Detection of appendicitis by measurement of orthohydroxyhippuric acid

• US 5,470,750 A
• Filed: 07/09/1992
• Issued: 11/28/1995
• Est. Priority Date: 11/15/1989
• Status: Expired due to Term

• Current Assignee: Ampio Pharmaceuticals, Inc.
• Original Assignee: Appenditech, Inc.
• Inventors: Bar-Or, David
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/911,159
• Time in Patent Office: 1,237 Days
• Field of Search: 435/4, 435/5, 435/6, 435/810, 435/10, 514/44, 514/1, 514/42, 436/501, 436/173, 436/8, 436/15, 436/63, 436/86, 436/106, 436/175, 436/178
• US Class Current: 436/63
• CPC Class Codes: G01N 2800/06 Gastro-intestinal diseases
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/523 the element being adapted for a specific analyte
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/105831 Protein or peptide standard or control [e.g., hemoglobin, etc.]
• CPC Class Codes: Y10T 436/17 Nitrogen containing
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

Appendicitis can be detected in human beings suspected of having appendicitis by determining a threshold level of 10 mg/liter of σ-hydroxyhippuric acid in the urine of such humans. This threshold level can be determined by qualitative, semiquantitative, or quantitative methods including HPLC (high pressure liquid chromatography), TLC (thin layer chromatography), radioimmunoassay, colorimetric tests, NMR (nuclear magnetic resonance), mass spectrometry, electrophoresis, monoclonal antibody tests, and enzymatic tests. The absence of appendicitis can be established by the presence of σ-hydroxyhippuric acid in concentrations less than about 10 mg/liter in a urine sample.

Claims

1. A method for diagnosing appendicitis, said method comprising:

obtaining a urine sample from a human being suspected of having appendicitis; and

determining, by a color-producing chemical reaction, that the concentration of σ

-hydroxyhippuric acid in the urine sample is at least about 10 mg/liter and thereby indicating the probable presence of appendicitis in said human being.

2. A method for diagnosing appendicitis, said method comprising:

obtaining a urine sample from a human being suspected of having appendicitis by virtue of nausea, vomiting or anorexia in association with abdominal pain, which is characteristically present in the periumbilical or right lower quadrant area; and

chemically extracting an σ

-hydroxyhippuric acid component from the urine sample; and

determining by a color-producing chemical reaction of the σ

-hydroxyhippuric acid component that the concentration of σ

-hydroxyhippuric acid in the urine sample was at least about 10 mg/liter and thereby indicating the probable presence of appendicitis in said human being.

3. A method for diagnosing appendicitis, said method comprising:

obtaining a urine sample from a human being suspected of having appendicitis by virtue of nausea, vomiting or anorexia in association with abdominal pain, which is characteristically present in the periumbilical or right lower quadrant area; and

determining by a color producing chemical reaction based upon use of ferric ion that the concentration of σ

-hydroxyhippuric acid in the urine sample is at least about 10 mg/liter and thereby indicating the probable presence of appendicitis in said human being.

4. A method for diagnosing appendicitis, said method comprising:

obtaining a urine sample from a human being suspected of having appendicitis by virtue of nausea, vomiting or anorexia in association with abdominal pain, which is characteristically present in the periumbilical or right lower quadrant area; and

chemically extracting an σ

-hydroxyhippuric acid component from the urine sample; and

determining by a color-producing chemical reaction based upon a reaction of ferric ion and the σ

-hydroxyhippuric acid component that the concentration of σ

-hydroxyhippuric acid in the urine sample was at least about 10 mg/liter and thereby indicating the probable presence of appendicitis in said human being.

5. A method for diagnosing appendicitis, said method comprising:

obtaining a urine sample from a human being suspected of having appendicitis by virtue of nausea, vomiting or anorexia in association with abdominal pain, which is characteristically present in the periumbilical or right lower quadrant area; and

verifying the presence of σ

-hydroxyhippuric acid in the urine sample in a concentration of at least 10 mg/liter by reacting the urine with an ion selected from the group consisting of Fe⁺⁺⁺ or Cu⁺⁺⁺ capable of entering into a color producing chemical reaction with the σ

-hydroxyhippuric acid; and

comparing the color of a product produced by the reaction of the ion selected from the group consisting of Fe⁺⁺⁺ or Cu⁺⁺⁺ and the σ

-hydroxyhippuric acid with a prepared color comparison chart having a color which correlates with a σ

-hydroxyhippuric acid concentration of at least 10 mg/liter and thereby indicating the probable presence of appendicitis in said human being.

