US3048475A - Method and diagnostic composition of detecting phenylketones - Google Patents

Description

United States Patent Ofilice 3,048,475 Patented Aug. 7, 1962 spasms METHOD AND DIAGNUSTIC COMPOSHTIGN @F DETECTING PHENYLKETUNES Chauncey 0. Rape, Elkhart, Ind, assignor to Miles Laboratories, inc Ellrhart, Ind, a corporation of Indiana No Drawing. Filed June 16, 1958, Ser. No. 741,993 6 Claims. (Ct. 23-430) 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.

The presence of 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.

Since early detection and treatment of this biochemical aberration gives promise of normality, it is desirable that the urine of every baby or young child be tested on several occasions. Additional study is indicated in siblings of patients known to have phenylketonuria or in children in which there is the slightest suspicion of mental retardation.

According to present practices in the art, 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.

It has been found, however, that several substances interfere with this testing procedure, such as bile, diacetic acid, salicylates, etc., and that the color formed in consequence of such procedure is unstable, rather indistinct and prone to fade rapidly. It would be desirable, therefore, to have a simple test composition in convenient strip form possessing qualities that would eifectively avoid the disadvantages just discussed.

Accordingly, it is an object of this invention to provide an improved diagnostic composition for detecting phenylketonuria which reliably indicates the presence of phenylpyruvic acid in urine and wherein the color is distinct and does not fade rapidly.

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.

The advantages of a diagnostic in this form will become at once obvious: ease and simplicity of test procedure, absence of cumbersome equipment, ease of disposal of the test device, rapidity of testing procedure, etc.

Other objects as well as features and advantages of the invention will become apparent from the following detailed description:

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. 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.

Prior art, as indicated above, teaches the use of soluble ferric chloride or ferric ammonium sulfate in the presence of sulfuric acid for detecting phenylpyruvic acid in urine. However, if such a solution were used to impregnate a bibulous strip, no practical diagnostic product would be obtained because of the instability of the ferric salt and the corrosive action of sulfuric acid on the carrier. Strips made with ferric chloride or ferric ammonium sulfate alone are likewise unstable and are not very sensitive.

It has been found that the addition of an organic acid, such as lactic, malic, succinic, cyclohexylsulfamic acids, greatly improves both the sensitivity and the stability of the strip. 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.

It has also been found that 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. Without addition of such an agent, 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. The presence of Mg++, A1+++, Ba or Ca++, however, obviates this drawback.

Although 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.

The invention will be illustrated, but is not limited, by the following examples:

EXAMPLE I Formulation of Preferred Impregnating Solution 3 vol. of ferric ammonium sulfate, 0.4 F.W. (formula weight/ liter of solution).

3 vol. of magnesium sulfate, 1.2 M (molecular weight/liter of solution).

dissolving 19.3 g. of reagent grade ferric ammonium sulfate FeNHASOQ 12H O or Fe (SO (NHQ SO 241-1 in distilled water and diluting this to a total volume of 100 ml. with distilled water.

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.

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. Thus, 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.

Preparation of 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.

Other specific embodiments of my invention are illustrated by the following examples:

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.

Preparation 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. Thus, 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.

The 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.

Again, the impregnating solution may be produced by first mixing the solid compounds and then dissolving them in water. Thus, 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.

For the preparation of the test strips the procedure described in Example I was followed.

Procedure of Testing In use, 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.

In addition to the compositions described in the above examples, various other combinations of ferric salts, or ganic acids, and salts of magnesium, aluminum, barium or calcium will give a satisfactory product. Chart 1 illustrates the possible combinations without, it is to be understood, limiting the scope of the invention to the compounds enumerated.

CHART 2 Component Operable Range Optimum Content 1. Iron Salt 0.012 FW/lUO 0.004 to 0.02 I'WV/lOO 0.008 to 0.05 moles/ 0.01 to 0.1 moles/100 2. Organic Acid 3. Phosphate ComplexingAgent.

0.024 moles/100 0.05 moles/100 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. If the pH is below 1.2, 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.

In summary, 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.

What is claimed is:

1. 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,

maleic, malonic, and succinic acids, and 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.

2. 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.

3. 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.

4. 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.

5. A diagnostic composition according to claim 3 wherein said pH is 1.5.

6. 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.

References Cited in the file of this patent UNITED STATES PATENTS 2,577,978 Nicholls et a1. Dec. 11, 1951 2,848,308 Free Aug. 19, 1958 2,862,796 Gomberg Dec. 2, 1958 OTHER REFERENCES Fleury: Clinica Chimica Acta, vol. 2, 1957, pages 424 to 428.

Rupe et al.: Clin. Chem, 1957, vol. 3, pages 716727.

Hawk: Practical Phys. Chem, 13th ed., 1954, pages 840841.

Kenp: Biochem. Z., vol. 326, pages 14 to 17, 1954.

Nash et al.: Lancet, vol. 266, April-June 1954, pages 801-803.

Fiegl, Chem. of Spec., Select. and Sensitive Reaction, 1949, pages and 91.

Claims (1)

1. 6. 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.