one feature of the instant invention resides in the novel analytical technique used to identify and quantify the nucleotide equivalents useful in this invention . analysis of certain starting materials , especially the protein , will determine the actual amount of nucleotides to be added . this analysis of the raw materials of the formula is critical to determine what nucleotides , if any , are contained in the starting materials . the analytical method is also critical to determine the proper ratios of the nucleotides to each other . the analytical method according to this invention will determine nucleotide equivalent levels in complex food matrices . the method in general exploits the enzymatic digestion of various forms of ribonucleic acids to the simple monomeric ribonucleosides and the ability of the cis - diol groups of ribonucleosides to form a ph dependent covalent complex with boronic acid . boronate derivatized polyacryl amide gel is used to very selectively prefractionate ribonucleoside directly from complex matrices . the isolated ribonucleosides are subsequently separated via low ph reverse phase / ion - pairing hplc using octanesulfonate as the ion - pairing agent . ribonucleosides are detected via uv absorbance , and the corresponding levels are determined by comparison to external standards . the method can be used to quantitate inherent levels of ribonucleoside in foods . because of the selective prefractionation , the method is essentially matrix independent . it should be understood that the novel analytical technique of this invention will not detect nucleosides from dna or any form of nucleic acid that does not contain the cis - diol groups of ribose . it has been used to determine ribonucleic acid types and levels in infant and medical nutritional products , human milk , protein commodities , and clinical and commercial animal chows . the following is an example of the analytical technique of this invention that can be used to determine the presence and ratios of the nucleotide equivalents . to a 10 ml reacti - therm vial with stir bar was placed 2 . 0 ml of similac ® with iron ( a non - fat milk protein infant formula produced by the ross products division of abbott laboratories , ready to feed form , 676 kcal per liter ) 3 . 0 ml of 50 mm sodium acetate at ph 5 . 1 , 50 μl of 10 mm zinc sulfate and 50 μl of the enzyme preparation nuclease p1 ( sigma chemical ). the enzyme preparation was 5 mg of dry enzyme powder , as received from sigma , and 4 ml of 50 μm sodium acetate at ph 5 . 1 . the mixture was heated to 37 ° c . and stirred for 16 hours . this reaction converted the polymeric rna to monomeric 5 &# 39 ; mono - nucleotides . to the same reaction vial was added 50 μl of 30 % ammonium hydroxide , 1 ml of 0 . 5m ammonium acetate ( ph 8 . 75 ), 50 μl of 1 . 0m magnesium chloride , 50 μl of bacterial alkaline phosphatase ( bap ) ( sigma chemical as a suspension ) and 50 μl of a nucleotide pyrophosphatase enzyme preparation ( sigma chemical ). the pyrophosphatase enzyme preparation was 5 mg of dry powder in 4 ml of 0 . 5m ammonium acetate buffer . the mixture was incubated at 37 ° c . for three hours . this reaction converted the nucleoside containing adducts and the nucleotides to the ribonucleosides . the reaction mixture was transferred to a 50 ml volumetric flask using 25 ml of 0 . 5m sodium phosphate , ph 10 . 5 . water was added to a final volume of 50 ml . the sample mixture was shaken and may be filtered to remove insoluble protein . 5 grams of dried affi - gel - 601 , boronate derivatized ( from bio - rad ) was hydrated in 50 ml of 100 mm phosphate buffer at ph 6 . 5 . to a 10 ml open column was added the hydrated affi - gel - 601 to obtain a packed volume of about 1 ml . the gel was converted to the basic form by washing with 5 ml aliquots of 0 . 25m sodium phosphate buffer , ph 10 . 5 , until the gel no longer swelled . the gel was now about 2 ml in volume . the gel was resuspended in the buffer to maintain adequate flow . to the prepared gel was added 10 ml of the sample that was previously treated with the enzymes and the eluant was discarded . at this point , the nucleosides are covalently attached , through the cis - diol groups , to the boronic acid gel . the gel was washed with 20 ml of 0 . 25m sodium phosphate , ph 10 . 5 and the eluant was discarded . the nucleosides were eluted and collected in a 10 ml volumetric flask by adding 2 ml of 1 . 0m phosphoric acid to the column followed by 5 ml of 0 . 