6. A method for diagnosing appendicitis, said method comprising:

obtaining a urine sample from a human being suspected of having appendicitis by virtue of nausea, vomiting or anorexia in association with abdominal pain, which is characteristically present in the periumbilical or right lower quadrant area;

chemically extracting an σ

-hydroxyhippuric acid component from the urine sample using a chemical extraction system based upon the use of σ

-hydroxyhippuric acid--extracting compounds selected from the group consisting of ferric silica, diethylether, butanol, a C18 bonded silica used to place the σ

-hydroxyhippuric acid component in an aqueous environment or a C18 bond silica used to place the σ

-hydroxyhippuric acid component in a methanol environment, in order to prepare the σ

-hydroxyhippuric acid component to undergo a color reaction; and

determining that the concentration of σ

-hydroxyhippuric acid originally in the urine sample was at least 10 mg/liter by subjecting said σ

-hydroxyhippuric acid component to thin layer chromatography and then noting, with the aid of ultraviolet light having a wave length from about 250 nm to about 360 nm, the presence of a blue fluorescent spot with a retention factor equal to a reference standard based upon known concentrations of σ

-hydroxyhippuric acid and thereby indicating the probable presence of appendicitis in said human being.

7. A method for diagnosing appendicitis, said method comprising:

obtaining a urine sample from a human being suspected of having appendicitis by virtue of nausea, vomiting or anorexia in association with abdominal pain, which is characteristically present in the periumbilical or right lower quadrant area;

verifying the presence of σ

-hydroxyhippuric acid in a first portion of the urine sample by reacting the first portion of the urine with an ion of Fe⁺⁺⁺ bound to a silica gel and which are collectively capable of entering into a color producing chemical reaction with the σ

-hydroxyhippuric acid;

chemically extracting a σ

-hydroxyhippuric acid component from the urine sample using extraction chemical systems selected from the group consisting of diethylether, butanol, bonded ferric silica, a C18 bonded silica used to place the σ

-hydroxyhippuric acid component in an aqueous environment or a C18 bonded silica used to place the σ

-hydroxyhippuric acid component in a methanol environment; and

determining that the concentration of σ

-hydroxyhippuric acid in the component correlates with a σ

-hydroxyhippuric acid concentration of 10 mg/L in the urine sample was at least 10 mg/liter by subjecting said component to thin layer chromatography and then subjecting a sample resulting from the thin layer chromatography to a staining procedure, the presence of a blue spot with a predetermined retention factor, and thereby indicating the probable presence of appendicitis in said human being.

8. A method for diagnosing appendicitis, said method comprising:

obtaining a urine sample from a human being suspected of having appendicitis;

performing a first test by reacting a σ

-hydroxyhippuric acid component of a first portion of the urine sample with a ferric silica material to produce a color reaction; and

performing a second test by;

(1) extracting an σ

-hydroxyhippuric acid component from a second portion of the urine sample, (2) subjecting the σ

-hydroxyhippuric acid component to thin layer chromatography, (3) developing a colored spot on a thin layer chromatography film and (4) comparing the colored spot to a control color chart to determine that the concentration of σ

-hydroxyhippuric acid in the urine sample was at least about 10 mg/L and thereby indicating the probable presence of appendicitis in said human being.

9. A kit for determining the presence of σ

-hydroxyhippuric acid in human urine in a concentration of at least 10 mg/liter, said kit comprising;

a vial containing a reagent capable of undergoing a color-producing reaction with σ

-hydroxyhippuric acid;

means for adding a measured amount of urine to the reagent in order to produce a color reaction; and

a color comparison chart having a color which correlates with a σ

-hydroxyhippuric acid concentration of at least 10 mg/liter to which the color of a product of the color-producing reaction is compared.

10. A kit for determining the presence of σ

-hydroxyhippuric acid in human urine in a concentration of at least 10 mg/L, said kit comprising;

(1) chemical means for extracting σ

-hydroxyhippuric acid from a sample of human urine,

(2) thin layer chromatography means,

(3) a reagent capable of undergoing a color-producing reaction with the σ

-hydroxyhippuric acid extracted by the chemical means, and

(4) a color comparison chart having a color which correlates with a σ

-hydroxyhippuric acid concentration of at least 10 mg/liter to which the color of a product of the color-producing reaction is compared.

11. A kit for determining the presence of σ

-hydroxyhippuric acid in human urine in a concentration of at least 10 mg/L, said kit comprising;

a vial containing a chemical capable of undergoing a color-producing reaction with an σ

-hydroxyhippuric acid component of the human urine;

a chemical reagent capable of extracting an σ

-hydroxyhippuric acid component of the human urine;

thin layer chromatography means for acting upon the σ

-hydroxyhippuric acid component;

thin layer chromatography means for developing a colored spot on the thin layer chromatography means; and

color chart means for comparing the colored spot to said color chart means to determine that the concentration of σ

-hydroxyhippuric acid in the urine is at least about 10 mg/L and thereby indicating the probable presence of appendicitis in said human being.

