was about 3 (50 and 100 milligrams dose) to 4 (100 milligram dose) hours. Peak concentrations of beta-carotene in the blood occurred earlier in dogs than in cats (see Examples 4 and 5 below). Also, the concentrations of beta-carotene in the plasma of dogs was approximately 10 to 16 times lower than that observed in cats after adjusting for differences in body weight.

Example 2 - Dogs - Assimilation in blood with repeated doses In this Example, dogs (n = 6 / treatment) were fed daily at eight hours for seven consecutive days with 0, 12.5, 25, 50 or 100 milligrams of beta-carotene . Beta-carotene was added in the meal and was administered in the morning meal. The blood was sampled once daily on day 0 (immediately before the first dose) and then six hours after each dosage (days 1 to 7). This blood sampling schedule was chosen based on the results obtained in Example 1 which showed peak concentrations of beta-carotene at six hours after a dose. Plasma was isolated and analyzed for beta-carotene concentrations. Daily dosing of dogs with beta-carotene for 7 days produced a dose-dependent increase (P <0.01) in circulating beta-carotene as illustrated in Figure 2. Dogs fed 100 milligrams of beta-carotene showed the sharper increase in plasma beta-carotene daily concentrations. Peak concentrations (18 μg / L) of beta-carotene in plasma on day 1 in dogs fed 100 milligrams of beta-carotene in this example was similar to that observed in Example 1 (Figure 1). Plasma beta-carotene concentrations after the last dose was generally 2.5 to 4 times higher than that observed after the first dose. The results of this example suggest that the dog can absorb beta-carotene from its diet. This finding contradicts previous studies that reported trace amounts, if any, of carotene in the blood, liver and milk of dogs. However, other studies have reported low to moderate concentrations of beta-carotene in the blood of dogs.