1m phosphoric acid . at this point the nucleosides have been isolated from the sample and are now ready to be characterized . the volumetric flask was brought to a final volume of 10 ml with water . the sample was then placed on a hplc for separation and quantification of nucleosides using external standards . the nucleosides were separated via low ph , reverse phase , ion pairing chromatography using an acetonitrile gradient . the nucleosides were detected by u . v . absorbance at 260 nm and 280 nm . nucleosides were quantified by reference to external standards and the results were converted to the corresponding monophosphate nucleotide value by multiplying the nucleoside value by the molecular weight ratio of the monophosphate nucleotide over the nucleoside . the results were expressed as mg / l , of mononucleotide . ______________________________________nucleotides in similac ® with iron______________________________________ uridine - 3 - 5 guanosine - trace adenosine - trace inosine - ( trace , & lt ; 0 . 5 ppm ) cytidine - 1 - 3______________________________________ it should be noted that some samples have been found to be active with respect to nucleic acid degradation . of particular concern is the enzymatic conversion of amp to imp . heat inactivation has proven to be effective in rendering the sample inactive . the procedure for heat inactivation is to heat the sample to over 100 ° c . for at least 15 minutes . after the sample has cooled , buffer , enzyme , and zinc are added and the first hydrolysis is carried out . this analytical technique was used on raw materials to determine base line nucleotide content and on final clinical product to confirm the presence and concentration of the four nucleotides used in the invention . on a commercial scale , a control and an experimental formula according to the invention were prepared having the compositions set forth in table ii . the two formula are as close as possible to being identical except for the nucleotide components . table ii______________________________________composition of study feedings per liter con nucnutrient ( control ) ( formula of the invention ) ______________________________________protein , g 14 . 0 14 . 4fat , g 36 . 5 38 . 3carbohydrate , g 77 . 1 75 . 5calcium , mg 544 . 4 532 . 5phosphorus , mg 295 . 0 316 . 2magnesium , mg 73 . 5 77 . 7sodium , mg 170 . 1 179 . 2potassium , mg 931 948 . 6chloride , mg 487 . 7 493 . 2iron , mg 14 . 0 14 . 0zinc , mg 5 . 1 5 . 1copper , mcg 608 608iodine , mcg 61 61manganese , mcg 34 34vitamin a , iu 2930 2970vitamin d , iu 405 405vitamin e , iu 24 . 6 24 . 8vitamin k , mcg 54 54vitamin c , mg 170 172β - carotene mcg 450 450selenium , mcg 23 23thiamin , mcg 1350 1360riboflavin , mcg 1014 1014pyridoxine , mcg 480 480vitamin b . sub . 12 , mcg 1 . 7 1 . 7niacin , mcg 7095 7095folic acid , mcg 101 101pantothenic acid , mcg 3041 3041biotin , mcg 30 30taurine , mg 45 45choline , mg 108 108inositol , mg 32 32energy , kcal 676 676cmp , mg 2 . 72 * 31 . 2ump , mg 4 . 19 * 17 . 7amp , mg 0 . 57 * 9 . 8gmp , mg 0 . 45 * 14 . 4______________________________________ * -- inherent levels from raw materials in this example , a 7711 kg batch of the formula according to the invention was prepared ( nuc ). the control formula ( con ) was prepared in a similar fashion except the addition of the nucleotides was omitted . the list of ingredients and amounts are found in table iii . table iii______________________________________ingredients and amounts for nuc formulaingredient amount______________________________________high oleic safflower oil 120 . 2 kgcoconut oil 85 . 7 kgsoy oil 80 . 3 kglecithin 2 . 92 kgmono - and diglyceride 2 . 92 kgoil soluble vit . premix 0 . 365 kgβcarotene 0 . 0137 kgcarrageenan 1 . 43 kgwhey protein concentrate 61 . 2 kglactose 476 . 3 kgpotassium citrate 4 . 6 kgmagnesium chloride 0 . 735 kglow heat condensed 821 kgskim milkcalcium carbonate 3 . 36 kgferrous sulfate 0 . 450 kgwater soluble vitamin 1 . 11 kgpremix trace minerals / taurinecholine chloride 0 . 600 kgadenosine 5 &# 39 ; monophosphate 0 . 113 kgguanosine 5 &# 39 ; monophosphate - na2 0 . 197 kgcytidine 5 &# 39 ; monophosphate 0 . 259 kguridine 5 &# 39 ; monophosphate - na2 0 . 216 kgascorbic acid 1 . 78 kg45 % koh 2 . 36 kgtotal yield 7711 kg______________________________________ the first step is the preparation of the oil blend . to an appropriately sized blend tank with agitation and heating soy oil , coconut oil and high oleic safflower oil were added . the mixture was heated to 73 . 8 °- 79 . 4 ° c . the lecithin and mono - and diglycerides ( myverol 18 - 06 ) were added to the blend tank with agitation . the oil soluble vitamin premix was added with agitation . the premix container was rinsed with the oil blend and transferred back to the blend tank to ensure complete delivery of the vitamin premix . the beta - carotene was added to the oil blend and the mixture agitated until the components were well dispersed . the beta - carotene container was rinsed with the oil blend and the contents returned to the blend tank to ensure complete delivery of the beta - carotene solution . lastly , the carrageenan was added to the oil blend and the mixture was agitated and held at 54 . 