12. A kit for determining the presence of σ

-hydroxyhippuric acid in human urine in a concentration of at least 10 mg/liter, said kit comprising;

a vial containing a ferric silica reagent to which a measured amount of urine is added;

means for adding a measured amount of urine to the ferric silica reagent to produce a color reaction; and

a color comparison chart having a color which correlates with a σ

-hydroxyhippuric acid concentration of at least 10 mg/liter to which the color of a product of the color reaction is compared.

13. A kit for determining the presence of σ

-hydroxyhippuric acid in human urine in a concentration of at least 10 mg/liter, said kit comprising;

a syringe containing a citrate buffer for receiving a measured amount of urine;

an extraction cartridge containing a C18 reagent and having means for receiving a fluid discharge from the syringe;

a vial of water for passing said water through the extraction cartridge containing the C18 reagent after the discharge from the syringe is passed through said extraction cartridge;

a vial of a solvent capable of extracting σ

-hydroxyhippuric acid from the C18 contained in the extraction cartridge;

a sample collection vial used to collect a solvent/σ

-hydroxyhippuric acid sample from the extraction cartridge;

a tank for holding the solvent/σ

-hydroxyhippuric acid sample;

a holder mounted thin layer chromatography plate having a first plate area designated to receive a sample of the solvent/σ

-hydroxyhippuric acid sample and a window area to receive a discharge from a swab impregnated with the solvent/σ

-hydroxyhippuric acid and dispensing at least a portion of said sample on a window area of said plate;

a swab; and

a control standard means having a color which correlates with a σ

-hydroxyhippuric acid concentration of at least 10 mg/liter comparing the location and color intensity of spots which appear in a window area of the plate with the control standard means.

14. A kit for determining the presence of σ

-hydroxyhippuric acid in human urine in a concentration of at least 10 mg/liter, said kit comprising;

a vial containing a ferric silica reagent to which a first measured amount of urine is added;

means for adding a measured amount of urine to the ferric silica reagent;

a color comparison chart;

a syringe containing a citrate buffer to receive a measured amount of a second measured amount of a urine sample;

an extraction cartridge containing a C18 reagent and having means for receiving a fluid discharge from the syringe;

a vial of water for passing said water through the extraction cartridge containing the C18 reagent after the discharge from the syringe is passed through said extraction cartridge;

a vial of a solvent capable of extracting σ

-hydroxyhippuric from the C18 contained in the extraction cartridge;

a sample collection vial used to collect a solvent/σ

-hydroxyhippuric acid sample from the extraction cartridge;

a tank for holding the solvent/σ

-hydroxyhippuric acid sample;

a holder mounted thin layer chromatography plate having a first plate area designated to receive a sample of the solvent/σ

-hydroxyhippuric acid sample and a window area to receive a discharge from a swab impregnated with the solvent/σ

-hydroxyhippuric acid and dispensing at least a portion of said sample on a window area of said plate;

a swab; and

a control standard means having a color which correlates with a σ

-hydroxyhippuric acid concentration of at least 10 mg/liter for comparing positions and color intensities of spots which appear in the window area of the plate with the said control standard means.

15. The kit of claim 14 wherein the vial containing the ferric silica reagent contains approximately 1 ml of ferric silica reagent.

16. The kit of claim 14 wherein the syringe containing the citrate buffer contains approximately 100 mg of citric acid as the citrate buffer and the solvent capable of extracting σ

-hydroxyhippuric acid from the C18 is an approximately 1 ml amount of methanol.

17. A method for diagnosing the absence of appendicitis, said method comprising:

obtaining a urine sample from a human being suspected of having appendicitis; and

determining, by a color-producing chemical reaction, that the concentration of σ

-hydroxyhippuric acid in the urine sample is less than about 10 mg/liter and thereby indicating the probable absence of appendicitis in said human being.

18. A kit for diagnosing the probable absence of appendicitis, said kit comprising:

chemical means for reacting σ

-hydroxyhippuric acid in human urine to produce a color reaction;

means for determining that the concentration of the σ

-hydroxyhippuric acid in the human urine is less than about 10 mg/liter; and

a color standard means having a color which correlates with a σ

-hydroxyhippuric acid concentration of less than about 10 mg/liter to which the color of a product of the color-producing reaction is compared;

thereby indicating the probable absence of appendicitis in said human being.