Патент USA US3063827код для вставки
United States Patent 0 3,%3,8l2 C6 Patented Nov. 13, 1962 1 '2 3,063,812 DETERMINATEUN 0F ALBUMEW IN LEQUIDS Galen F. (Iollins, Ellrhart, Ind, assignor to Miles Labora tories, Inc., Eiiriiart, Ind, a corporation of lndiana No Drawing. Filed Apr. 2, 1957, Ser. No. 650,068 17 (Ilaims. ((11. 23-230) This invention relates to diagnostic compositions and, smaller proportions of dye may be used satisfactorily although with the smaller proportions the color is weak ened rendering the test correspondingly less sensitive, and with greater proportions the excess of dye is for the most part wasted. Inasmuch as the function of the cellulose in the com positions is to adsorb albumin on the surface of the tablet where it may be readily detected during the test, I prefer more particularly, to new and improved compositions for to use cellulose in such an amount that it accounts for the detection and quantitative estimation of albumin in 10 at least 35% of the weight of the total composition. In aqueous solutions such as urine. general, I prefer the cellulose component of my composi it is an object of the invention to provide improved diagnostic compositions in convenient tablet form which may be used with con?dence, even by unskilled persons, to detect and give a reliable quantitative estimation of albumin in urine and in other aqueous solutions. A further object is to provide diagnostic compositions for the determination of albumin in aqueous liquids which may be easily prepared from readily available materials tions to comprise from about 45% to about 55% by weight of the tablet; however, greater or lower percentages of cellulose powder may be used if desired, compositions having as little as about 25% and as much as about 75% by weight of cellulose based on the weight of the total composition giving satisfactory results. Four indicators which I have found to work very well in my improved compositions and which are preferred are brom phenol which is capable of detecting as little as about 5 mg. blue and brom cresol green, tetra brom phenol blue and percent (50 parts per million) of albumin in urine, and 20 tetrabromphenolphthalein ethyl ester. Another indica which may be stored over long periods of time without losing its sensitivity in testing for albumin. Other objects and advantages of the invention will be apparent from the following description thereof. The basis of the present invention is the phenomenon of “protein error” exhibited by certain indicators where tor which ‘will react as desired, but which gives a weak color during the test, is m(p~anilinophenylazo) benzene sulfonic acid sodium salt. The function of the acid reacting substance is to lower the pH of the urine below the acid-to-basic color change point of the indicator, which for brom phenol blue is pH 3.0, for brom cresol green is pH 3.8, for tetra brom go their characteristic acid-to-base color change at a phenol blue is pH 3.0, and for tetrabromphenolphthalein lower pH value than that at which they would change 30 ethyl ester is pH 4.0‘. When urine is the liquid tested, color in the absence of protein. That is to say, an indica by in solutions containing protein such indicators under tor which exhibits “protein error” will, in solution contain ing protein, under certain conditions show its basic color although the pH of the solution may actually be well be low that at which the color change normally occurs, and the extent to which the color of the solution is changed is the acid forms a buffer with the alkalinity of the urine which assists in maintaining the pH of the liquid below the color change point of the dye. Many solid acid reacting substances, including a large group of organic acids and a number of inorganic acid rea‘cting salts’, may be employed for this purpose, those having an ionization constant between about 10-5 and The mechanism by which the presence of protein enables about 10%, preferably about lO—3, and which are either certain dyes to exhibit their basic color in solutions having a pH below the acid-to-base color change point is not 4.0 slightly soluble or slowly soluble in' water being preferred. Suitable acid reacting substances are citric, tartaric, giy clearly understood but it is known that protein is limited collie, malic, fuma'ri'c, phthalic, malonic, ma'ndeli'c, glu in its range and capacity for bringing about this effect. taric, aconitic, mal‘eic,‘ benzoic, adipic, salicylic, gallic and Speci?cally, this color-change of the indicator, due to the gentisic acids, and- aluminum sulfate and aluminum chlo presence of protein, may be eifected only if the solution ride. Of these I prefer to use salicylic, fumaric or being tested has a ?nal pH not substantially lower than phthalic acid. about 1.25 pH units below the pH at which the normal an indication of the amount of protein in the solution. color change of the indicator occurs. in accordance with the invention, a diagnostic compo The acid used‘ must not be too strong, i.e., it must not have an ionization constant greater than 10"2 since such sition for determining albumin in liquids comprises a an acid would tend to‘ rapidly reduce the pH of the al which exhibits “protein error,” the mixture being dis The cellulose powder used in my compositions