4 °- 60 ° c . until used . the carbohydrate , mineral and csm ( condensed skim milk ) protein slurry was prepared next . to water heated to 68 . 3 °- 73 . 8 ° c . the lactose was added and the mixture agitated until the lactose was well dissolved . potassium citrate was then added followed by potassium chloride , sodium chloride and magnesium chloride . the condensed skim milk ( csm ) was then added . tri - calcium phosphate was added , the mixture agitated and held at 54 . 5 °- 60 ° c . until used . the protein - in - water ( piw ) slurry was then prepared . the whey protein concentrate was added to water at 54 . 5 °- 60 ° c . under mild agitation . the piw slurry was held under mild agitation until needed . also contemplated in this invention is the use of protein - in - fat ( pif ) slurries , wherein an appropriate amount of protein is admixed with all or a portion of the oil component . the piw slurry was then added to the prepared oil blend . the required amount of the carbohydrate , mineral and csm slurry was then added to the oil blend . the ph of the mixture was then determined and if below specification it was adjusted using koh to a ph of 6 . 75 to 6 . 85 . the mixture was then held at 54 . 4 °- 60 ° c . under agitation for at least 15 minutes . the mixture was then heated to 68 . 3 °- 73 . 8 ° c . and deaerated under vacuum . the mixture was then emulsified through a single stage homogenizer at 6 . 21 to 7 . 58 mpa . after emulsification , the mixture was heated to 120 °- 122 ° c . for 10 seconds and then 149 °- 150 ° c . for 5 seconds . the mixture was then passed through a flash cooler to reduce the temperature to 120 °- 122 ° c . and then through a plate cooler to reduce the temperature to 71 . 1 °- 79 . 4 ° c . the mixture was then passed through a two stage homogenizer at 26 . 89 to 28 . 27 mpa and 2 . 76 to 4 . 14 mpa . the mixture was held at 73 . 9 ° to 83 . 2 ° c . for 16 seconds and then cooled to 1 . 1 ° to 6 . 7 ° c . at this point , samples are taken for microbiological and analytical testing . the mixture was held under agitation . a calcium carbonate solution may be prepared for use in adjusting the calcium level of the mixture if outside of specification . a vitamin stock solution was prepared . to water heated to 37 . 8 ° to 65 . 6 ° c . was added potassium citrate and ferrous sulfate . the vitamin premix was then added and the mixture agitated . the choline chloride was added and then the required amount of this vitamin mixture was added to the batch . the nucleotide solution was then prepared . the following nucleotides were added to water with mild agitation in the following order : amp , gmp , cmp , ump . agitation was continued for about 10 minutes to dissolve the nucleotides . the nucleotide solution was then added to the batch . this is one critical aspect of the invention . it is extremely important that the nucleotides be added after the homogenizations and heat treatments . numerous experiments have been conducted that have shown the addition of the nucleotides at any other point will result in degradation of the nucleotides and thereby change the specific levels and ratios as claimed . it is believed that amp is converted to imp through the presence of adenosine deaminase in the raw materials , especially the protein components . lastly , an ascorbic acid solution was prepared and added slowly to the batch with agitation for at least 10 minutes . final dilution with water to meet specified levels of solids and caloric density was completed . the batch was then packaged in 32 ounce metal cans and sterilized using conventional technology . the purpose of the clinical investigation was to determine the effect of a nucleotide - fortified formula according to the present invention on the development of the neonatal immune system in infants as measured by the antibody response to childhood vaccines . this was a 12 - month , randomized , controlled , blinded , multi - site trial of term infants . infants enrolled into the study received human milk ( hm ) or one of two clinically labelled formulas : 1 ) control formula ( con ) or 2 ) con formula supplemented with nucleotides ( nuc ). the analyzed composition of each formula is set forth in table ii . a total of 311 infants completed the study ( 107 con , 101 nuc , 103 hm ). infants followed the immunization schedule recommended by the american academy of pediatrics with single lots of hib titer ® hemophilus influenzae type b conjugate vaccine ( diphtheria crm 197 and tetanus protein