NO315184B1 - Use of a composition based on fish milk for the preparation of a preparation which increases immunoglobulin production - Google Patents

Description

Field of the invention

The present invention relates to a new use of a preparation

based on fresh or preserved fish milk which is useful in the preparation of a preparation that increases the immunoglobulin level in serum, especially the IgG level and thereby strengthens the immune system.

Background for the invention

The health and nutritional status of children in developing countries is far from satisfactory. Malnutrition in all its forms and manifestations remains a global challenge and a major public health problem in several countries in both industrialized and developing countries. The size and severity of its consequences are alarming and devastating for better living conditions, living standards and economies in Africa, Asia and Latin America. In the industrialized countries, malnutrition is primarily seen among young children, elderly people who live alone and individuals who are addicted to drugs and alcohol.

It is well known that malnutrition affects the immune system and causes immunodeficiency. Although considerable progress has been achieved in the knowledge of the immune system and its function in recent years, little is still known about the interaction and mechanism between nutrition and the immune system. Development and understanding of nutritional needs and the role of nutrition in immune function is essential in the prevention and treatment of nutrition-related immune compromise (Krenitsky, 1996, Nutrition and the immune system, AACN-Clin-Issues, 7(3): 359-69). Consequently, there is a significant need for new nutritional supplements to combat malnutrition and strength

the immune system.

It is believed that a good response to vaccines and other drugs depends on a functioning immune system. In addition to malnutrition, there are also several conditions and diseases where a strong immune system is very important in terms of reducing the development and increasing the chances of healing the condition or disease. Illustrative, but not limiting, conditions or diseases such as AIDS, various types of benign and/or malignant tumors, bacterial or viral infections, transplant rejection can be mentioned. Accordingly, there is a need for dietary supplements and methods to improve the response to vaccines and strengthen the immune system in individuals suffering from or at risk of developing conditions or diseases as mentioned hereinabove.

There is also a need for nutritional supplements to strengthen the immune system in individuals who have a need for a strong immune system as a result of e.g. extreme stress, such as athletes.

During the last few years, many preparations based on fish milk have been reported, see e.g. respectively European patent no. EP-A1-729710, Russian patent no. RU 2091073, US patent no. 5 552 889. Until today, however, nothing is known about how the immunoglobulin level in serum can be increased, because thereby e.g. to provide improved nutritional status and response to vaccines and other medications.

Neuman et al., (1972) examined the quantitative levels of immunoglobulins in several malnourished, moderately malnourished and control children from Ghana compared to age-matched US standards. The levels of all Igs were higher in the severely malnourished children than in the moderately malnourished and control children. Compared to age-matched US standards, even the control children from Ghana had higher levels of all Igs. Similar studies

reported in the literature yielded comparable higher levels of

. immunoglobulins in malnourished children than in normal children (Bell et al., 1976, "Serum and small intestinal immunoglobulin levels in undernourished children, Am. J. Clin, Nutrl., 29: 392-97 and Suskind et al., 1976, " Immunoglobulins and antibody response in children with protein-calorie-manutrition", Am. J. Clin. Nutrl. 29: 836-41).

Revillard and Cozon, 1990, "Experimental models and mechanisms of immune defenses of nutritional origin", Food Additives and Contaminants, Vol. 7, Suppl No. 1, pages S82-S86 report that specific humoral immunity is altered in several ways by malnutrition and that serum levels are IgM, IgG and IgA, and immunoglobulin synthesis is usually normal or increased. Revillard and Cozon, 1990, also indicate that the catabolic rate of gamma globulins is increased through infections. However, the increased catabolic rate is compensated for by increased synthesis. Furthermore, immunoglobulin levels return to normal after nutritional rehabilitation and antimicrobial therapy.

In a series of studies conducted on children from Ghana, it was also shown that supplementation of local cereal-based diets with fish powder resulted in an increase in serum IgG, IgM and IgA concentrations (Tetteh-Tuwor, 2000; Adu-Affawuah, 1998). .

Furthermore, Navarro et al (1999), BioFactors, vol. 10, pages 67-76 examined the effect of nucleotide supplements in a milk composition on the level of lymphocytes and the level of IgG, IgM and IgA in prematurely born children. The children were given a supplement comprising ribonucleotides (CMP, AMP, UMP, GMP and IMP) and an attempt was made to mimic the amount of acid-soluble nucleotides found in human breast milk. The results from this investigation showed that ribonucleotide supplementation did not produce any significant difference in the IgG level.

