we first developed a standard assay procedure ( the “ standard test ”). we used human breast milk collected and pooled from a newborn nursery as the β - glucuronidase source . the samples were stored at − 20 ° c . the milk was thawed and diluted 10 × with 0 . 2 m sodium acetate / glacial acetic acid ( naac ) buffer ph 4 . 5 just prior to being used . a 4 . 0 mm stock solution of 4 - methylumbelliferyl β - d - glucuronide ( mug , sigma m - 9130 lot # 75h3777 ) was prepared in 0 . 2 m naac buffer ph 4 . 5 . triplicate test and blank tubes contained a reaction mixture consisting of 50 μl diluted breast milk and 50 μl of an inhibitor solution diluted with 0 . 2 m naac buffer ph 4 . 5 . controls were prepared that contained only 50 μl diluted breast milk and 50 μl 0 . 2 m naac buffer ph 4 . 5 . the assay was started by adding 25 μl of the mug ( 4 . 0 mm , 0 . 800 mm final concentration ) to the test sample to start the reaction . the final volume of the reaction was 125 μl . both blank and test samples were vortexed and incubated at 37 ° c . for 1 hour ( or for shorter times if indicated ). the reaction was quenched in both the test and blank samples with 3 . 0 ml of glycine - carbonate ( 320 mm : 200 mm ) buffer ph 10 . 0 . the blank samples had 25 μl of the mug ( 4 . 0 mm , 0 . 800 mm final ) added to them and all tubes were vortexed . the resulting fluorescence was read using a perkin elmer ls5 - b fluorescence spectrophotometer ( 360 nm excitation , 445 nm emission , autorange scale ). the amount of 4 - methylumbelliferone ( 4 - mu ) released by the β - glucuronidase was calculated using a 4 - mu quadratic standard curve and the mathplot software . the velocity was calculated as pmoles of 4 - mu formed per hour per ml undiluted breast milk . we diluted pooled breast milk 20 × with 0 . 2 m naac buffer ph 4 . 5 . we diluted 2 . 2 % w / v solution of amicase ( sigma cat . # a2427 — a mixture of free amino acids with virtually no unhydrolyzed peptides , only minimal inorganic components are present , total nitrogen ˜ 12 . 8 %, amino nitrogen ˜ 9 . 8 %) with buffer 5 ×. we then incubated 50 μl milk with 50 μl buffer and amicase . we then added 25 μl mug to start the reaction . we then quenched with na - carbonate buffer ph 10 . 0 and read fluorescence at 360 nm ex 445 nm em . autorange scale . the mixture of amino acids was compared to controls and nutramigen ® as follows : we then studied the effect of specific amino acid mixtures on β - glucuronidase activity . we diluted pooled breast milk 10 × with 0 . 2 m naac buffer ph 4 . 5 and diluted 50 × stock essential amino acid solution ( sigma cat # m7020 = l - arginine . hcl 6 . 32 g / l , l - cystine 1 . 2 g / l , l - histidine . hcl . h20 2 . 1 g / l , l - isoleucine 2 . 625 g / l , l - leucine 2 . 62 g / l , l - lyscine . hcl 3 . 625 g / l , l - methionine 0 . 755 g / l , l - phenylalanine 1 . 65 g / l , l - threonine 2 . 38 g / l , l - tryptophane 0 . 51 g / l , l - tyrosine 1 . 8 g / l , l - valine 2 . 34 g / l ). we also used a non - essential aa solution = sigma cat # m7145 = l - alanine hcl 0 . 89 g / l , l - asparagine . h20 1 . 50 g / l , l - aspartic acid 1 . 33 g / l , l - glutamic acid 1 . 47 g / l , glycine 0 . 75 g / l , l - proline 1 . 15 g / l , l - serine 1 . 05 g / l . we 5 × diluted 100 × stock essential aa solution . we incubated 50 μl diluted milk with 50 μl buffer ( control ), or the essential aa solution , or the non - essential aa solution , or nutramigen ® rtu , added 25 μl mug to start the reaction , quenched with na - carbonate buffer , and read fluorescence ( 360 nm excitation : 445 nm emission ). the results are as follows : we then analyzed inhibitory effect of each of the seven amino acids in the non - essential amino acid mixture . we diluted pooled breast milk 10 × with 0 . 2 m naac buffer ph 4 . 5 , and diluted nutramigen ® rtu ( n ) and nutramigen ® lipid - free supernatant ( lfs ) 5 fold with 0 . 2 m naac buffer ph 4 . 5 . we prepared stock solutions of each of the non - essential amino acids l - alanine ( ala ), l - asparagine ( asn ), l - aspartic acid ( asp ), l - glutamic acid ( glu ), glycine ( gly ), l - proline ( pro ), and l - serine ( ser ). all solutions were at ph 4 . 5 . the final concentration of each amino acid in the assay was the same as when the amino acids were analyzed as a mixture . we then added 50 μl of diluted breast milk to 50 μl of stock solutions , added 25 μl mug to start the reaction , quenched with na - carbonate buffer ph 10 . 0 , and read fluorescence at 360 nm ex 445 nm em . the results are as follows : we concluded that the only one of these seven amino acids with significant β - glucuronidase inhibitory activity was l - aspartic acid . this inhibition was approximately the same as when these seven amino acids were tested for inhibitory activity together . we then tested the main carbohydrates in casein for their effect . we diluted pooled breast milk 10 × with 0 . 