conjugate sold by lederle , inc .) and diphtheria and tetanus toxoids and pertussis vaccine adsorbed , sold by lederle , inc . infants were full - term with a gestational age of 38 - 42 weeks , at or above the 5th percentile for weight , length , and head circumference and were enrolled between 2 and 10 days of age . all subjects were healthy with no indication of systemic disease and did not receive any medications , mineral , or vitamin supplements . the primary outcome variable investigated was vaccine response at 6 , 7 , and 12 months of age . also investigated were differential white blood cell count , lymphocyte subset analysis , nk activity , and lymphoblast transformation in response to specific and non - specific stimuli at 2 , 6 , 7 , and 12 months of age . secondary outcome variables included intake , anthropometry , and indices of tolerance ( stool characteristics and incidence of spit - up ). also investigated was the antioxidant status of infants fed the formula according to the present invention which contained the novel antioxidant system of : 10 - 30 iu of r , r , r , α - tocopherol per liter of formula , 375 - 575 μg of β - carotene per liter of formula and 14 - 32 mcg of selenium per liter of formula . during infancy , as in adulthood , the body has a number of antioxidant systems to protect against injury from free radicals , the products of oxidation . the antioxidant system of this invention was clinically proven to promote the antioxidant status of the infant greater than currently available infant formula . this improved antioxidant status was demonstrated as a function of increased levels of plasma vitamin e , reduced levels of plasma lipid peroxides , and increased free radical trapping capacity . at 2 , 4 , and 6 months of age dpt and hib vaccines were administered . blood samples were obtained by venipuncture at 2 , 6 , 7 , and 12 months of age . when vaccines were administered the blood sample was obtained before the inoculation . parents of the infants agreed to feed the infant only study formula until 4 to 6 months of age when table foods were added to supplement the study formula . the hm fed group were exclusively breast fed up to 2 months of age and a mixture of hm and similac ® with iron ( ross products division of abbott laboratories ) after 2 months , if necessary . weight , length and head circumference were measured at 21 days of age and at 2 , 4 , 6 , 7 , and 12 months of age . three - day records of formula intake , frequency of spit - up and vomiting and the frequency , color and consistency of stools were used to assess tolerance . blood samples ( 2 ml ) were drawn at 2 months of age and transferred directly into a heparin - containing tube , and gently inverted . at 4 , 6 , 7 , and 12 months of age 5 ml of blood was collected . two and a half ml were transferred to heparinized tubes and 2 . 5 ml to a plain tube without an anti - coagulant . tubes of blood were carefully packed in thermally insulated containers and shipped to the laboratory for analysis . radial immunodiffusion assays were performed using standard kits purchased from the binding site , inc ( 5889 oberlin drive , suite 101 , san diego , calif . 92121 ) for the measurement of serum or plasma igg and iga . the detection of tetanus and diphtheria igg was accomplished as follows . tetanus toxoid antigen ( connaught ) was diluted in 0 . 05m carbonate buffer ( ph 9 . 6 ) to 2 μg / ml , added to the wells of microtiter plates at 200 μl per well , and incubated at room temperature for 1 hour . diphtheria toxoid antigen ( connaught ) was diluted in the same manner to 15 μg / ml . the coated plates were washed three times in pbs containing 0 . 05 % chicken egg albumin and 0 . 1 % tween 20 . samples and positive control tetanus and diphtheria toxoid immune globulin , were diluted in pbs / albumin / tween , added to triplicate wells at 200 μl / well , and incubated at room temperature for 1 hour . pbs alone was also added to triplicate wells to provide a blank . plates were again washed three times in pbs / albumin / tween . affinity purified horseradish peroxidase - conjugated goat anti - human igg ( the binding site , inc ) was diluted in pbs / albumin / tween , added to the microtiter plates , and again incubated at room temperature for 1 hour . tetramethylbenzidine ( tmb ) substrate ( kirkegaard and perry laboratories ) was added to all wells at 100 μl / well , and incubated at room temperature for 10 minutes . the substrate reaction was stopped by adding 100 μl of 1m phosphoric acid per well . optical density of each well was measured using a wavelength of 450 nm . sample units were calculated based on the tetanus and diphtheria toxoid immune globulin standards . see sedgurch and bolton ; j clin microbiol . 