Also Jyonouchi (1994) J. Nutrition, vol. 124, pages 138-143 and Martinez-Augustin et al. (1997), Biol. Neon., vol. 71, pages 215-223 have sought to investigate the importance of ribonucleotides for the immune system in children born prematurely.

The present inventors have now surprisingly found that dietary supplements comprising a preparation based on fish milk used according to the present invention provide increased serum levels of immunoglobulins. Furthermore, the present inventors have found that said dietary supplements provide higher serum levels of immunoglobulin G (IgG).

Accordingly, the addition of a preparation comprising a ■ composition based on fish milk used according to the present invention to the diet of malnourished people provides a better immune defense shown by stronger immune response and higher serum levels of immunoglobulins such as IgG. Furthermore, the addition of said preparation according to the present invention to the diet of malnourished people provides an improved response to vaccines and other pharmaceutical preparations.

The increase in the immunoglobulin level after regular consumption of a protein-rich diet supplemented with a preparation based on fish milk used according to the present invention is surprising since

the immunoglobulin level decreased after consumption of the same diet without such supplementation, and considering that this last result is what the expert in the field would have expected with reference to the previously published literature (Revillard and Cozon (1990)).

Definitions

The term "composition based on fish milk" as used herein means a composition comprising substantially pure DNA or fragments thereof and physiologically and pharmaceutically acceptable salts thereof.

The term "DNA powder" used herein below means a composition based on fish milk as defined above.

Detailed description of the invention

The present invention provides in one embodiment a new use of a composition based on fish milk for the preparation of a preparation for increasing immunoglobulin production in non-premature mammals.

In a preferred embodiment, the invention comprises the use of a composition based on fish milk for the production of a preparation to increase IgG production in non-premature mammals.

In another embodiment according to the present invention, a new application is provided in which the animal's general state of health/nutritional state is improved.

In yet another embodiment according to the present invention, a new application is provided in which the animal's weight and development are regulated.

In yet another embodiment according to the present invention, a new application is provided in which the animals' response to vaccines or other pharmaceutical preparations is increased.

In yet another embodiment according to the present invention, a new application is provided in which said preparation preferably contains mainly pure DNA or fragments thereof. Furthermore, the preparation according to the present invention preferably comprises approximately 0.5% DNA sodium salt. It is further preferred that said DNA sodium salt is substantially pure and/or contains at least no more than 2% protein.

In yet another embodiment according to the present invention, a new application is provided in which the preparation is preferably a pharmaceutical preparation, respectively a dietary supplement, a food additive or a pre-additive. In yet another embodiment of the present invention, a new application is provided in which the mammal is a human.

Examples

1. Materials and methods

1. 1 DNA sources

The DNA natural serum salt is obtained from naturally occurring sources such as spleen or milk from fish. DNA can be extracted from fish milk by means of isolation procedures known in the art which gives a substantially pure product containing DNA sodium salt with less than 2% protein. The product is a white or cream colored powder. Purity measured by spectrometry gives OD260/OD28O= min. 1.7. The DNA sodium salt has a molecular weight distribution of 4 x 10<4>- 1 x 10<6>Daltons and a hyperchromicity of OD2«>(denatured)/OD26o(native) = max. 1.05.

In the study described below, 0.5% DNA sodium salt produced by Bjørge Biomarin A/S, Norway was used to prepare the DNA dietary supplement according to the present application according to the invention.

Study design

The study was a simple blind randomized population-based study. The study site was Nima, a surrounding urban slum near Accra, the capital of Ghana. The study subjects were children aged 3-5 years selected by taking weight and height measurements. Bam who participated in the study had weight-for-height z-scores below or equal to -1SD. The eligible children were divided into two groups using random stratification using a random number table. Stratification was performed for gender and age. The two different groups were based on the two different diets used indicated in the table below. Group 1 consisted of 74 bam and group 2 of 70 bam. Bjørge Biomarin A/S, Norway produced the fish protein and Na-DNA powders used according to the present invention.

Equal portions of tomato, onion, pepper and spices were fried in equal portions

of vegetable oil to prepare the sauce for the two different groups. The only difference was that the first group only had fish powder while group 2 had DNA in addition to fish powder. The diets were prepared for i

at least providing the recommended daily allowance (RDA) of protein for the baby so that the supplemented food would provide the daily requirement if they ate no protein at all during the day. Bama was given food for lunch ad libitum. The feeding lasted for 7 weeks.