Example 3 - Dogs - Assimilation by blood leukocytes This example was designed to study the assimilation of beta-carotene in dogs by blood lymphocytes. Dogs (n = 8 / treatment) were fed 0, 50 or 100 milligrams of beta-carotene daily for 30 days. Blood was sampled from all dogs via the jugular vein on days 10, 20 and 30. Lymphocytes and neutrophils were separated by density gradient centrifugation. The number of cells was listed. The lymphocytes and neutrophils were resuspended in phosphate buffered solution containing 3% sodium ascorbate as an anti-oxidant. An aliquot of the cell suspension was sonified for 30 seconds to break the cells. The leukocyte homogenates were extracted by high performance liquid chromatography analysis of beta-carotene. On day 30, a larger aliquot of blood was taken and leukocyte suspensions were prepared as described above for subsequent subcellular fractionation. The cells were disrupted by sonification of 20 minutes in five volumes of 0.25 M sucrose. Sodium ascorbate was added as the anti-oxidant. The homogenate was centrifuged (600 x g for 10 min at 4 ° C) and the nuclear agglomerate was separated from the supernatant. The post-nuclear supernatant was centrifuged (17, 300 x g for 20 min at 4 ° C) to separate the mitochondrial fraction. The post-mitochondrial supernatant was centrifuged (102,000 x g for 60 min at 4 ° C) to separate the microsomal fraction from the cytosolic. Each subcellular fraction was analyzed to determine the content of beta-carotene by high performance liquid chromatography. On day 0 (before supplementation with beta-carotene), beta-carotene concentrations in peripheral blood lymphocytes were undetectable in all dogs as illustrated in Figure 3. Also, beta-carotene in lymphocytes of dogs without supplement remained undetectable throughout the study. In contrast, beta-carotene concentrations in lymphocytes of dogs fed beta-carotene generally increased (P <; 0.01) in a time-dependent manner. There was no significant treatment difference in beta-carotene concentrations in lymphocytes when compared with dogs fed between 50 versus 100 milligrams of beta-carotene. The concentrations of beta-carotene in the lymphocytes of dogs in this example is 20 to 30 times lower than that observed in cats (see examples 4 and 5 below). Figures 4 to 8 illustrate the assimilation of beta-carotene by the subcellular lymphocyte and neutrophil fractions. Beta-carotene was not detectable in the different subcellular fractions of the lymphocytes obtained from dogs without supplement (Figure 4). In contrast, beta-carotene was assimilated by all subcellular fractions of blood lymphocytes isolated from dogs supplemented with beta-carotene. The cytosol fraction formed from 52 to 62% of the total beta-carotene in the lymphocytes (Figure 5) while the nucleus contained the lowest amount (from 6 to 8%) of the total beta-carotene. The mitochondria (14 to 17%) and the microsomes (from 16 to 23%) were intermediate between the cytosol and the nucleus. The dose of dietary beta-carotene did not have a significant influence on the assimilation of beta-carotene by the subcellular fractions on day 30 of feeding. The results show that beta-carotene was assimilated by all the subcellular fractions of the lymphocyte. However, beta-carotene was higher in the cytosol of dogs, but was higher in the mitochondria of cats (see Examples 4-6 below). Also, beta-carotene concentrations in all subcellular fractions of dog lymphocytes were substantially lower than those reported in cats (see Examples 4-6 below). As with lymphocytes, blood neutrophils similarly assimilated beta-carotene (Figure 6). However, unlike lymphocytes, maximum assimilation occurred on day 10, with no further increase in beta-carotene concentrations in neutrophils observed on day 30. Cytosol, mytochondria and microsomes of blood neutrophils they also showed significant assimilation of beta-carotene (Figure 7). In contrast, beta-carotene was not detected in the nucleus. As with the subcellular fractions of the lymphocyte, beta-carotene was higher (61 to 68) in the cytosolic fraction of blood neutrophils (Figure 8). No significant effect of dose was observed. The results indicate that the dog is able to absorb beta-carotene in the diet. This result is surprising since previous studies have found only trace amounts of beta-carotene in the liver and milk of dogs, and ~ only from traces to moderate amounts in the blood of dogs. In addition, it has been found that circulating beta-carotene is significantly absorbed by both lymphocytes and peripheral blood neutrophils in the dog. Beta-carotene is distributed in the different subcellular organelles. Beta-carotene in the various organelles of leukocytes is thought to (1) protect these cells from attack by oxygen free radicals and / or (2) directly regulate nuclear events. Thus, feeding dogs with effective amounts of beta-carotene, will result in the presence of beta-carotene in important cellular sites in the body tissues which will provide improved health in these dogs.