1983 ; 18 : 104 - 109 . serum igg directed against haemophilus influenzae type b capsular polysaccharide ( hib ) antigen was detected using a modified version of the procedure described by anthony et al ; j clin microbiol 1982 ; 16 : 350 - 354 . the modifications are described in granoff , et al ; j infect dis 1986 ; 154 : 257 - 264 . concentrations of total serum antibody to the hib antigen were measured by a radioactive antigen - binding assay ( hib farr ) using the procedure described by granoff et al ; j infect dis 1986 ; 154 : 257 - 264 . the hib antigen was purified and labeled with iodine . a reference serum pool from the us bureau of biologics ( rockville , md .) was used to standardize the assay . the smallest amount of immunoglobulin detectable was 0 . 025 μg / ml serum , as determined with this reference pool . natural killer cell ( nk cell ) activity was measured using histopaque - purified peripheral blood lymphocytes . the cytotoxicity of the nk cells was measured using procedure described by wierda et al ., j immunol . methods 1989 ; 122 : 15 - 24 . the immunological variables were analyzed in two different ways . for the variables relating directly to vaccine response ( hib farr , hib igg , tetanus , diphtheria , total igg and iga ) the variables were transformed by taking logarithm base 10 and doing analysis of variences ( anovas ). the procedure is commonly used in the vaccine literature . anthropometric data were analyzed for each gender separately . analysis of varience ( anova ) was done at birth , initial visit , 2 , 4 , 6 , 7 , and 12 months of age for weight , length and head circumference . weight gain , length gain and head circumference gain were also analyzed by anovas . intake data were ranked and analyzed by anovas ( number of feedings , volume intake , percent of feedings with spit - up , vomits or both ). stool variables were ranked and analyzed with anovas ( number of stools , mean rank consistency and percent of stools with gas or unusual odor ). substantial amounts of data were collected on each of the 311 infants enrolled in this clinical investigation . disclosure of all this information is outside the scope of this document , however , the following is a summary of the information that supports the novel and unobvious features of the instant invention . vaccine antibody response data was statistically analyzed by two methods . table iv shows the medians of the variables in the original units . the anova was performed on medians of ranked data . table v shows geometric means . for this analysis , the variables were transformed by taking logarithm base 10 , and the anova compared the mean of the logs . the mean of the logs converted back to the original units is the geometric mean . use of geometric means is commonly used in the vaccine literature . at 7 months of age , infants in the nuc group had a higher antibody ( p & lt ; 0 . 05 ) response than the con or hm group to hib vaccine ( geometric mean of 7 . 24 vs 4 . 05 or 4 . 21 μg ig / ml , respectively by the hib farr assay ). the nuc group had a higher response than the hm group to diphtheria toxoid vaccine ( geometric means of 1 . 77 vs 1 . 29 u diphtheria toxoid specific igg / ml , respectively ). the enhanced antibody response to hib vaccine persisted through 12 months of age as seen in table v . there were no differences in nk activity at any time , and the differential white count , lymphocyte subsets , and lymphoblast transformation was very similar among all groups . the primary differences were at 12 months of age , when infants fed hm had more white blood cells , monocytes , lymphocytes , cd3 , and cd19 cells than con ( p & lt ; 0 . 05 ). the nuc group was intermediate and not statistically different . infants fed hm had greater numbers of nk cells ( cd3 -, cd16 +, cd56 +) than formula - fed ( con or nuc ) infants ( p & lt ; 0 . 05 ). the nuc group had a higher percent cd4 cells than hm - fed infants ( p & lt ; 0 . 05 ) throughout the study . growth of infants was similar in all three groups . tolerance and intake was similar for the two formula groups . the similarity in growth and tolerance among all infants demonstrated that both formulas are acceptable . likewise , the similarity in measures of immune system components among infants fed formulas or hm demonstrates that all feedings promote development of the immune system within normal ranges , however , for the first time an immune enhancement as measured by vaccine response to h . influenzae b and diphtheria toxoid is reported for infants consuming infant formula ( nuc ). the consistently enhanced vaccine response of infants fed nuc vs con suggests that nucleotides play an important function in immunological development of the infant . vaccine response data are provided in table iv as reported from the assays and table v as geometric means . the antibody response to the hib vaccine was measured as hib farr ( μg ig / ml ). nuc - fed infants had significantly higher levels of hib farr antibody than infants fed hm at 6 months ( 0 . 