The protein content of the fish powder (group 1) and the mixture of fish and Na-DNA powders (group 2) was 88.96% and 84.68% respectively on a dry matter basis. The protein content of the diet of group 1 was more (18.06%)

than in group 2 (17.39) because the fish powder for group 2 had lower

protein content as a result of the addition of 5% DNA. DNA in the diet contributes to the nitrogen content in general but not the protein content of the diet.

There were no statistically significant differences in the intake of the diets in the two groups.

1. 3 Immunological analysis of blood samples

Venous blood up to 10 ml was collected from the right or left arm of each child by five well-trained and qualified blood samplers and one physician from the University Hospital, "Legon and Nugouchi Memorial Research Institute", Legon, Ghana. The blood samples were immediately centrifuged and the harvested serum samples were transported under ice packs in an ice chest to "the Department of Nutrition and Food Science", University of Ghana, Legon, where they were frozen. At the end of the study, all serum samples were sent to Broegelman's research laboratory for immunology and

microbiology, University of Bergen, Norway, under, dry ice in an ice chest and stored at -20°C until analyzed.

Approximately 300 serum samples were sent to the laboratory for analysis. The samples were analyzed for various immunoglobulins such as IgG, IgA, IgM and IgE. In all cases, a method well known to the person skilled in the art, Enzyme Linked Immuno-Sorbent Assay, ELISA was used.

Using a computer program (Softmax for Macintosh, version 2.3, "Molecular Devices Corporation", Sunnyvale, CA, USA) it was possible to plot a standard curve in four parameter mode and optical density results were converted to immunoglobulin values. In this way, the serum immunoglobulins were expressed in mg/ml. All samples were analyzed in duplicate for each immunoglobulin. Samples with coefficient of variation between duplicates of 15 and above were repeated to ensure accurate results.

2. Results

2. 1 Effect on serum levels of immunoglobulins

IgG, IgM and IgA levels in serum samples from children in said study at baseline were within the range reported for normal Ghanaian infants (Adu-Affawuah, 1998 and Neuman et al., 1975, "Immunologic response in malnourished children" , Am. J. Clin. Nutr., 28: 89-104). Since Neuman's study involved children up to 6 years of age, which includes the age range used in the present study, the results of the serum levels of the lgs mentioned above in the examined children at baseline can be said to be reliable. This is without regard to the fact that the ELISA method used, like any other analytical laboratory method, was believed to have its own or associated errors, but since great care was exercised throughout the analysis, any such errors were reduced to almost a minimum. At baseline, the mean serum IgG concentration of children in the group receiving only fish powder was found to be 12.087/ml + 2.68, and 10.91 mg/ml + 4.23 for the children receiving fish and DNA powder. However, a significant Student's t-test showed that the difference was not statistically significant (p>0.05). The mean serum concentrations of IgM and IgA were almost comparable for both groups.

The general health of the ewes in both groups improved dramatically throughout the period of supplementary feeding. The number of children who were reported sick to the clinics decreased as the number of weeks of supplementation increased. There was a drastic decrease in the number of children with skin rashes and runny noses at the end of the supplementary feeding. There is therefore no doubt that supplementing rice with both fish powder and the mixture of fish and DNA powder improved the health status of the children.

It is important to note that since the serum concentrations of the various immunoglobulins determined by the ELISA procedure at baseline were found to be reliable, it can be said with certainty that the results after supplementation are similarly reliable and therefore can be used to draw conclusions. Since the bama in the two groups were also not significantly different (statistically) with regard to serum immunoglobulin levels at baseline, any difference in their levels of immunoglobulins after supplementation could be caused by the difference in the supplemented diets.

As mentioned above previously, it has been shown in series of studies carried out with bam from Ghana that supplementation of local cereal-based diets with fish powder resulted in a decrease in serum IgG, IgM and IgA concentrations (Tetteh-Tuwor, 2000Adu-Affawuah, 1998).

In the study reported here, supplementation with fish powder alone was found to result in a significant decrease in IgG concentration and this is in accordance with the results of previous studies mentioned above.

The effect of supplementation on the serum level of immunoglobulins in group 1 is summarized in table 2 below.

In the group that only received fish powder (group 1) there was a significant decrease (p<0.05) on average in the IgG level, corresponding to 14.3% in

.compared to the baseline level, a significant increase in the mean IgM level (p=0.03) and a non-significant increase in the mean

IgA and IgE levels (p>0.05).