Example 4 - Dogs - Effect on the immune response Beagle dogs (4 to 5 months old) were supplemented daily with 0, 25, 50 or 100 milligrams of beta-carotene to study the role of beta-carotene in the diet to increase The immune systems mediated by the dog's cell and humoral. The following parameters were assessed in all animals or peripheral blood lymphocytes: (1) delayed type hypersensitivity (DTH) against PHA (non-specific immunity) and vaccine (specific immunity), (2) lymphocyte proliferation, (3) ) populations of lymphocytes and (4) immunoglobulins (Ig). Supplementation of beta-carotene increased plasma beta-carotene concentrations in a dose-dependent manner as shown in Figure 9 but did not influence plasma retinol or α-tocopherol. These changes generally reflected the delayed-type hypersensitivity response to both the specific (vaccine) and non-specific (PHA) antigens as shown in Figures 11 and 10, respectively. The greatest response to the PHA threat was observed in dogs fed 50 milligrams of beta-carotene while dogs fed either 20 or 50 milligrams of beta-carotene showed significantly greater response to delayed-type hypersensitivity to the vaccine. Hypersensitivity of the delayed type is strictly a cellular reaction involving the cells that present T antigen without involving an antibody component. The antigen-presenting cells (e.g., macrophages) present the antigen or the allergen to the T cells which is activated and releases lymphokines. These lymphokines activate macrophages and cause them to become voracious scavengers of foreign invaders. Therefore, the data show an increased cell-mediated response in dogs fed beta-carotene. Feeding with beta-carotene also produced significant changes in the subsets of lymphocytes. Compared to controls, dogs fed 20 to 50 milligrams of beta-carotene gave a high CD4 + cell population (week 8) as shown in Figure 12. Dogs fed 20 milligrams of beta-carotene also had high population of CD8 cells at weeks 2 and 4. T cells can be classified according to the expression of CD4 membrane molecules. CD4 functions as an adhesion molecule and as a co-signaling co-receptor. It plays a role in the activation of T cells. CD4 + T lymphocytes recognize the antigen in association with MHC class II molecules and function - largely as helper cells. The increase in the population of helper T cells in this study may explain the corresponding increase in the delayed-type hypersensitivity response in dogs fed 20 to 50 milligrams of beta-carotene. The concentrations of IgG, IgM and total IgG (Figure 10) increased significantly in dogs fed beta-carotene as early as one week after dietary supplementation. Increases in immunoglobulins were dose dependent for dogs fed 0 to 20 milligrams of beta-carotene. The highest level of beta-carotene (50 milligrams) does not produce an additional increase. Dogs fed 20 milligrams of beta-carotene consistently had the highest antibody response for both immunoglobulins. One of the main functions of the immune system is the production of antibodies, > which circulate freely to protect the body against foreign materials. Antibodies serve to neutralize toxins, immobilize certain micro-organisms, neutralize viral activity, agglutinate micro-organisms or antigen particles and precipitate soluble antigens. Feeding with beta-carotene did not influence the lymphocyte-induced lymphocyte blastogenesis and the production of interleukin-2. Lymphocytes are involved in cell-mediated immunity. After recognizing an antigen, the lymphocytes will divide quickly, cloning by this in preparation to combat a potential invasion. In the humoral immune response, interleukin-2 stimulates both T helper cells and B cells to proliferate in response to antigens. This is required for the clonal expression of T cells activated by antigen or mitogen. In the cell-mediated immune response, interleukin-2 activates natural killer cells, stimulates the proliferation of thymocytes and induces the cytotoxic activity of the T cell. It is surprising that these two immune parameters are not influenced by feeding with beta- carotene while numerous others do. Based on the results of these experiments, the dog absorbs a significant amount of beta-carotene from the diet and transfers the beta-carotene to the subcellular organelles of immune cells and phagocytes. In these cells, beta-carotene appears to enhance the dog's immune system through enhanced cell-mediated immune responses (delayed-type hypersensitivity responses, change in lymphocyte subsets) and humoral response (production of IgG and IgM) . In this way, dietary supplemental beta-carotene promotes the immunological health of dogs, which probably translates into improved overall health.

Example 5 - Cats - Assimilation in blood after a single dose Mature Tabby cats with short hair (7 to 8 months of age, from 1.5 to 2.0 kilograms of body weight) were used for Examples 4-6 and fed with a Basal diet (The Iams Co., Lewisburg, OH) that meets or exceeds the requirements of all essential nutrients. The animals were housed in groups indoors in rooms with controlled light and temperature. A test was conducted to study the assimilation profile of beta-carotene after a single oral dose of beta-carotene. To study the uptake of oral beta-carotene in cats given a single dose of beta-carotene orally, cats were administered (n = 6 / treatment) once orally 0, 10, 20 or 50 milligrams of beta-carotene (dissolvable at 10% in cold water, - BASF Corp., Ludwigshafen, Germany). The appropriate dose of beta-carotene was dissolved in 0.6 milliliter of water and fed orally using a feeding syringe. In order to establish the appropriate sampling times, two cats were used in a preliminary study. These cats were fed once with 50 milligrams of beta-carotene and blood was sampled at 0 (immediately before feeding with beta-carotene), 3, 6, 10, 16, 24, 30 and 36 hours. The blood plasma was separated by centrifugation and the beta-carotene concentrations were analyzed using high performance liquid chromatography as described above. The results of this example are illustrated in Figure 14 and show peak concentrations of beta-carotene occurring generally between 10 and 16 hours after dosing. Subsequently, the blood of the remaining cats was sampled at 0, 12, 24, 30, 36, 42, 48 and 72 hours after the dose. The plasma was separated in a similar manner and analyzed. Plasma beta-carotene concentrations were undetectable in non-supplemented cats at all time periods studied. In contrast, plasma beta-carotene in cats given an oral dose of beta-carotene generally increased (P <0.01) in a dose-dependent manner (Figure 14). The concentrations were higher (P <0.01) in cats fed 50 milligrams of beta-carotene. The plasma concentrations of beta-carotene in cats fed 10 to 20 milligrams of beta-carotene were similar (P> 0.01). Peak concentrations were observed at 12 hours in cats given 10 to 20 milligrams of beta-carotene, while those fed 50 milligrams of beta-carotene showed peak concentrations at 24 hours. Concentrations declined (P <0.01) subsequently in all supplemented animals to undetectable levels (group of 10 and 20 milligrams) at 72 hours. However, beta-carotene in plasma was still detectable (over 100 μg / ml) at 72 hours in cats fed 50 milligrams of beta-carotene. The half-life of beta-carotene in plasma was 12 to 18 hours for cats fed 10 to 20 milligrams of beta-carotene but was approximately 24 hours in the group of cats fed 50 milligrams of beta-carotene.