43 vs 0 . 30 , p & lt ; 0 . 05 ) higher than infants fed con or hm at 7 months ( 7 . 7 vs 3 . 62 and 5 . 40 , respectively , p & lt ; 0 . 05 ) and at 12 months ( 1 . 35 vs 0 . 68 and 0 . 82 , respectively , p & lt ; 0 . 05 ). hib response was also measured as hib specific igg , and the results paralleled the hib farr values at 6 and 7 months . this parameter was not measured at 12 months . response to the diphtheria vaccine was measured as diphtheria toxoid specific igg . there were no differences between groups at 6 or 12 months , but at 7 months infants fed nuc had a significantly ( p & lt ; 0 . 05 ) higher response ( 1 . 77 u / ml ) than infants fed hm ( 1 . 29 u / ml ). see table v ). there were no differences at any time point for tetanus specific igg . table iv______________________________________vaccine response median ( n ) nuc con hm______________________________________6 monthshib farr ( μg ig / ml ). sup . 1 0 . 43 ( 93 ). sup . a 0 . 36 ( 96 ). sup . a , b 0 . 30 ( 97 ). sup . bhib igg ( mg / ml ) 0 . 06 ( 94 ). sup . a 0 . 06 ( 101 ). sup . a , b 0 . 03 ( 99 ). sup . bdiphtheria igg ( u / ml ) 0 . 47 ( 78 ) 0 . 32 ( 85 ) 0 . 36 ( 80 ) tetanus ( igg ( u / ml ) 0 . 71 ( 80 ) 0 . 72 ( 82 ) 0 . 53 ( 80 ) 7 monthshib farr ( μg ig / ml ) 7 . 70 ( 94 ). sup . a 3 . 62 ( 101 ). sup . b 5 . 40 ( 99 ). sup . bhib igg ( mg / ml ). sup . 2 1 . 25 ( 93 ) 0 . 63 ( 101 ) 0 . 60 ( 97 ) diphtheria igg ( u / ml ) 0 . 70 ( 85 ). sup . a 1 . 53 ( 89 ). sup . a , b 1 . 42 ( 90 ). sup . btetanus igg ( u / ml ) 5 . 01 ( 86 ) 4 . 47 ( 90 ) 4 . 75 ( 91 ) 12 monthshib farr ( μg ig / ml ) 1 . 35 ( 89 ). sup . a 0 . 68 ( 94 ). sup . b 0 . 82 ( 95 ). sup . bhib igg ( mg / ml ) nd . sup . 3 nd nddiphtheria igg ( u / ml ) 0 . 30 ( 82 ) 0 . 24 ( 87 ) 0 . 30 ( 84 ) tetanus igg ( u / ml ) 0 . 92 ( 83 ) 0 . 84 ( 87 ) 0 . 90 ( 85 ) ______________________________________ . sup . 1 values in the same horizontal row with different superscripts ( a o b ) are significantly different , p & lt ; 0 . 05 . . sup . 2 p & lt ; 0 . 05 , no pairwise differences nd = not determined table v______________________________________vaccine response geometric mean ( n ). sup . 1 nuc con hm______________________________________6 monthshib farr ( μg ig / ml ) 1 . 30 ( 93 ). sup . a 1 . 24 ( 96 ). sup . a , b 1 . 23 ( 97 ). sup . bdiphtheria igg ( u / ml ) 0 . 36 ( 78 ) 0 . 28 ( 85 ) 0 . 33 ( 80 ) 7 monthshib farr ( μg ig / ml ) 7 . 24 ( 94 ). sup . a 4 . 05 ( 101 ). sup . b 4 . 21 ( 99 ). sup . bdiphtheria igg ( u / ml ) 1 . 77 ( 85 ). sup . a 1 . 38 ( 89 ). sup . a , b 1 . 29 ( 90 ). sup . b12 monthshib farr ( μg ig / ml ) 1 . 41 ( 89 ). sup . a 0 . 76 ( 94 ). sup . b 0 . 85 ( 95 ). sup . bdiphtheria igg ( u / ml ) 0 . 33 ( 82 ) 0 . 25 ( 87 ) 0 . 27 ( 84 ) ______________________________________ . sup . 1 values in the same horizontal row with different superscripts ( a o b ) are significantly different ; p & lt ; 0 . 05 . . sup . 2 p & lt ; 0 . 05 , no pairwise differences it is generally accepted that a hib farr level of antibody greater than 1 μg of ig / ml one month after immunization imparts protection to the infant . the percent of infants who had this level of protection was determined from the data set and is set forth in table vi . the infants fed the nuc formula consistently had a 10 % greater protection rate than infants in the other two groups . table vi______________________________________hib protection rate (% of subjects with & gt ; 1 μg anti - hib ig ( ml ) nuc con hm______________________________________ 6 months 28 % 18 % 16 % 7 months 90 % 80 % 80 % 12 months 55 % 44 % 45 % ______________________________________ natural killer ( nk ) cell activity was similar in all three groups . hm group had significantly higher numbers of nk cells ( p & lt ; 0 . 05 ) than nuc at 2 , 6 , and 12 months and con at 2 , 7 , and 12 months . formula - fed infants had a higher percent cd4 cells at 2 months ( nuc , con & gt ; hm ; p & lt ; 0 . 005 ), 7 months ( con , nuc & gt ; hm ; p & lt ; 0 . 01 ), and 12 months ( nuc & gt ; hm ; p & lt ; 0 . 05 ). the nk activity data are presented in table vii . table vii______________________________________nk activity . sup . 1 nuc con hm______________________________________ 2 months 11 . 2 8 . 0 9 . 0 6 months 9 . 0 12 . 6 9 . 0 7 months 13 . 9 14 . 3 13 . 012 months 19 . 4 21 . 3 21 . 4______________________________________ . sup . 