Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/64—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving ketones
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/20—Oxygen containing
  • Y10T436/200833—Carbonyl, ether, aldehyde or ketone containing

Definitions

• This invention relates to a new and improved diagnostic composition and to a method of testing for phenylketonuria. Particularly, this invention is concerned with a diagnostic composition incorporated upon a bibulous strip which is useful for the qualitative detection and quantitative estimation of phenylketones in body fluids and especially phenylpyruvic acid in urine.
• phenylpyruvic acid in urine is indicative of phenylketonuria, a serious metabolic disorder resulting from the incomplete metabolism of aromatic amino acids.
• Pathologically phenylketonuria manifests itself as a syndrome identified by severe mental retardation, decreased pigmentation, epileptic seizures, dematoses and certain neurologic disorders, like poor motor control.
• Biochemically phenylketonuria indicates the failure of the body tissues to properly metabolize phenylalanine, which is an essential amino acid required for the synthesis of most proteins. In patients with phenylketonuria the excessive phenylalanine undergoes oxidative deamination to phenylpyruvic acid (phenylpyruvate) which is excreted in the urine.
• One method of treating this disease is directed towards decreasing the intake of phenylalanine, since the mental retardation in phenylketonuria is due to intoxication by phenylalanine or one of its metabolites.
• a diet low in phenylalanine is now commercially available and is in current use, viz. Ketonil, etc.
• urine specimens of suspects are universally screened by means of a test involving several steps and utilizing a ferric chloride or a ferric ammonium sulfate solution.
• the appearance within minutes of a deep green or blue-green color (sometimes an opaque deep blue-gray, owing to precipitated phosphates) after addition of a few drops of a 5% or 10% ferric chloride or a 10% ferric ammonium sulfate solution to either the fresh urine of suspects or such urine acidified by sulfuric acid or acetic acid may indicate the presence of phenylpyruvic acid. This likelihood is then ordinarily verified by subsequent reaction of such a urine specimen with a 2,4-dinitrophenylhydrazine and isolation and identification of the hydrazone formed.
• Another object of this invention is to provide a diagnostic composition for detecting phenylpyruvic acid in urine, which is in the form of a bibulous strip or stick.
• a diagnostic composition according to the present invention comprises an iron salt as a source of ferric ions for the chromogenic reaction with phenylpyruvic acid, an organic acid to impede the hydrolysis of the iron salt, and an additional salt as a phosphate complexing agent, all of these ingredients being carried by a bibulous base material or carrier, such as strips of filter paper.
• a bibulous base material or carrier such as strips of filter paper.
• I prefer to prepare my diagnostic composition by dissolving these components in a suitable solvent or mixture of solvents and impregnating a bibulous body (e.g., a strip of filter paper) with the resulting solution, thereafter drying the impregnated bibulous body.
• an organic acid such as lactic, malic, succinic, cyclohexylsulfamic acids
• the organic acid in the composition reacts with ferric ions to form salts, such as ferric lactate, ferric malate, ferric succinate, etc., which are less-readily hydrolyzed, thus checking the undesirable formation of ferric hydroxide.
• the color reaction may be further improved by addition of a phosphate complexing agent, such as magnesium sulfate, aluminum sulfate, aluminum ammonium sulfate, barium chloride, or calcium chloride.
• a phosphate complexing agent such as magnesium sulfate, aluminum sulfate, aluminum ammonium sulfate, barium chloride, or calcium chloride.
• phosphates and other constituents of urine tend to bind ferric ions so that fewer are available for the chromogenic reaction with phenylpyruvic acid in consequence of which the color indications are less distinct.
• Mg++, A1+++, Ba or Ca++ obviates this drawback.
• the impregnating solution can be prepared by dissolving the components in water only, it has further been found that an alcohol content of up to 20%, supplied by addition of ethanol, aids in producing a test strip which gives more uniform distribution of the reagent throughout the impregnated area of the strip, and consequently more uniform color indications.
• N-propanol and isopropanol in concentrations of up to 10% as well as N-butanol in a concentration of up to 1% may be substituted for ethanol with similar effects.