Example 6 - Cats - Assimilation in blood with repeated doses In this example, cats (n = 6 / treatment) were dosed daily at 8 hours for 6 consecutive days with 0, 1, 2, 5 or 12 milligrams of beta-carotene . The blood was sampled once daily on day 0 (immediately before the first dose) and subsequently at 12 hours after each dosage (days 1 to 6).

This blood sampling time was chosen based on the results obtained in Example 5. The plasma was isolated and analyzed for the amounts of beta-carotene. Daily dosing of cats with beta-carotene for 6 days resulted in a dose-dependent increase (P <0.01) in circulating beta-carotene as shown in Figure 15. Cats fed 10 milligrams of beta-carotene showed the sharpest increase in daily changes in plasma beta-carotene. In this example, the concentration of beta-carotene in plasma at 12 hours after the first dose (192 ± 58 μg / L) and is similar to that observed in Example 5 (230 ± 26 μg / L; see Figure 14). Plasma beta-carotene concentrations after the last dose was generally 1.5 to 2 times higher than that observed after a dose. Based on these results from this example, plasma beta-carotene concentrations may continue to increase with continuous supplementation. The results of this example suggest that the domestic cat readily absorbs beta-carotene from the diet. This finding contradicts previous reports that indicate that domestic cats are unable to absorb oral beta-carotene. Cats do not possess the intestinal enzyme needed to convert beta-carotene to vitamin A. This has been suggested by some researchers as an explanation for the presence of high concentrations of beta-carotene in the general circulation. However, it is very unlikely that this physiological difference has a direct relationship with the cat's ability to absorb beta-carotene since pigs and rodents have very low concentrations of beta-carotene although they possess intestinal beta-carotene dissociation enzymes. Thus, the ability of cats to absorb beta-carotene from the diet as demonstrated in this example is more likely due to the presence of a mechanism of beta-carotene transport in the intestinal mucosa.