1 values are % target cells killed at effector : target ratio of 50 : 1 . part of the impetus for this study and evidence that different ratios and levels of nucleotides impact on different physiological parameters was the report by carver et al . ( pediatrics 1991 ; 88 : 359 ) that infants fed nucleotide - fortified sma ® ( infant nutritional sold by wyeth , inc . believed to contain 21 mg cmp ; 6 . 0 mg amp ; 6 . 0 mg ump ; 6 . 0 mg amp and 3 . 0 mg imp per liter of formula ) had significantly higher nk activity than those fed unfortified sma . the present study , using the formula according to the instant invention , shows no effect of nucleotides on nk activity at 2 months and in fact no difference , among any of the groups at any time . given the small number of infants in the carver study ( 42 degrees of freedom at 2 months ) compared to this study ( 255 degrees of freedom at 2 months ), it would seem likely the carver data are an aberration due to small sample size or , the addition of nucleotides does not increase number of nk cells or , the types and levels of nucleotides used by carver produced only a cellular response as opposed to the humoral response seen in this invention . the anthropometric measurements indicate that growth was comparable among all infants in the study . the fact that even before controlling for birth values there were no differences among males for weight , length , or head circumference gives assurance that growth was acceptable among all groups . the higher stool frequency and number of feedings per day of hm - fed infants compared to formula - fed infants during the first 2 months is well established . softer stools of hm - fed infants are also common , although only the nuc group was different at 2 months and by a small amount . overall , the measures of tolerance among all groups were very similar through 4 months when half the infants were still being exclusively breastfed . these data demonstrate both formulas were extremely well tolerated and are set forth in table ix . table ix______________________________________intake and tolerance mean ( sem ). sup . 1______________________________________2 months nuc con hm 100 107 103______________________________________feedings (#/ day ) 6 . 2 ( 0 . 1 ) 6 . 4 ( 0 . 1 ) 7 . 7 ( 0 . 2 ) intake ( ml / day ) 831 ( 19 ) 823 ( 18 ) ndspit - up (% of feedings ) 8 ( 2 ) 18 ( 2 ) 20 ( 2 ) stool frequency (#/ day ) 1 . 6 ( 0 . 1 ) 1 . 4 ( 0 . 1 ) 2 . 7 ( 0 . 2 ) stool consistency . sup . 2 2 . 0 ( 0 . 1 ) 1 . 9 ( 0 . 1 ) 1 . 7 ( 0 . 1 ) ______________________________________4 months nuc con hm 98 107 103______________________________________feedings (#/ day ) 5 . 9 ( 0 . 1 ) 6 . 0 ( 0 . 1 ) 6 . 6 ( 0 . 2 ) intake ( ml / day ) 987 ( 33 ) 926 ( 17 ) ndspit - up (% of feedings ) 22 ( 2 ) 18 ( 2 ) 20 ( 2 ) stool frequency (#/ day ) 1 . 4 ( 0 . 1 ) 1 . 4 ( 0 . 1 ) 1 . 5 ( 0 . 1 ) stool consistency . sup . 2 2 . 0 ( 0 . 1 ) 2 . 1 ( 0 . 1 ) 2 . 1 ( 0 . 1 ) ______________________________________ . sup . 1 values in the same row with different superscripts are significantly different ; p & lt ; 0 . 05 . . sup . 2 mean rank consistency , where 1 = watery , 2 = mushy , 3 = soft , 4 = formed , 5 = hard . the differential white counts and lymphocyte subset numbers of all infants receiving the formula according to this invention were well within normal ranges throughout the first year of life . the vaccine response in this study was intended to be an immunological probe or indicator as to the responsiveness of the immune system in general . on the humoral side , tetanus toxoid vaccine was selected because it is a strong antigen , diphtheria toxoid was selected as a vaccine containing a weaker antigen , and hib vaccine was selected as a very weak antigen that requires conjugation to a carrier protein to achieve a t - cell dependent immune response to the hib polysaccharide component of the vaccine to be effective . it was thought that if nutritional intervention could evoke a difference in response that could be measured , that difference would more likely occur with the weaker antigens . while all infants would be expected to respond well to a strong antigen , like tetanus toxoid , a less vigorous response would be expected to a weak antigen . the lederle hib titer ® was selected specifically because the literature indicated that infants responded rather weakly after the first and second immunizations . furthermore , the protein used as the conjugate in this vaccine , the crm 197 protein ( a non - toxic mutant diphtheria toxin ), is antigenically very similar to diphtheria toxoid . diphtheria toxoid vaccination also represents a response to a moderately weak antigen and correlates with immune response to the h . influenzae conjugate vaccine with the crm 197 protein carrier . the vaccine response at 6 months is taken from blood drawn immediately before the 6 - month vaccination and represents the response 2 months after the second immunization given at 4 months of age . already at that time point the hib response was significantly higher in nuc than hm for both anti - hib igg and hib farr antibody . at 7 months , one month after the third immunization , nuc is significantly higher than con and hm for hib farr . hib igg is higher at 7 months , and although there are not pairwise differences , the nuc group is double con and hm ( 1 . 