• Cyclohexylsulfamic acid solution was prepared by placing 21.5 g. in about 75 ml. of water, gently heating until solution was complete and then diluting it to a total volume of 100 ml. with distilled water.
• Magnesium sulfate solution was prepared by dissolving 29.5 g. of reagent grade magnesium sulfate (MgSO -7H O) in distilled water and diluting it to a total volume of 100 ml. with distilled water.
• MgSO -7H O reagent grade magnesium sulfate
• the impregnating solution was then prepared by mixing in 250 ml. Erlenmeyer flask at room temperature 30 ml. of the ferric ammonium sulfate solution with 30 ml. of the magnesium sulfate solution, ml. of the cyclohexylsulfamic acid solution measured out while the solution was warm (70 C.), since a 1.2 molar concentration cannot be obtained at room temperature (3.), and 20 ml. of special denatured ethyl alcohol, 2B. These solutions were thoroughly admixed by swirling the flask upon the addition of each solution. The mixture thus prepared remains stable without any precipitation.
• An equally operable impregnating solution may be prepared by weighing out solid chemicals and dissolving them in water or in a solution containing 20 ml. of special denatured alcohol, 213, per 100 ml. of solution.
• a solution containing 20 ml. of special denatured alcohol, 213, per 100 ml. of solution 5.8 g. of ferric ammonium sulfate 8.9 g. of magnesium sulfate (MgSO -7H O), 6.5 g. of cyclohexylsulfamic acid were dissolved in water containing 20 ml. of special denatured alcohol, 2B, per 100 ml. of solution. Then the solution was diluted to a total volume of 100 ml. with the same alcohol-water solution.
• the impregnating solution was poured into a shallow flat bottom dish to a depth of about inch.
• Test Strip Bibulous strips, such as filter paper cut into narrow strips, small sticks of wood, or other porous or absorbing material with a Water impervious barrier of ethyl cellulose /2 inch from tip were dipped into the solution so that through the process of submersion and capillary attraction the entire /2 inch of the strip up to the barrier was completely impregnated. The dipped strips were then placed in drying racks with the clipped ends up. The strips were allowed to stand on the laboratory desk at room temperature for 15 to 20 minutes and then placed in a hot air oven at 90110 C. for 15 minutes.
• EXAMPLE II Formulation 50 ml. of ferric ammonium sulfate, 0.4 F.W. 20 ml. of gluconic acid, 1.2 M. 27.0 g. of aluminum ammonium sulfate. ml. of distilled water.
• the ferric ammonium sulfate solution was prepared by dissolving 19.3 g. of reagent grade ferric ammonium sulfate [FeNH (SO -12H O OI Fe (SO 3 804 in distilled water and diluting it with distilled water to a total volume of 100 ml. the gluconic acid, by dissolving 23.5 g. of a-glucono-lactone in distilled water and diluting it with distilled water to a total volume of 100 ml. Because of its limited solubility, aluminum ammonium sulfate [AlNH (SO --12H O, 27.0 g.] was i added in solid form directly to the impregnating mixture along with 30 ml. of distilled water. Heat was then gently applied till the solution was completed.
• the impregnating mixture may also be produced by first mixing the solid compounds and then dissolving them in water.
• 9.7 g. of ferric ammonium sulfate, 27.0 g. of aluminum ammonium sulfate and 4.7 g. of 6-glucono-lactone were weighed into a 250 ml. Erlenmeyer flask, about 75-80 ml. of Watcr added, and the components dissolved by the gentle application of heat. V lhen the solution was complete, distilled water was added to a total volume of ml. The solution was kept at about 70 C. until the strips were dipped.
• test strips were then made as described in Example 1.
• EXAMPLE III Formulation 50 ml. of ferric sulfate, 0.2 M 30 ml. of magnesium sulfate, 1.2 M 20 ml. of fi-glucono-lactone Preparation
• the ferric sulfate solution was prepared by dissolving 10 g. of reagent grade Fe (SO -xI-I O in distilled water and diluting it with water to a total volume of 100 ml.; the magnesium sulfate solution, by dissolving 29.6 g. of reagent grade MgSO '7I-I O in distilled water and diluting it with distilled water to a total volume of 100 ml.; and the gluconic acid solution was prepared as described in Example Ii.
• the impregnating solution may be produced by first mixing the solid compounds and then dissolving them in water.
• 5 g. of ferric sul- 8.9 g. of magnesium sulfate (MgSO -7H O) and 4.7 g. of fi-glucono-lactone were weighed into a 250 ml. Erlenmeyer flask, dissolved in distilled water and diluted with distilled water to a total volume of 100 ml.