Example 7 - Cats - Assimilation by peripheral blood lymphocytes This example studied the assimilation of beta-carotene by lymphocytes in the blood in cats. The cats (n = 8 / treatment) were fed with 0, 5, 10 milligrams of beta-carotene daily for 14 days. Blood was sampled on days 7 and 14 with the aid of a blood collection kit Vacutainer (Becton Dickenson, Franklin Lakes, NJ) in sedated animals (10 milligrams of ketamine and 0.1 milligram of acepromazine / kilogram of body weight). Lymphocytes and neutrophils in the blood were separated by density gradient centrifugation. The cell numbers were counted. The lymphocytes were resuspended in phosphate buffered solution containing 10% sodium ascorbate as an anti-oxidant. An aliquot of the cell suspension was sonified for 30 seconds to break the cells. The lymphocyte homogenate was extracted for analysis by high performance liquid chromatography of beta-carotene. Unable to obtain adequate amounts of neutrophils; therefore, there are no data available to quantify the assimilation of beta-carotene by circulating neutrophils. A larger aliquot was used to prepare fractions of subcellular lymphocytes. The lymphocytes were broken by sonification for 20 seconds in 5 volumes of 0.25 M sucrose. Sodium ascorbate was added as an i-oxidant. The homogenate was centrifuged (600 x g for 10 minutes at 4 ° C) and the crude nuclear agglomerate was separated from the supernatant. The post-nuclear supernatant obtained above was centrifuged (17,300 x g for 20 minutes at 4 ° C) to separate the mitochondrial fraction. The post-mitochondrial supernatant was centrifuged (102,000 x g for 60 minutes at 4 ° C) to separate the microsomal fraction from the cytosolic. Each subcellular fraction was analyzed to determine the content of beta-carotene by high performance liquid chromatography. On day 0 (prior to supplementation with beta-carotene), beta-carotene concentrations in peripheral blood lymphocytes were undetectable in all cats (Figure 16). On day 7, blood lymphocytes showed significant assimilation (P <0.01) of beta-carotene, with no additional increase observed on day 14. Cats supplemented with 10 milligrams of beta-carotene had no higher accumulation of beta-carotene in lymphocytes. Therefore, the maximum assimilation of dietary beta-carotene occurred on day 7 and with an oral dose of 5 milligrams or less. After subcellular fractionation of peripheral blood lymphocytes, it was observed that beta-carotene accumulated in all cell fractions as illustrated in Figures 17 and 18. Beta-carotene concentrations were higher in the mitochondria (40 to 52%), intermediate in microsomes (20 to 35%) and cytosol (15 to 34%), and lower in the nucleus (1.5 to 6%) as illustrated in Figures 19 and 20. These relative profiles of beta -carotene in the subcellular fractions of lymphocytes were generally not influenced by the dose of the treatment or the duration of supplementation. However, the assimilation of beta-carotene in the nucleus is still significant in the lymphocytes of the cats supplemented with beta-carotene compared to the non-supplemented controls. Cats fed 10 milligrams of beta-carotene (Figure 20) did not have higher beta-carotene concentrations than those fed 5 milligrams (Figure 19). There was also no additional accumulation of beta-carotene in all fractions on day 14 (Figure 18) compared to that of day 7 (Figure 17). These results are generally in agreement with the data of the whole lymphocytes (Figure 16) showing that the maximum assimilation of beta-carotene occurred around day 7 of the feeding with beta-carotene and that the oral dose of beta-carotene of 5 milligrams it is adequate to produce maximum assimilation by lymphocytes. The maximum assimilation of beta-carotene in the cat lymphocytes in this example was also observed around day 7, and the mitochondria also contain the highest proportion of total beta-carotene. These results show that the Tabby domestic cat is able to absorb dietary beta-carotene. In addition, circulating beta-carotene is significantly absorbed by peripheral blood lymphocytes and distributed in different subcellular organelles, especially in the mitochondria. Beta-carotene in subcellular lymphocyte organelles is thought to (1) protect lymphocytes from oxygen free radical attack and / or (2) directly regulate nuclear events. In this way, feeding domestic cats with effective amounts of beta-carotene will result in the presence of beta-carotene at important cell sites in the body tissue which can result in increased immune function and improved health in these cats.

Although certain embodiments and representative details have been described for purposes of illustration of the invention, it will be apparent to those skilled in the art that various changes in the methods and apparatus described herein may be born without departing from the scope of the invention, which it is defined in the appended claims.

Claims (4)

1. A composition for increasing the immune response and improving overall health in a pet comprising a diet including a protein source, a source of fat, a source of dietary fiber, and from about 6 to about 315 milligrams of beta-carotene per kilogram of the diet.

2. A composition as claimed in claim 1 wherein the pet is a dog.

3. A composition as claimed in claim 1 wherein the pet is a cat.

4. A composition as claimed in claim 1 wherein the diet comprises about 30% crude protein, about 20% fat, and about 10% dietary fiber.