may be manufactured from‘ different source materials such as mixture of a major proportion of a solid acid reacting 50 bumin causing a rapid disappearance of the basic color of the dye characteristic of the‘ “positive” test. substance and a minor proportion of an indicator dye persed throughout an inert carrier or matrix of ?nely wood, cotton, etc’. I have found the‘ following speci?c divided cellulose, and all of these ingredients being com pressed into tablets of convenient size, e.g-., 7A5” in di 55 powdered cellulose products satisfactory for my purposes: B quality coarse grade cellulose powder, B quality stand ameter and weighing about .30 gram. As will be pointed and‘ grade cellulose powder (both products manufactured out in greater detail hereinafter, the test for albumin using by W 82R BalstonLiLtd‘J‘, and alpha cellulose (a product such a tablet requires only that a drop of the liquid speci manufactured by Brown Company and sold under the men to be tested, e.g.,~ urine, be placed on the tablet to produce any color reaction due to the presence of albumin. 60 trade name of Solka Floc‘ BW-lOO')‘. The‘ products do not necessarily need to be white'g‘howeven'the lighter the If albumin is not present, the color of the tablet remains the acid color of the indicator. However, if albumin is present in the specimen it will be adsorbed by thecel color, the‘ easier it' is to observe the color changes; Example] lulose and remain on the surface of the tablet where .it contacts the dye which is present there and causes the 65 ‘A, preferred for diagnostic compositions dye to change color while the remaining liquid portion comprises the following ingredients in the proportions of the specimen is absorbed by the tablet. indicated: . As would be expected, the proportion of dye in the present compositions is very small, good results being obtained using between about .02% and about .3%_ by 70 weight of dye based on the weight of tablet. This range of proportions is not critical, however, and greater or 7 Parts by weight Salicylic .acidug ____________________________ __ 600 Brom. phenol blue ___________________________ __ 1 Powdered wood- cellulose ____________________ _'__ 500 Corn starch_ ' ' __ 26 3,063,812 3 in liquids comprising a mixture of a major proportion of a member of the group consisting of salicylic, fumaric and phthalic acids and a minor proportion of an indicator dye selected from the group consisting of brom phenol blue, brom cresol green, tetra brom phenol blue, tetra tablets of about 716” diameter, having a weight of about .30 gram. The cornstarch serves as a binder to assist in tableting. If desired, other protein-free binders such as sucrose and dextrose may be used instead of the corn starch. 4 7. A diagnostic composition for determining albumin The foregoing ingredients in the quantities given are granulated, dried, intimately mixed and compressed into bromphenolphthalein ethyl ester and m(p-anilinophenyl , azo) benzene sulfonic acid sodium salt, and a carrier for Example 2 The following ingredients in the proportions given, said mixture consisting essentially of ?nely divided cel lulose, said ingredients being intimately mixed together tableted as in Example 1, provide a modi?cation of the 10 and compressed in tablet form. compositions of my invention: 8. A composition according to claim 7 wherein said indicator is brom phenol blue. 9. A composition according to claim 7 wherein said Parts by weight Sodium citrate-2H2O ________________________ __ 70 Citric acid anhydrous ________________________ .._ 180 ‘Tetrabromphenolphthalein ethyl ester __________ _.. 0.1 15 indicator is brom cresol green. 10. A composition according to claim 7 wherein said indicator is tetra brom phenol blue. 11. A composition according to claim 7 wherein said Cellulose powder (whatman “B” quality) _______ __ 300 Sucrose 26 indicator is tetrabromophenolphthalein ethyl ester. In testing a liquid specimen for albumin one drop of 12. A diagnostic composition for determining albumin the specimen is placed on a tablet prepared as described 20 in liquids comprising a mixture of a major proportion of above. After this drop is absorbed, two drops of Water salicylic acid and a minor proportion of brom phenol are placed on the tablet. If albumin is present in the blue, and a carrier for said mixture consisting essentially specimen it is adsorbed on the surface of the tablet while of ?nely divided cellulose, said ingredients being inti the liquid (e.g., urine) soaks into the mass of the tablet. The two added drops of water wash all but the albumin 25 mately mixed together and compressed in tablet form. 13. A diagnostic composition for determining albumin and reacted dye from the surface of the tablet so that in liquids comprising about 600 parts by weight of any color change due to the presence of albumin stands salicylic acid, about one part by weight of brom phenol out in detail. The presence of albumin is indicated by blue, about 560 parts by Weight of cellulose powder and the basic color of the indicator being observed on the about 26 parts by weight of corn starch, said ingredients surface of the tablet. In the absence of albumin, the ‘being intimately mixed together and compressed in tablet color of the tablet does not change