2 m naac buffer ph 4 . 5 and prepared stock solutions ( 25 mm , 2 . 5 mm and 0 . 25 mm ) of the 3 major carbohydrate constituents of casein *: d (+) galactose ( sigma cat # g6404 ), n - acetyl - d - galactosamine ( sigma cat # a2795 ), and n - acetylneuraminic acid ( sigma cat # a0812 ). we diluted nutramigen ® rtu ( n ) 5 × with 0 . 2 m naac buffer ph 4 . 5 , and added 50 μl of diluted breast milk to 50 μl of stock solutions , nutramigen ® or controls . controls ( c ) received buffer in place of test stock solutions . we added 25 μl mug to start the reaction , quenched with na - carbonate buffer ph 10 . 0 , and read fluorescence at 360 nm ex 445 nm em . the results are as follows for the following carbohydrates : n - acetyl - d - galactosamine ( nag ) ( 0 . 1 , 1 , 10 mm ) 98 - 100 %; n - acetylneuraminic acid ( 0 . 1 , 1 , 10 mm ) 99 - 100 %; galactose ( 0 . 1 , 1 , 10 mm ) 99 %. we then tested the effect of l - versus d - aspartic acid . we diluted pooled breast milk 10 × with 0 . 2 m naac buffer ph 4 . 5 and diluted nutramigen ® rtu 5 ×. we then prepared stock solutions of the amino acids , added 50 μl of diluted breast milk to 50 μl of stock solutions , and added 25 μl mug to start the reaction . we then quenched with na - carbonate buffer ph 10 . 0 and read fluorescence at 360 nm ex 445 nm em . the stock solutions were 2 . 5 × the final solution concentration . the results are as follows : l - aspartic acid showed significant β - glucuronidase inhibitory activity at all concentrations great than 10 μm . further β - glucuronidase inhibition by l - aspartic acid showed a dose response curve with maximum inhibition being at a level of only 8 . 4 percent of control at the highest concentration studied . d - aspartic acid was significantly inhibitory to β - glucuronidase only at the highest concentration , 10 , 000 μm . the inhibition of β - glucuronidase at this concentration was approximately equal to that of l - aspartic acid at 100 μm . thus , l - aspartic acid was approximately 100 times more potent than d - aspartic acid in β - glucuronidase inhibition . we also ran experiments testing the effect of ph on the inhibition by l - aspartic acid of β - glucuronidase . maximum inhibition was at about ph 5 . by similar experiments , we determined that l - malic acid also contributed to the inhibition of β - glucuronidase at similar concentrations to l - aspartic acid . we therefore believe that supplementing breast milk with l - malic acid would have similar effects . it is desirable to administer l - aspartic acid and / or l - malic acid to infants ( particularly neonates suffering from infant jaundice ). preferably , this is done via oral administration of breast milk mixed with exogenously supplied l - aspartic acid and / or l - malic acid . alternatively , other liquid carriers can be used such as water with 3 : 1 k : na at concentrations of potassium and sodium similar to breast milk . it is preferable to administer between 1 g and 5 g ( preferably between 1 g and 3 g ) of l - aspartic acid and / or l - malic acid per day to a neonate ( e . g . for the first seven days of the baby &# 39 ; s life ). typical unsupplemented breast feeding supplies about 400 mg of l - aspartic acid daily to a neonate . this can therefore be supplemented with about another 1100 mg . for example , each day the newborn can be supplied with six doses of 5 ml of supplement , with each supplement containing 180 mg of l - aspartic acid . alternatively , a standard casein hydrolysate infant formula ( liquid or dry powder ) can be supplemented with amounts of l - aspartic acid and / or l - malic acid that will deliver these amounts to the infant ( taking into account such amounts of l - aspartic acid and / or l - malic acid as may already be in the hydrosylate and such levels of dilution as are recommended when normally using the formula ). the present invention is not to be limited to the specific examples described above . in this regard , there are other modifications that are meant to be within the scope of the invention . thus , the claims should be looked to in order to judge the full scope of the invention . the present invention provides compounds useful for developing infant formulas and supplements , and methods of administering such compounds .
disclosed herein are infant dietary supplements containing l - aspartic and / or l - malic acid . the supplements are designed to inhibit β - glucuronidase activity in breast feeding babies , and thereby suppress serum bilirubin levels and the incidence of neonate jaundice . in one form l - aspartic acid is mixed with human breast milk and fed to the neonate on the same schedule that breast feeding would otherwise normally occur . in another form the l - aspartic acid is delivered in an aqueous solution supplemented with sodium and potassium .