• an impregnated strip made in accordance with the invention, is dipped into the liquid specimen to be tested. It will give a positive reaction evidenced by a bluish-gray or green color within 10 to 20 seconds when contacted with urine containing as little as 100 ppm. of phenylpyruvic acid. Negative tests do not give this characteristic color but remain practically color-less or light butt in color. The intensity of the color varies with the content of phenylpymvic acid; from a light blue-gray at 100 ppm. to a dark bluish-gray at 3000 p.p.rn., depending on the composition of the test strip. This phenomenon may be utilized for preparing a convenient color chart.
• Chart 1 illustrates the possible combinations without, it is to be understood, limiting the scope of the invention to the compounds enumerated.
• a satisfactory diagnostic composition i.e., one that is stable and sensitive and gives a positive reaction when only 100 ppm. of phenylpyruvic acid are present in urine, may be obtained by regulating the ratio of the equivalents of ferric ions to that of the organic acid to about 3 to 2 in the impregnating solution.
• This ratio of ferric salt to organic acid is tantamount to a solution with a pH of 1.0 to 1.8. For optimum testing results a pH of 1.5 is especially preferred.
• the test strip becomes too brittle and fragile, and the color produced by a positive test fades rapidly; if the pH is above 1.8, the composition turns brown and becomes insensitive owing to the formation of ferric hydroxide and to the chelation of the ferric ions by the organic acid used.
• this invention pertains to a diagnostic composition for the detection of phenylketones in body fluids, especially phenylpyruvic acid in urine, which comprises a bibulous carrier or strip that has been impregnated With a composition consisting of an iron salt, an organic acid, and an additional salt which acts as a phosphate complexing agent, all of which are dissolved in a water-alcohol solution.
• the composition containing ferric ammonium sulfate, cyclohexylsulfamic acid and magnesium sulfate constitutes the specially preferred embodiment of this invention.
• the ingredients of the composition are so adjusted as to give a pH of 1.0 to 1.8 of the impregnating solution; a pH of 1.5 is especially preferred.
• a diagnostic composition for the detection of phenylketones in body fluids which comprises a bibulous carrier impregnated with a solution consisting essentially of a source of ferric ions selected from the group consisting of ferric sulfate, ferric ammonium sulfate and ferric chloride, an organic acid as an iron stabilizing agent selected from the group consisting of cyclohexylsulfamic,
• a phosphate complexing agent selected from the group consisting of magnesium sulfate, aluminum sulfate and aluminum ammonium sulfate, said solution having a pH Within a range of from 1.6 to 1.8.
• a diagnostic composition for the detection of phenylketones in body fluids which comprises a bibulous carrier impregnated with a solution consisting essentially of ferric ammonium sulfate, cyclohexylsulfamic acid and magnesium sulfate, said constituents being so adjusted as to result in a solution having a pH within a range of from 1.0 to 1.8.
• a diagnostic composition for the detection of phenylketones in body fluids which comprises a bibulous carrier in strip form impregnated with a solution comprising from 1.9 to 9.7 wt. percent of ferric ammonium sulfate, from 1.6 to 9.0 Wt. percent of cyclohexylsulfamic acid and from 2.5 to 24.7 wt. percent of magnesium sulfate, said solution having a pH within a range of from 1.0 to 1.8.
• a diagnostic composition for the detection of phenylpyruvic acid in urine which consists essentially of a bibulous strip which has been impregnated upon at least a portion thereof with a solution which consists essentially of about 5.8 wt. percent of ferric ammonium sulfate, about 4.3 wt. percent of cyclohexylsulfamic acid and about 13.3 wt. percent of magnesium sulfate, said solution having a pH of 1.8.
• a method of detecting phenylketones in a body fluid which comprises introducing into said liquid a diagnostic composition consisting of a bibulous carrier which has been impregnated upon at least a portion thereof with a solution which consists essentially of about 5.8 Wt. percent of ferric ammonium sulfate, about 4.3 Wt. percent of cyclohexylsulfamic acid and about 13.3 wt. percent of magnesium sulfate, said solution having a pH of 1.8.

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Citations (3)

• 0
  • IT IT609831D patent/IT609831A/it unknown
  • NL NL238977D patent/NL238977A/xx unknown
  • NL NL101097D patent/NL101097C/xx active
  • BE BE578901D patent/BE578901A/xx unknown
• 1958
  • 1958-06-16 US US741993A patent/US3048475A/en not_active Expired - Lifetime
• 1959
  • 1959-04-27 CH CH7258259A patent/CH377557A/de unknown
  • 1959-05-15 GB GB16798/59A patent/GB886709A/en not_active Expired
  • 1959-05-15 FR FR794831A patent/FR1230687A/fr not_active Expired
  • 1959-05-25 SE SE496059A patent/SE178407C1/sv unknown
  • 1959-06-05 DE DEM41743A patent/DE1169699B/de active Pending

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