25 vs 0 . 63 and 0 . 60 , respectively ). the hib farr value was still significantly higher for nucs at 12 months . for this weak antigen , a difference was first seen at 6 months . the difference became stronger at 7 months when the maximum response was expected and was maintained through 12 months of age . in response to the moderately weak antigen diphtheria vaccine , there were no differences at 6 months , but at 7 months the nuc group was significantly higher than hm . by 12 months this difference was no longer present . for the moderately weak antigen , the direction of the present difference was the same as with the weak antigen ( hib ) but was different only at the point of highest response . for the strong antigen , tetanus , there were no differences among feeding groups at any time point . these data strongly support the instant invention of specific nucleotide equivalents at specific levels and ratios to enhance the immune system . in this example and in commercial production of enteral formulas according to the invention , background levels of nucleotide equivalents are determined and then the formula would be supplemented with appropriate commodities , such as cmp , amp , ump and gmp , to the claimed levels and ratios . it should be remembered that by nucleotide equivalents is meant ribo - nucleotides , ribo - nucleosides , rna , and ribo - nucleotide adjuncts , such as activated sugars . the sum of all these elements determine the total potentially available ribo - nucleotide equivalents . two additional pieces of data strongly support that the formula according to this invention provides an unexpected result . the number of subjects who have achieved protective levels of anti - hib immunoglobulin as shown in table vii is consistently 10 % higher in the nuc group . the three - way comparison does not show a statistical difference . however , a two - way comparison between the nuc and con formula groups at 7 months is significant ( p & lt ; 0 . 05 ). an additional piece of data comes from two of the clinical sites which chose to collect morbidity data . as part of the study the incidence of diarrhea was determined at the two clinical study sites . of 26 infants fed the nuc formula , only two reported diarrhea while 10 of 29 reported diarrhea in the con formula . the χ 2 analysis comparing the incidence of diarrhea in infants fed the two formulas is significant ( p & lt ; 0 . 05 ). in summary , the improved response to vaccination , the higher percent of subjects who have protective levels of antibodies , and the reduced incidence of diarrhea show that infants consuming the nucleotide - fortified formula according to this invention achieve enhanced immunological development as compared to those consuming the control formula . the results from these experiments demonstrate that the enteral formula of this invention is effective in enhancing the immune system and treating diarrhea . the medical community is constantly searching for nutritional formulas that will benefit the infant . the present invention can clearly fill that need . the nucleotide equivalent level of the formula in the study is about the minimum for efficacious effect . additionally , the formula is nutritionally complete as an infant formula . the manufacture of the formula utilizes conventional equipment and may be readily accomplished . while the infant formula and method of making said formula herein described constitute a preferred embodiment of this invention , it is to be understood that the invention is not limited to this precise formulation or method and that changes may be made therein without departing from the scope of the invention which is defined in the appended claims .
an improved enteral nutritional formula containing ribo - nucleotide equivalents at a level of at least 10 mg / 100 kcal of formula is disclosed . the formula comprises carbohydrates , lipids , proteins , vitamins and minerals and four ribo - nucleotide equivalents at specific levels and ratios . the invention also discloses novel methods of production and analytical techniques . this invention also provides a dietary formula that enhances the immune system and alleviates diarrhea .