In group 2, a significant increase in the serum IgG concentration was demonstrated. This corresponds to 25% compared to the baseline level. This significant increase in the IgG concentration and the increase in the levels of the other Igs in the serum samples of the children (although they were not statistically significant) fed the diet supplemented with a preparation comprising mainly DNA from fish milk can undoubtedly be said to be due to the presence of DNA. The exact mechanism that explains this phenomenon is not yet known.

The effect of the supplementation on the serum levels of immunoglobulins i

group 2 is summarized in table 3 below.

For all the immunoglobulins analyzed in this study, there was a higher increase in the levels in the serum samples from children fed the DNA powder supplemented diet (group 2) than those fed the non-DNA powder diet (group 1). A more dramatic observed difference, which was highly statistically significant,

was the decrease in the IgG level in group 1 while there was an increase of the lg in group 2.

The significant difference between IgG levels in the two groups after supplementation must be caused by the additional supplementation of the diet for group 2 including DNA from fish milk.

In the group that received DNA powder (group 2) there was a significant increase in the IgG level (p>0.05) and increases in IgM, IgA and IgE levels that were not statistically significant.

While there was a decrease in the IgG concentration in serum samples from children in group 1, there was an increase in group 2. The non-significant increase of IgA, IgM and IgE may be caused by low sample material. However, the increase of IgA concentration in children in group 2 had a p-value of 0.0577. This means that one can say with 94% certainty that the increase in IgA concentration in the DNA group was significant.

Table 4 shows average changes in serum

immunoglobulin concentrations in the children in the two groups after supplemented feeding. It is important to note that while there was a significant decrease in the IgG level in the non-DNA group there was a significant increase in the level of the same immunoglobulin in the DNA group.

A Student's t-test of the differences between the two groups showed a very high significant p-value that was below 0.01. Since there was no difference between the two groups at baseline with regard to IgG, this means that the difference after supplemented feeding is caused by the differences in the diets. The increase in the concentrations of the other lg's (IgM, IgA and IgE) after supplemented feeding was still greater in group 2 than in group 1, although the differences were not statistically significant in all three cases (p>0.05). The increased IgA level in the children in group 2 had a p-value of 0.0577, corresponding to a confidence level of 94%.

However, it is worth mentioning that the addition of the DNA powder to the diet caused an effect in the immunoglobulin levels and the differences between the two groups could not be statistically detected in the case of the other three immunoglobulins probably due to low sample material.

Consequently, feeding bam with a resulted

fish protein powder supplemented diet for 7 weeks in a significant decrease in their serum IgG concentration, while feeding bam a fish protein powder plus a supplemented diet comprising DNA powder isolated in fish milk resulted in a significant increase in their serum IgG level;

the latter indicates that DNA supplementation improves the body's ability to synthesize antibodies and indicates an improved defense against infections.

The results presented herein support that the present application of a preparation based on fresh or preserved fish milk according to the invention is useful for increasing the immunoglobulin level in serum according to it. present invention and consequently strengthen the immune system and the response to vaccines and/or other pharmaceutical preparations. In particular, these results confirm the applicability of the present invention in increasing the serum IgG level.

Claims (14)

1. Use of a composition based on fresh or preserved fish milk for the production of a preparation for increasing immunoglobulin production in non-premature mammals. 2. Use of a composition based on fresh or preserved fish milk for the production of a preparation for increasing IgG production in a mammal. 3. Use according to claims 1 and 2, whereby the animal's general state of health/nutritional state is improved. 4. Use according to claims 1 and 2, whereby the animal's growth and development are regulated. 5. Use according to claims 1 and 2, whereby the response to vaccines or other pharmaceutical agents is increased. 6. Use according to claims 1-5, wherein said composition preferably contains mainly pure DNA or fragments thereof. 7. Use according to claims 1-6, wherein said composition comprises approximately 0.5% DNA sodium salt. 8. Use according to claim 7, where said DNA sodium salt is essentially pure. 9. Use according to claim 8, wherein said DNA sodium salt does not contain more than approximately 2% protein. 10. Use according to claims 1-10, where the preparation is a pharmaceutical preparation. 11. Use according to claims 1-11, where the preparation is a dietary supplement. 12. Use according to claims 1-11, where the preparation is a food preservative. 13. Use according to claims 1-12, where the preparation is an additive. 14. Use according to any of the preceding claims where the animal is a human.