from the acidic color form. of the dye. 14. A diagnostic composition for determining albumin I claim: in liquids comprising about 70 parts by weight of sodium 1. A diagnostic composition comprising a mixture of a major proportion of a solid organic acid and a minor 35 citrate dihydrate, about 180 parts by weight of anhydrous proportion of an indicator dye which exhibits “protein error,” said mixture being distributed throughout a matrix of powdered cellulose compressed in tablet form. 2. A diagnostic composition for determining albumin in liquids comprising a mixture of a major proportion 40 of an acid-reacting substance selected from the group con sisting of solid organic acids and acidic salts and a minor proportion of an indicator dye which exhibits “protein error,” and a carrier for said mixture consisting essentially of ?nely divided cellulose, said ingredients being inti lmately mixed together and compressed in tablet form. 3. A diagnostic composition in accordance with claim 2 including a protein-free binder for said ingredients se lected from the group consisting of corn starch, sucrose and dextrose. ' r citric acid, about 0.1 part by weight of tetrabromphenol phthalein ethyl ester, about 300 parts by weight of cellu lose powder and about 26 parts by weight of sucrose, said ingredients being intimately mixed together and com pressed in tablet ‘form. l5. In a method of determining the presence of albu min in a liquid wherein an acid reacting substance and an indicator dye exhibiting “protein error” serve as the determining media, the improvement which comprises adsorbing the albumin contained in the liquid on cellulose in the presence of said determining media, and separat ing the non-albumin constituents of the liquid from the adsorbed albumin by washing the latter. 16. The method of determining the presence of albu 50 min in a liquid comprising placing a drop of the liquid 4. A diagnostic composition for determining albumin in liquids comprising a mixture of a major proportion of 'on a tablet containing in intimate admixture: a major proportion of an acid-reacting substance selected from the a solid organic acid having an ionization constant between about 10*5 and about 10-2 and a minor proportion of an indicator dye which exhibits “protein error,” and a a minor proportion of an indicator dye which exhibits :“protein error,” and a carrier for said acid-reacting sub carrier for mixture consisting essentially of ?nely divided cellulose, said ingredients being intimately mixed together and compressed in tablet form. 5. A diagnostic composition comprising an intimate mixture, compressed in tablet form, of a solid acid react ing substance having an ionization constant between about 10-5 and about 10-2, an indicator dye which exhibits group consisting of solid organic acids and acidic salts, stance and dye consisting of ?nely divided cellulose; and, 'after said drop is absorbed in said tablet, placing thereon two drops of water, the presence of albumin being indi cated by the display of the basic color of the indicator on the surface of said tablet. 17. The method of determining the presence of albu min in a liquid comprising adsorbing the albumin from a drop of said liquid on a quantity of compressed powdered “protein error,” and powdered cellulose, the dye being cellulose in the presence of a protein determining medium present in an'arnount between about .02% and about 3% by weight, said powdered cellulose being present in an 65 consisting essentially of (a) a major proportion of an acid-reacting substance selected from the group con amount between about 25% and about 75% by weight sisting of solid organic acids and acidic salts, and (b) a based on the weight of the total composition. > minor proportion of an indicator dye which exhibits “pro 56. A diagnostic composition comprising an intimate tein error,” and separating the non-albumin constituents mixture compressed in tablet form, of a solid acid reacting substance having an ionization constant between about 70 of the liquid from the adsorbed albumin by washing the 10-5 and about 10*, an indicator dye which exhibits latter with two drops of water, the presence of albumin “protein‘error” and powdered cellulose, the dye being being indicated by the display of the basic color of the present in an amount between about .02% and about 3% indicator on the surface of the cellulose. by weight, said powdered cellulose forming at least 25% of the weight of said composition. 75 ., (References on following page) 5 8,068,812 6 References Cited in the ?le of this patent UNITED STATES PATENTS 2,171,961 2,178,550 2,281,758 2,314,336 2,331,573 2,387,244 2,854,317 2,912,309 OTHER REFERENCES Abstracts of Papers, 131st Meeting Am. Chem. Soc., Fortune ______________ __ Sept. 5, 1939 March 25, 1957, pages 750 and 76C. Acree ________________ __ Nov. 7, 1939 Fiegl: Microchim. Acta, v01. 2, pages 107-110, 1937. Kolmer et aL: “Approved Lab Technic,” 5th Ed. 1951, page 142. Galat ________________ __ May 5, 1942 Goodale _____________ __ Mar. 23, Sheftel _______________ __ Oct. 12, Compton ____________ __ Oct. 23, Free et a1 _____________ __ Sept. 30, 1943 1943 1945 1958 Free ________________ __ Nov. 10, 11959 Free: “Gastroenterology,” v01. 24, No. 3, July 3, 1953, pages 414 et seq.